We reported elsewhere that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine, and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the α2 adrenergic receptor (α2-AR) antagonist, idazoxan or BRL44408, and was mimicked by the α2-AR-selective agonist, UK14304. A low concentration of Ba2+ inhibited NA-induced hyperpolarization, which suggests that activation of G protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by α1-AR antagonist, prazosin, which suggests the existence of α1-ARs on the orexin neurons along with α2-AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons; however, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the α2-AR, because the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via α1-ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner.
Orexin A and orexin B (also called hypocretin-1 and hypocretin-2) are a pair of neuropeptides implicated in the regulation of sleep/wakefulness and energy homeostasis (de Lecea et al. 1998; Sakurai 2005; Sakurai et al. 1998). Orexin neurons are exclusively located in the lateral hypothalamic area (LHA) and project to almost all parts of the brain (Nambu et al. 1999; Peyron et al. 1998). Especially dense projections are observed in monoaminergic nuclei such as the noradrenergic locus coeruleus (LC), serotonergic raphe nuclei, histaminergic tuberomammillary nucleus (TMN) and the dopaminergic ventral tegmental area (VTA). The activities of monoaminergic neurons in the brain stem and hypothalamus are reportedly synchronized and strongly associated with behavioral states: they fire tonically during wakefulness, less during non-REM sleep, and not at all during REM sleep. This regulation might be influenced by orexin neurons, which are also wake-active (Lee et al. 2005; Mileykovskiy et al. 2005), because orexin neurons project to and excite histaminergic neurons in the TMN, noradrenergic neurons in the LC, and serotonergic neurons in the dorsal raphe (DR). The presence of orexin receptor 1 (OX1R) in the LC and OX2R in the TMN and both receptors in the DR have been confirmed. Furthermore, orexins activate isolated cells from these nuclei in vitro (Brown et al. 2002; Eggermann et al. 2001; Hagan et al. 1999; Horvath et al. 1999; Nakamura et al. 2000; Takahashi et al. 2002; Yamanaka et al. 2002).
Mice lacking the orexin gene (prepro-orexin knockout mice) or orexin neurons (orexin/ataxin-3 transgenic mice) have phenotypes remarkably similar to the human sleep disorder narcolepsy (Chemelli et al. 1999; Hara et al. 2001). Consistent with these findings, human narcolepsy is accompanied by a loss of orexin neuropeptide production and specific destruction of orexin neurons (Nishino et al. 2000; Peyron et al. 2000; Thannickal et al. 2000). The involvement of orexin neurons in narcolepsy supports the theory that these neurons have important roles in the normal regulation of sleep/wakefulness states (Hungs and Mignot 2001; Kilduff and Peyron 2000; Sakurai 2005; Siegel 2004; Sutcliffe and de Lecea 2002; Taheri et al. 2002; Willie et al. 2001).
Electrophysiological and histological studies have shown that orexin neurons excite monoaminergic neurons (Hagan et al., 1999; Horvath et al., 1999; Bourgin et al., 2000; Nakamura et al., 2000; Brown et al., 2001; Eriksson et al., 2001; Brown et al., 2002; Yamanaka et al. 2003b). Conversely, serotonin (5-HT) and catecholamines, including noradrenaline (NA) and dopamine (DA), inhibit orexin neurons, whereas histamine has little effect on these cells (Yamanaka et al., 2003b). These observations suggest the possibility that orexin neurons receive direct inhibitory inputs from serotonergic and catecholaminergic neurons. Elsewhere we showed that activation of 5-HT1A receptors and G protein-activated inward rectifier potassium (GIRK) channels are involved in 5-HT-induced hyperpolarization of orexin neurons (Muraki et al., 2004). Li and van den Pol (2005) reported that orexin neurons are inhibited by noradrenaline through α2 adrenergic receptors (α2-AR) and GIRK channels in mice. Recent reports using rats, however, showed an intriguing result that orexin neurons are activated by NA but are inhibited when rats were sleep deprived for a few hours (Bayer et al. 2005; Grivel et al. 2005). Thus, the effects of catecholamines on the orexin neurons is still controversial. Here we report the effect of catecholamines on orexin neurons in detail by slice patch clamp and calcium imaging of orexin neurons using transgenic mice in which orexin neurons specifically express enhanced green fluorescent protein (EGFP) or a calcium-sensing protein (YC2.1). Electrophysiological experiments revealed that NA activates nonselective cation channels (NSCCs) and GIRK channels on the orexin neurons through α1-AR and α2-AR, respectively. The direct effects of NA on orexin neurons, however, are a balance between an α1-AR-mediated depolarization and α2-AR-mediated hyperpolarization. In addition, NA indirectly inhibited orexin neurons through an α1-AR-mediated increase in GABAergic inhibitory inputs and through an α2-AR-mediated decrease in glutamatergic excitatory inputs. On the other hand, NA inhibited both miniature EPSCs (mEPSCs) or IPSCs through the α2-AR, which is located on the presynaptic membrane of these glutamatergic or GABA-ergic interneuron. Calcium imaging experiments revealed that the NA effect on the orexin neurons was not altered by either sleep deprivation (SD) or circadian time in mice.
All experimental procedures involving animals were approved by the University of Tsukuba Animal Resource Center and were in accordance with National Institutes of Health guidelines. All efforts were made to minimize animal suffering or discomfort and to reduce the number of animals used.
Male and female orexin/EGFP mice (3–4 wk old) in which the human prepro-orexin promoter drives expression of EGFP (lines E2 and E7; Muraki et al. 2004; Yamanaka et al. 2003a; Yamanaka et al. 2003b), were used for experiments. The mice were deeply anesthetized with fluothane (Takeda, Osaka, Japan) and then decapitated. The brains were isolated in ice-cold bubbled (100% O2) physiological solution containing (mM): sucrose 280, KCl 2, MgCl2 10, CaCl2 0.5, HEPES 10, glucose 10, pH 7.4 with NaOH. Brains were cut coronally into 300-μm slices with a microtome (VTA-1000S, Leica, Germany). Slices containing the hypothalamus were transferred for ≥1 h to an incubation chamber at room temperature (RT; 24–26°C) filled with physiological solution containing (mM): NaCl 140, KCl 2, CaCl2 1, MgCl2 1, HEPES 10, glucose 10, pH 7.4 with NaOH. Some experiments were also conducted in physiological bicarbonate buffer containing (mM): NaCl 125; KCl 2.0; CaCl2 1; MgCl2 1; NaHCO3 26; NaHPO4 1.25; glucose 10. For electrophysiological recording, the slices were transferred to a recording chamber (RC-26G, Warner Instrument, Hamden, CT) at a controlled temperature of 34°C on a fluorescence microscope stage (BX51WI, Olympus, Tokyo). The slices were superfused with physiological solution that was warmed by an in-line heater (Warner Instrument) to 34°C before entering the recording chamber at a rate of 3 ml/min using a peristaltic pump (Dynamax, Rainin Instruments, Oakland, CA). The fluorescence microscope was equipped with an infrared camera (C2741-79, Hamamatsu Photonics, Hamamatsu, Japan) for infrared differential interference contrast (IR-DIC) imaging and a charge-coupled device (CCD) camera (IK-TU51CU, Olympus) for fluorescent imaging. Each image was displayed separately on a monitor (Gawin, EIZO, Tokyo) and was saved on a Power Macintosh G4 computer (Apple, Cupertino, CA) through a graphic converter (PIX-MPTV, Pixcela, Osaka, Japan).
Patch pipettesgips prepared from borosilicate glass capillaries (GC150-10, Harvard Apparatus, Holliston, MA) with a micropipette puller (P-97, Sutter Instruments, Pangbourne, UK). The pipettes were filled with an internal solution containing (mM): KCl 145; MgCl2 1; EGTA-Na3 1.1; HEPES 10; MgATP 2; NaGTP 0.5; pH 7.2 with KOH. Osmolarity of the solution was checked by a vapor pressure osmometer (model 5520, Wescor, Logan, UT). Pipette resistance was 4–10 MΩ. The series resistance during recording was 10–25 MΩ and was not compensated. The osmolarities of the internal and external solution were 280–290 and 320–330 mOsm/l, respectively. The liquid junction potential of the patch pipette solution and perfused HEPES solution was estimated to be 3.9 mV and was applied to the data. Recording pipettes were advanced toward individual cells in the slice while under positive pressure. On contact, tight seals on the order of 0.5–1.0 GΩ were made by negative pressure. The membrane patch was then ruptured by suction, and membrane current and potential were monitored using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA). Depolarizing and hyperpolarizing current pulses were applied to cells at durations of 200 ms at 20 pA steps at 2-s intervals from the resting membrane potential (−60 mV) set by varying the intensity of a constantly injected current. The reference electrode was an Ag-AgCl pellet immersed in bath solution. All current clamp recordings were made in Axopatch 200B fast mode. The membrane capacitance was calculated by dividing the time constant by the input resistance. Input resistance was calculated from the slope of the current-voltage relationship. The output signal was low-pass filtered at 5 kHz and digitized at 10 kHz. Data were recorded on a computer through a Digidata 1322A A/D converter using p-Clamp software version 8.2 (Axon Instruments). The trace was processed for presentation using Origin 6.1 (Origin Lab Corporation, Northampton, MA) and Canvas 9.0 (ACD Systems, Miami, FL) software. Miniature PSCs (mPSCs) were recorded in the presence of TTX (1 μM) in the extracellular solution. Spontaneous PSCs (sPSCs) were recorded in the absence of TTX. Spontaneous EPSCs (SEPSCs) and miniature EPSCs (mEPSCs) were recorded using KCl-based pipette solution containing the sodium channel blocker QX-314 (1 mM) to inhibit action potentials in the recording neuron and in the presence of picrotoxin (100 μM) in the bath to block GABAA receptor-mediated neurotransmission. To block NMDA and AMPA ionotropic glutamate receptor–mediated neurotransmission, sIPSCs and miniature IPSCs (mIPSCs) were recorded using the same pipette solution but in the presence of AP-5 (50 μM) and CNQX (20 μM), respectively. The frequency and amplitude of EPSCs or IPSCs was analyzed by mini analysis software (Synaptosoft, Fort Lee, NJ); only those events with amplitudes >10 pA were used.
Adult male mice C57BL/6J (20–25 g, Charles-River, Kanagawa, Japan) were anesthetized with sodium pentobarbital (50 mg/kg, ip) and perfused sequentially with 20 ml chilled saline and 20 ml chilled 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, trimmed, and immersed in the same fixative solution for 12 h and were then immersed in 30% sucrose solution for 2 days at 4°C. The brains were quickly frozen in optimum cutting temperature (O.C.T.) compound (Sakura Finetechnical, Tokyo). Cryostat sections (40 μm) were stained by the avidin-biotin-peroxidase method. Brain sections were incubated for 40 min in Tris-buffered saline containing 0.3% H2O2 to inactivate endogenous peroxidase. Sections were transferred into Tris-buffered saline containing 0.25% Triton X-100 and 1% bovine serum albumin fraction V (TBS-BX) for 30 min and then incubated with monoclonal anti-tyrosine hydroxylase (TH) antibody (CHEMICON Temecula, CA) diluted 1/400 in TBS-BX overnight at 4°C. Sections were then incubated with biotin labeled anti-mouse IgG goat antibody (Vector Laboratories, Burlingame, CA) for 1 h at RT followed by incubation with avidin and biotinylated peroxidase complex solution for 30 min at RT. Bound peroxidase was visualized by incubating sections with 0.01 M imidazole acetate buffer containing 0.05% 3,3′-diaminobenzidine tetrahydrochloride, 0.005% hydrogen peroxide and 2.5% nickel ammonium sulfate, which resulted in a black reaction product. For double-labeling, sections were incubated overnight at 4°C with rabbit anti-orexin antibody (Nambu et al. 1999) diluted 1/2000 in TBS-BX. Sections were then incubated with biotin labeled anti-rabbit IgG goat antibody (Vector) for 1 h at RT. Bound peroxidase was visualized as described above without nickel sulfate, resulting in a golden-brown reaction product. The sections were mounted and examined with a microscope (AX-70, Olympus). To confirm the specificity of antibodies, incubations without primary antibody were conducted as a negative control in each experiment and no signal was observed.
Calcium imaging of orexin neurons
Male orexin/YC2.1 mice (8 wk old, 20–24 g) in which orexin neurons specifically express calcium sensing protein (Tsujino et al. 2005), were housed under controlled lighting (12 h light-dark cycle; light-on 8:00 A.M.–8:00 P.M.) and temperature (22°C) conditions. Orexin/YC2.1 mice were sleep deprived for 2 or 4 h in a slow motor-driven drum (60 cm diameter) rotating at a rate of 3 rpm. During SD, the mice were constantly observed and received a gentle nudge when they stopped moving. SD was initiated at Zeitgeber Time 1 (ZT1) and ended at ZT5 for 4 h SD and was initiated at ZT3 and ended at ZT5 for 2 h SD. Brain slices (350 μm thickness) were made at 13:00 (ZT5) by the same method as described in the electrophysiological studies. To evaluate possible circadian effects, brain slices were made at ZT5 (light period) and ZT14 (dark period) without SD. Optical recordings were performed on a fluorescence microscope (BX51WI, Olympus) equipped with a cooled CCD camera (Cascade 650, Roper Scientific, Tucson, AZ) controlled by Meta Fluor 5.0.7 software (Universal Imaging, West Chester, PA). YC2.1 was excited through a 440DF20 filter and its fluorescent image was subjected to dual emission ratio imaging through two emission filters (480DF30 for ECFP and 535DF26 for EYFP) controlled by a filter changer (Lambda 10–2, Sutter Instruments, Novato, CA). Images were captured at a rate of 1 Hz (300–500 ms exposure time) with 2 × 2 binning through a 20 × UMPlanFI water immersion objective (Olympus).
Drugs and drug application
The drugs used were tetrodotoxin, barium chloride (Wako, Osaka, Japan), noradrenaline, dopamine, adrenaline (± arterenol), idazoxan, prazosin, QX-314, 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), d-2-amino-5-phosphono-pentanoic acid (AP-5), picrotoxin, UK14304 (Sigma, St Louis, MO) and BRL44408 (TOCRIS, Northpoint, UK). In the electrophysiological experiments, drugs were dissolved in HEPES-buffered solution and applied either by bath application or local application by gravity flow through a thin polyethylene tube (diameter 100 μm) positioned near the cells being recorded. Agonists were dissolved in the extracellular solution. The solution was switched by a valve perfusion control system (VC-6, Warner Instrument). Selective receptor antagonists were applied by bath application using a peristaltic pump (Dynamax, Rainin Instruments) at a rate of 3 ml/min. In the calcium imaging experiments, both agonists and antagonists were applied by bath application at a rate of 3 ml/min. In both experiments, the same recording chamber (RC-26G, Warner Instrument Corp.; 180 μl volume) was used.
Data were analyzed by two-way ANOVA followed by Fisher's protected least significant difference test using the Stat View 4.5 software package (Abacus Concepts, Berkeley, CA). Probability (p) values <0.05 were considered statistically significant.
Orexin-ir neurons are in apposition to TH-ir nerve endings
Orexin neurons have been well established to be localized to the LHA. To determine whether these cells receive catecholaminergic input, coronal sections through the region containing these neurons were studied using double immunostaining for orexin (brown) and for TH (black). Figure 1 demonstrates TH immunoreactivity (TH-ir) in the region of the orexin neurons. The majority of orexin-ir neurons were located within a field of dense TH-ir axons (Fig. 1A). TH-ir varicosities were closely apposed to orexin-ir cell bodies (Fig. 1, B, and C, arrowhead).
Catecholamines hyperpolarize orexin neurons in the presence or absence of TTX
To study the effect of catecholamines on orexin neurons, whole-cell current clamp and voltage clamp recordings were made on acute slice preparations of orexin/EGFP transgenic mice. In current clamp mode, NA application hyperpolarized the membrane potential of all EGFP-positive neurons (orexin neurons) tested in the presence or absence of TTX (Fig. 2A, top and middle, n = 80). In the presence of TTX, NA (30 μM) application significantly decreased membrane resistance to 47% of control values; membrane resistance of orexin neurons before and after NA application was 613.1 ± 24.8 and 289.6 ± 16.9 MΩ (n = 5), respectively. At a holding potential of –60 mV under voltage clamp, NA (30 μM) induced an outward current in orexin neurons in the presence of TTX (19.0 ± 3.8 pA, n = 4; Fig. 2A, bottom). Figure 2C demonstrates that NA hyperpolarized orexin neurons in a concentration-dependent manner: Emax was 17.3 ± 0.5 mV at 100 μM, IC50 was 6.7 ± 0.7 μM (n = 4–6). Adrenaline also induced hyperpolarization of orexin neurons in a concentration-dependent manner (Fig. 2C); the effect was more potent than that of NA (Emax was 24.2 ± 0.4 mV at 30 μM; IC50 was 2.4 ± 0.2 μM, n = 4–6).
DA also induced hyperpolarization of orexin neurons but was much less potent than either NA or adrenaline: although the efficacy of the DA-induced response was similar to that of NA (Emax = 17.5 ± 0.6 mV), the potency was much lower (IC50 = 141.5 ± 21.9 μM, n = 4–6; Fig. 2C). In current clamp mode, DA hyperpolarized orexin neurons in the presence of 1 μM TTX (Fig. 2B, top trace). In the presence of TTX, DA (300 μM) application decreased membrane resistance to 67.0% of control values; membrane resistance of orexin neurons before and after DA application was 749.1 ± 118.4 and 502.3 ± 49.1 MΩ (n = 4), respectively. At a holding potential of −60 mV in voltage clamp, DA (300 μM) induced an outward current in the presence of TTX (5.8 ± 1.7 pA, n = 4; Fig. 2B, bottom trace).
Activation of the α2 adrenergic receptor (α2-AR) is involved in NA-induced hyperpolarization
To identify the subtype of the adrenergic receptor involved in NA-induced hyperpolarization of orexin neurons, preferential adrenergic receptor antagonists were used. Idazoxan, a selective α2-AR antagonist, inhibited the hyperpolarization induced by 30 μM NA in a concentration-dependent manner (Fig. 3). NA-induced hyperpolarization was partially blocked by bath application of 0.1 μM idazoxan and completely blocked by 1 μM idazoxan (Fig. 3A, -B). Pretreatment of slices with 0.01 and 0.1 μM idazoxan for 1.5 min inhibited 30-μM NA-induced hyperpolarization to 65.5 ± 10.7% (n = 4) and 27.0 ± 15.8% (n = 6), respectively, compared with before antagonist treatment. Involvement of the α2-AR in this NA-induced hyperpolarization was further confirmed by use of the α2-AR selective agonist, UK14304 (Fig. 3C). UK14304 application induced hyperpolarization in the orexin neurons in a concentration-dependent manner: 1 μM and 10 μM of UK14304 hyperpolarized orexin neurons by 38.3 ± 8.0% (n = 4) and 72.1 ± 6.3% (n = 4), respectively, compared with prior application of NA (30 μM). To determine which subtype of the α2-AR is involved in this response, the α2A receptor selective antagonist, BRL44408 (Young et al., 1989), was tested. BRL44408 (3 μM) almost completely inhibited NA-induced hyperpolarization to 6.3 ± 2.5% of control values (n = 3), which suggests that the α2A receptor subtype may mediate this response. We also found that NA induces a slight depolarization of orexin neurons in the presence of 1 μM idazoxan. The depolarization became more prominent at membrane potentials more negative than −50 mV; 1, 10, and 30 μM NA induced 1.9 ± 0.9, 12.0 ± 2.5, and 17.5 ± 3.0 mV (n = 6) depolarization, respectively, when membrane potential was adjusted at −70 mV before the experiment (Fig. 3, D and E). NA-induced depolarization was eliminated by coapplication of the selective α1-AR antagonist prazosin (n = 6, Fig. 3B). Isoproterenol (100 μM, n = 6), a β-adrenergic receptor agonist, had little effect on orexin neurons (Fig. 3C). In addition, propranolol (20 μM, n = 6), a β-adrenergic receptor antagonist, did not influence the NA-induced response (Fig. 3C), suggesting that the β-adrenergic receptor is not involved in the NA-induced response in orexin neurons.
DA-induced hyperpolarization was also inhibited by idazoxan; 0.1, 1, and 10 μM idazoxan suppressed the DA (300 μM) response to 78.2 ± 8.6%, 63.0 ± 1.5% and 38.4 ± 4.4% (n = 4) of the pretreatment level, respectively, which suggests that this response is also mediated by α2-ARs. This is consistent with our observation that potency of DA-induced hyperpolarization was much lower than that of NA or adrenaline.
NA-induced hyperpolarization of orexin neurons is mediated by activation of GIRK potassium channels
Figure 4 presents evidence that the NA-induced decrease in input resistance occurs through an increase in potassium conductance. The reversal potential estimated from the I-V relationship was −106 mV (n = 6) in normal external solution containing 2 mM K+ (Fig. 4, A and B). This value is similar to the theoretical K+ equilibrium potential (−116 mV) calculated from the Nernst equation (dotted line in Fig. 4C) by using the K+ concentration of the external and pipette solutions. Similar results were obtained when recording in the physiological bicarbonate buffer: reversal potential of the NA-induced response was −110.2 ± 5.9 mV (n = 5) in this condition. As the extracellular K+ concentration ([K+]o) was increased to 10 mM, the reversal potential (Erev) shifted to –66.6 ± 4.9 mV (n = 5; Fig. 4C). The slope of the Erev values for a 10-fold change in [K+]o was 43.6 mV. Erev of the DA-induced response (110.3 ± 14.4 mV; n = 5) was also similar to the theoretical K+ equilibrium potential.
We used the GIRK channel inhibitor Ba2+ to evaluate the possible involvement of the GIRK channel in NA-induced hyperpolarization. Pretreatment with Ba2+ for 2 min inhibited NA-induced hyperpolarization in a concentration-dependent manner (Fig. 4, D and E). Incubation with 30 μM and 300 μM Ba2+ inhibited 30-μM NA-induced hyperpolarization to 55.2 ± 8.8% (n = 5) and 26.4 ± 7.2% (n = 5), respectively, compared with the response before Ba2+ treatment (Fig. 4E). The inhibition by Ba2+ was reversible; NA-induced hyperpolarization recovered after washout for 10 min (data not shown).
In the presence of idazoxan, NA induced activation of orexin neurons through α1-ARs
Although NA 30 μM induced 22.5 ± 5.7 pA (n = 9) of outward current in voltage clamp experiments (Figs. 2A and 5A), in the presence of idazoxan, NA induced 13.7 ± 2.5 pA (n = 15) of inward current (Fig. 5A). Therefore we studied the NA-induced inward current in the presence of idazoxan in detail. This inward current was robustly enhanced in the extracellular calcium ion–free (Ca2+-free) solution (see Fig. 6, A and B). Thus the concentration dependency of this inward current was tested in the Ca2+-free solution. NA induced an inward current in a concentration-dependent manner in the presence of idazoxan and in the Ca2+-free solution (Fig. 5B). Emax and EC50 were 165.6 ± 5.2 pA and 10.7 ± 0.7 μM (n = 6), respectively. This inward current was inhibited by prazosin, a selective α1-AR antagonist, in a concentration-dependent manner (Fig. 5, C and D). Prazosin inhibited NA-induced inward current to 45.2 ± 9.0% (0.01 μM prazosin, n = 6) and 4.8 ± 1.9% (0.1 μM prazosin, n = 6) of control levels (Fig. 5D). The α1-AR selective agonist, phenylephrine, mimicked an inward current (Fig. 5E), which supports the results obtained with the α1-AR selective antagonist. Phenylephrine-induced inward current was 16.0 ± 2.2% (10 μM, n = 7) and 46.6 ± 6.1% (100 μM, n = 7) of the 30-μM NA-induced inward current in the presence of idazoxan (Fig. 5F).
An activation of NSCCs is involved in the NA-induced inward current
NA-induced inward current in the presence of idazoxan was robustly potentiated by removing extracellular calcium ions. This inward current increased approximately 14-fold in calcium-free solution, which suggests that the NA-induced inward current was suppressed by extracellular calcium ions. Thirty-micrometer NA-induced current in calcium-free extracellular solution was 195.8 ± 73.3 pA (n = 8; Fig. 6A). This value is larger than the Emax value, which was obtained from the concentration-response curve (Fig. 5B), because this potentiated inward current in the calcium-free solution has the tendency to dampen by repeated activation. To examine what types of channels are involved in the NA-induced inward current, the reversal potential was determined by means of a ramp protocol. Reversal potential of the NA-induced current in the presence of idazoxan in calcium-free extracellular solution (mM: 140 NaCl, 2 CsCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 10 glucose) was near 0 mV (4.5 ± 1.4 mV, n = 5) when measured using a CsCl pipette solution (mM: 145 CsCl, 1 MgCl2, 10 HEPES, 1.1 EGTA, and 0.5 Na2GTP) (Fig. 6, C and D). This reversal potential is midway between negative Cs+ and positive Na+, which suggests the involvement of NSCCs. NA-induced inward current is inhibited by a NSCC blocker, SKF96365, in a concentration-dependent manner (Fig. 6, E and F). Thirty-micrometer NA-induced inward current was inhibited to 74.4 ± 5.8% (3 μM SKF96365) and 28.6 ± 5.3% (30 μM SKF96365) of control levels. These results suggest that an activation of NSCCs through α1-AR is involved in the NA-induced inward current observed in the presence of idazoxan.
NA influences EPSCs and IPSCs in orexin neurons
To examine the possibility that NA also affects synaptic inputs to orexin neurons, EPSCs and IPSCs were recorded in orexin neurons under whole-cell voltage clamp mode at a holding potential of −60 mV. sEPSCs or sIPSCs were recorded in the absence of TTX. sEPSCs were recorded using KCl-based pipette solution containing the sodium channel blocker QX-314 (1 mM) to inhibit action potentials in the recording neuron and in the presence of the GABAA receptor antagonist, picrotoxin (100 μM), in the bath solution. NA (30 μM) application significantly decreased sEPSC frequency to 38.2 ± 6.7% of control values (n = 6, P < 0.0001, ANOVA) (Fig. 7A, -C). sEPSC frequency recovered partially after NA washout. sEPSC amplitude was not altered by NA application. Mean amplitude of before and after NA application was 24.3 ± 2.9 pA and 26.5 ± 3.1 pA, respectively (n = 6, P = 0.31, not significantly different). When the ionotropic glutamate receptor antagonists AP-5 (50 μM) and CNQX (20 μM) were added to the bath solution, sEPSCs were completely abolished, suggesting that they were due to the activation of ionotropic glutamate receptors (data not shown). The 30-μM NA-induced decrease in sEPSC frequency was inhibited in the presence of the α2-AR antagonist, idazoxan (1 μM, n = 6, P < 0.05) (Fig. 7C).
We also examined the effect of NA on the sIPSCs. sIPSCs were recorded using KCl-based pipette solution containing QX-314 (1 mM) in the presence of AP-5 (50 μM) and CNQX (20 μM) in the bath solution. The IPSC is recorded as an inward current because the high intracellular chloride concentration used results in a reversal potential, which is more positive than the holding potential used. NA (30 μM) application dramatically increased IPSC frequency by 398.1 ± 78.6% (n = 6, P < 0.0001) (Fig. 7, B and D). sIPSC amplitude was also increased by 340 ± 100% (n = 6) of control value. sIPSCs returned to basal levels after NA washout and were abolished by coapplication of picrotoxin (100 μM) (data not shown). The 30-μM NA-induced increase in sIPSCs was abolished in the presence of the α1-AR antagonist, prazosin (1 μM, n = 6, P < 0.0001, ANOVA) (Fig. 7D). The α1-AR agonist, phenylephrine (100 μM), significantly increased sIPSC frequency to 531.6 ± 173.3% (n = 5, P < 0.05) of control levels. On the other hand, the α2-AR agonist UK14304 (10 μM) significantly decreased sIPSC frequency to 24.9 ± 6.8% (n = 5, P = 0.0002) of control values. NA did not induce further increase in sIPSC frequency in the presence of idazoxan. In the presence of idazoxan, NA application increased sIPSC frequency to 408.7 ± 92.7% of control levels (n = 6).
To reveal whether these NA effects on the PSCs are mediated via a presynaptic mechanism or via neuronal somata, the effect of NA on the mEPSCs and mIPSCs were studied in the presence of TTX (1 μM). NA application decreased mEPSCs frequency to 38.5 ± 8.9% of control levels (n = 6, P < 0.001, ANOVA) (Fig. 8, A-C); however, in contrast to the NA effect on the sIPSCs, NA induced a decrease in mIPSC frequency: mIPSCs decreased to 32.6 ± 4.1% of control levels (n = 7, P < 0.0001, ANOVA) (Fig. 8, B-D). mEPSC and mIPSC frequency were recovered by NA washout. The amplitude of both mEPSCs and IPSCs was not altered by NA (30 μM) application. mEPSC and mIPSC amplitude during NA application was 89.1 ± 5.5% (n = 5) and 99.8 ± 4.9% (n = 5), respectively, compared with basal amplitude. Although the frequency of mEPSCs or mIPSCs was not significantly altered in the presence of idazoxan (1 μM), it abolished the NA-induced decrease in mEPSCs and mIPSCs. This observation suggests an involvement of the α2-AR in these responses (Fig. 8, C and D). These data suggest a possibility that the NA-induced increase in sIPSCs is mediated through the α1-AR localized on the somata of GABAergic interneurons, which innervate orexin neurons. On the other hand, α2-ARs might exist on both GABAergic and glutamatergic terminals that synapse onto orexin neurons.
To confirm this hypothesis, the effects of NA on the electrically evoked EPSCs (eEPSCs) or IPSCs (eIPSCs) were also examined. Electrical stimuli (100–200 μA, 0.1 ms, 0.1 Hz) were generated using bipolar stimulation electrodes placed within the LHA. eEPSCs or eIPSCs were recorded from orexin neurons voltage clamped at −60 mV by use of a KCl-based pipette solution. In the presence of picrotoxin (100 μM), eEPSCs with an amplitude of 226.0 ± 22.6 pA (n = 5) were recorded. NA (30 μM) application rapidly reduced the eEPSC amplitude to 41.3 ± 8.0% (n = 5, P < 0.0001, ANOVA) as compared with before NA application (Fig. 9, A–C). eEPSCs recovered within 10–18 min after NA washout. Recovered eEPSCs were completely abolished by coapplication of AP-5 (50 μM) and CNQX (20 μM) in the bath solution, which suggests that they were attributable to the activation of ionotropic glutamate receptors (Fig. 9C).
In the presence of AP-5 (50 μM) and CNQX (20 μM), we observed an eIPSC with an amplitude of 589.8 ± 277.7 pA (n = 5). NA application decreased the eIPSCs to 27.8 ± 10.9% (n = 5) as compared with before application (Fig. 9, D–E). eIPSCs were partially recovered after NA washout. Recovered eIPSCs were completely blocked by coapplication of picrotoxin (100 μM, data not shown). These observations suggest that NA inhibits both glutamatergic and GABAergic inputs to orexin neurons through a presynaptic mechanism.
Effect of NA on the calcium current
To examine the possibility that NA also affects calcium current in orexin neurons, calcium current was recorded under whole-cell voltage clamp. AP-5 (50 μM), CNQX (20 μM), picrotoxin (100 μM), and TTX (1 μM) were added to the bath solution to block synaptic activity. BaCl2 was substituted for CaCl2 in the bath solution to increase the conductance of the calcium channels. Voltage ramps from −60 mV to 40 mV for 2 s induced −258.9 ± 26.0 pA (n = 6) inward current, which was inhibited by the calcium channel inhibitor, Cd2+ (200 μM) and Ni2+ (100 μM) (Fig. 10). NA (100 μM) decreased Ba2+ current by 197 ± 19.0 pA (n = 6), (P < 0.001, ANOVA). Ba2+ current was completely recovered after NA washout. This result suggests that NA inhibits calcium channels on the orexin neurons.
Effect of SD on the NA responsiveness of orexin neurons
Recent reports using rats showed that orexin neurons are activated by NA but are inhibited when the rats were sleep deprived for a few hours before the experiments (Bayer et al. 2005; Grivel et al. 2005). In contrast, we and Li and van den Pol (2005) observed that mouse orexin neurons showed NA-induced hyperpolarization. To examine whether SD alters the response of mouse orexin neurons to NA, we performed calcium imaging using hypothalamic slices from orexin/YC2.1 transgenic mice in which orexin neurons specifically express calcium-sensing protein. We previously reported that this system detects both increases and decreases in intracellular calcium concentration (Tsujino et al. 2005). In addition, this system allows us to examine the effect of biologically active substances on orexin neurons in adult mice for more than several hours, whereas only very young mice are usable for patch clamp recording. First, we confirmed that this system can detect NA-induced inhibition or activation of orexin neurons. Sequential application of 30-μM NA induced the same amplitude of decrease in YFP/CFP ratio (Fig. 11, A and B). The ratio alteration induced by the first and the second NA application was 0.091 ± 0.013 (n = 7) and 0.094 ± 0.013 (n = 7), respectively. In the presence of idazoxan, 30 μM NA induced an increase in YFP/CFP ratio (0.053 ± 0.007, n = 5, Fig. 11C and D). These results suggest that this system detects both NA-induced inhibition and activation.
Orexin/YC2.1 mice of 6 wk of age were sleep deprived for 2 or 4 h before experiments in a motor-driven drum (60-cm diam) rotating at a rate of 3 rpm. To control for circadian effects, all slices were prepared at the circadian time 13:00 (ZT 5). NA (30 μM) application decreased the YFP/CFP ratio in orexin neurons in both control and sleep-deprived mice (Fig. 11E). No NA-induced activation of orexin neurons was observed in the sleep-deprived mice. The mean YFP/CFP ratio alteration induced by NA application in control mice and those sleep deprived for 2 h and 4 h was 0.069 ± 0.006 (n = 19), 0.063 ± 0.008 (n = 22, P = 0.52, ANOVA) and 0.065 ± 0.007 (n = 23, P = 0.64, ANOVA), respectively. Neither the amplitude nor the time course of the NA-induced response was distinguishable between control and sleep-deprived mice (Fig. 11E).
We also examined the effect of circadian timing on NA-induced response of orexin neurons by preparing slices in the dark period at 22:00 (ZT 14). NA induced a decrease in the YFP/CFP ratio of the same magnitude in the orexin neurons: the NA-induced ratio was 0.065 ± 0.007 (n = 9, P = 0.75, ANOVA). These results indicate that NA-induced inhibition of orexin neurons was altered by neither SD nor circadian time in mice.
Previous studies have demonstrated that NA (Li et al. 2002; Li and van den Pol 2005; Yamanaka et al. 2003b) and DA (Yamanaka et al. 2003b) show inhibitory effects on orexin neurons. The present study confirms these results, extends these observations to adrenaline and establishes that the hyperpolarization induced by catecholamines involves α2A-ARs and, subsequently, an activation of GIRK channels. Furthermore, we demonstrate that orexin neurons express α1-ARs as well as α2A-ARs and that NA activates NSCCs through an α1-AR-mediated mechanism. Last, catecholamines were found to indirectly influence the activity of orexin neurons by modulating both glutamatergic and GABAergic neurotransmission onto these cells.
Catecholamines directly inhibit orexin neurons through α2A-AR-mediated activation of GIRK channels
Multiple lines of evidence suggest that the direct catecholaminergic inhibition of orexin neurons is mediated through activation of α2A-AR. As indicated in Fig. 2, the potency order for hyperpolarization of the orexin neurons by the three catecholamines tested was adrenaline > NA ≫ DA (IC50 values 2.4 ± 0.2 μM, 6.7 ± 0.7 μM and 141.5 ± 21.9 μM, respectively). Of these three catecholamines, adrenaline is known to have the highest affinity for the α2-ARs. NA has a higher affinity for α1A-AR than adrenaline, which might cause the differences of maximal response (Watson and Abbott 1991). NA-induced hyperpolarization was blocked by the α2-AR antagonist idazoxan and was mimicked by the α2-AR agonist UK14304 (Fig. 3, B and C). BRL44408, a selective α2A receptor antagonist (Young et al., 1989), inhibited the NA-induced hyperpolarization. Together, these observations suggest involvement of α2A-ARs in the catecholamine-induced hyperpolarization of orexin neurons. On the other hand, at the highest concentration of idazoxan used (1 μM), NA induced a slight depolarization of orexin neurons. This depolarization became more prominent in membrane potentials more negative than −50 mV and was eliminated by coapplication of the selective α1-AR antagonist prazosin (Fig. 3B), which suggests that the response of orexin neurons to catecholamines may involve both α1-AR-mediated depolarization and α2-AR-mediated hyperpolarization. This means that NA-induced hyperpolarization is a balance between α1-AR-mediated depolarization and α2-AR-mediated hyperpolarization. As a result, NA acts on α2-AR like a partial agonist. Although we could not completely exclude the presence of DA receptors on the orexin neurons, it seems likely that the DA-induced hyperpolarization is mostly mediated by α2-ARs rather than by DA receptors since a very high concentration of DA is necessary to show hyperpolarization. The fact that DA-induced hyperpolarization is inhibited by the α2-AR antagonist idazoxan supports this idea. These results obtained by electrophysiological experiments are in agreement with recent immunohistochemical studies using adult rats by Modirrousta et al. (2005) showing that orexin neurons express both α1A- and α2A-ARs.
The data in Fig. 4 strongly suggest that the membrane conductance change induced by NA involves an α2-AR-mediated activation of GIRK currents. NA has been reported to increase potassium conductance through GIRK channels via activation of α2-ARs in LC neurons (Arima et al. 1998; Williams et al. 1985). Although three α2-AR subtypes are well known, the concentration-response curves for NA-induced GIRK current activation do not allow distinction among these receptor subtypes. However, BRL44408, a selective α2A receptor antagonist (Young et al. 1989), almost completely inhibited the NA-induced hyperpolarization. This suggests that an α2A receptor is involved in this NA-induced hyperpolarization of orexin neurons. GIRK current activation has also been proposed to underlie inhibitory effects on orexin neurons that are mediated by Gi-coupled receptors, including the 5-HT1A receptor (Muraki et al. 2004) and the neuropeptide Y1 receptor (Fu et al. 2004).
Both α1-AR and α2-AR are localized on orexin neurons
In the presence of an α2-AR antagonist, NA induced depolarization or inward current in orexin neurons. This NA-induced depolarization or inward current was inhibited by an α1-AR antagonist, which suggests that α1-ARs are involved in this response. Electrophysiological studies revealed an involvement of NSCCs in NA-induced inward current. Elsewhere, we reported that cholecystokinin activates orexin neurons through extracellular calcium ion sensitive NSCCs (Tsujino et al. 2005). NA-induced inward current was also enhanced by removing extracellular calcium ions. In addition, both inward currents are inhibited by SKF96365, a NSCC blocker, which suggests that same NSCC might be involved. Why is hyperpolarization a dominant response to NA on orexin neurons? One possibility is that the hyperpolarization mediated through α2-AR and GIRK channel is faster than the depolarization mediated through α1-AR and NSCC because the former response does not need an intracellular signal cascade. In addition, we reported that NSCC on the orexin neurons activated by CCK through the CCKA receptor showed voltage dependency (Tsujino et al. 2005). NSCC would be inactivated by the hyperpolarization. The physiological significance of α1-AR mediated activation of NSCC in the orexin neurons is not clear because α1-AR-mediated activation of orexin neurons is only evident in the presence of idazoxan.
Bayer et al. (2005) reported completely opposite effects of NA on orexin neurons using immature rat hypothalamic slice preparation: they observed NA-induced depolarization in orexin neurons. Furthermore, Grivel et al. (2005) recently reported using immature rats that the action of NA on orexin neurons changes from excitation to inhibition after a short 2-h period of total SD. In the current study, we performed electrophysiological analyses using orexin/EGFP mice (immature mice: 2–3 wk old) and concluded that all orexin neurons are inhibited by NA in mice (Fig. 11). It is possible, however, that the type of response to NA on orexin neurons is altered by circadian time or SD, because all electrophysiological experiments were performed during the light period without SD. To address this issue, we performed calcium imaging experiments with orexin/YC2.1 transgenic mice. This system enabled us to analyze several orexin neurons simultaneously and study both young and adult mice. Calcium imaging experiments revealed that the type of response to NA is not dependent on circadian time. This inhibitory response was not altered by SD for 2 or 4 h. It seems likely that catecholamines inhibit orexin neurons in adult mice regardless of circadian time or SD. We and Modirrousta et al. (2005) found that orexin neurons express both α1-AR as well as α2-AR. It is possible that the populations of orexin neurons that express these adrenergic receptors differ between mice and rats.
Indirect effects of catecholaminergic systems on orexin neurons
Catecholamines such as NA not only elicited direct inhibition of orexin neurons through α2A-AR, but also showed an indirect influence on orexin neurons by modulating both IPSCs and EPSCs. NA application resulted in an increase in sIPSC amplitude and an increase in sIPSC frequency. The α1-AR antagonist prazosin inhibited the NA-induced increase in sIPSC frequency and α1-AR agonist phenylephrine mimicked NA response (increased sIPSC frequency), which suggests that the α1-AR is involved in this response. This idea is consistent with a report that showed NA increases sIPSCs in GABAergic neurons in the hypothalamic paraventricular nucleus through α1-ARs (Chong et al., 2004). Li and van den Pol (2005) have also reported that orexin neurons were directly inhibited by NA through α2-ARs and that NA indirectly inhibited orexin neurons by facilitating GABAergic transmission at presynaptic sites through α1-ARs. Our results, however, suggest that GABAergic neurons, which innervate orexin neurons, separately express α1-ARs and α2-ARs in somata and presynaptic terminals, respectively. In addition, the NA-induced increase in sIPSCs was mediated through α1-ARs located on somata membrane, not by a presynaptic mechanism, because mIPSCs frequency and eIPSCs amplitude were both inhibited by NA. On the other hand, the NA-induced decrease in mEPSCs is inhibited by idazoxan, which suggests an involvement of α2-ARs in this response. Both mEPSC frequency and eEPSC amplitude were also decreased by NA, which suggests that a presynaptic inhibitory mechanism through α2-ARs is involved in this response. Li et al. (2002) showed the existence of glutamatergic local circuitry that positively modulates the activity of orexin neurons in the hypothalamus. Furthermore, we recently reported that orexin neurons are innervated by the local interneurons located in the LHA (Sakurai et al., 2005). Thus, it is possible that catecholamines influence the activity of orexin neurons through glutamatergic or GABAergic interneurons in the LHA, as well as through direct action. In the presence of idazoxan (to block presynaptic inhibition through α2-AR), NA did not induce a further increase in sIPSC frequency (408.7 ± 92.7%) compared with NA alone (398.1 ± 78.6%). This result might suggest that presynaptic α2-ARs are expressed on GABAergic neurons, which innervate orexin neurons from outside the hypothalamic slice rather the interneurons located near the orexin neurons.
Physiological significance of catecholamine-mediated inhibition of orexin neurons
Although it may appear surprising that waking-active NA neurons would inhibit orexin neurons, which are likely to also be waking-active (Lee et al. 2005; Mileykovskiy et al. 2005), we found that catecholamines also indirectly modulate both GABAergic and glutamatergic inputs to orexin neurons, which suggests a complex physiological role for catecholaminergic influences on regulation of the orexin neurons. Monoaminergic cell groups such as the NA-containing cells of the LC are generally known to be waking-active neurons (Chu and Bloom 1974; Hobson et al. 1975). Recent studies, however, suggest that orexin neurons do not receive direct synaptic input from the LC in mice or in rats (Sakurai et al. 2005; Yoshida et al. 2006). Rather, orexin neurons might receive noradrenergic input from neurons outside the LC, such as the C1/A1 region, whose discharge rate in relation to behavioral states is currently unknown; however, because these tracing studies are not likely to label secondary or higher-order afferents, indirect effects of NA on orexin neurons might exist. Because NA appears to both directly and indirectly inhibit orexin neurons by modulating local interneurons, the net effect of the noradrenergic system on orexin neurons is very complex.
What are the consequences of the inhibitory actions of NA for orexin neuron activity? The orexin neurons appear to lack the property of spike frequency adaptation and can follow stimulus frequencies as fast as 333 Hz (Li et al. 2002; Yamanaka et al. 2003b). Hyperpolarization of the orexin neurons by catecholamines and 5-HT during wakefulness may have important functional consequences to balance excitatory drive onto these cells. The inhibitory action of NA on orexin neurons might work as a negative feedback system that maintains orexin neuronal activity within appropriate ranges during each behavioral state. Alternatively, this might play an important role in the presynaptic inhibition of orexin neurons at projection sites such as the LC because NA concentration in the LC increases in proportion to the neuronal activity of noradrenergic LC neurons. This idea is supported by the fact that NA also inhibited calcium current in the orexin neurons (Fig. 10), which is involved in the release of neurotransmitters.
In conclusion, catecholamines directly and indirectly inhibit orexin neurons. The mechanism of direct inhibition is an α2A-AR-mediated activation of GIRK channels in the orexin neurons. The indirect inhibitory mechanism involves both an increase in IPSCs and a decrease in EPSCs in these cells. These direct and indirect influences on orexin neurons by catecholaminergic neurons, summarized in Fig. 12, likely have an important role in both the physiological regulation of orexin neuronal activity and the regulation of sleep and wakefulness.
This study was supported by a grant-in-aid for scientific research (S), (B) and Grant-in-Aid for Scientific Research on Priority Areas [Elucidation of neural network function in the brain] from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17023007), Kanae Foundation, a grant for anorexia nervosa research from the Japanese Ministry of Health, Labor, and Welfare, and by National Institutes of Health grants RO1MH61755 and RO1AG020584. Thanks to C. Jones for proofreading the manuscript.
↵* A. Yamanaka and Y. Muraki contributed equally to this work.
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- Copyright © 2006 by the American Physiological Society