Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa

Accepted Manuscript Title: Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa Author: Younghoon Jeon Ki Bum Park Rokeya Pervin Tae Wan Kim Dong-ho Youn PII: DOI: Reference:

S0304-3940(15)30071-9 http://dx.doi.org/doi:10.1016/j.neulet.2015.08.001 NSL 31474

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

23-5-2015 22-7-2015 3-8-2015

Please cite this article as: Younghoon Jeon, Ki Bum Park, Rokeya Pervin, Tae Wan Kim, Dong-ho Youn, Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2015.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa Younghoon Jeon1, Ki Bum Park2,*, Rokeya Pervin3, Tae Wan Kim3 and Dong-ho Youn4,*

1

Department of Anesthesiology and Pain Medicine and 4Department of Oral Physiology, School of

Dentistry, Kyungpook National University, Daegu 700-706, R. O. Korea 2

Department of Anesthesiology and Pain Medicine, School of Medicine, Kyungpook National

University, Daegu 700-721, R. O. Korea 3

Department of Physiology, College of Veterinary Medicine, Kyungpook National University, Daegu

702-701, R. O. Korea

*Corresponding authors. Tel.: +82-53-660-6841; fax: +82-53-421-4077. E-mail addresses: [email protected] (D. Youn) or [email protected] (K. Park)

Highlights 1. Orexin-A depresses primary afferent-evoked excitatory synaptic transmission. 2. The orexin A-induced depression was exclusively mediated by OX1R. 3. Orexin-A reversibly increases spontaneous EPSC frequency through both OX1R and OX2R. 4. Orexin-A induces oscillation and inward current through both OX1R and, to a great extent, OX2R.

ABSTRACT Although intrathecal orexin-A has been known to be antinociceptive in various pain models, the role of orexin-A in antinociception is not well characterized. In the present study, we examined whether orexin-A modulates primary afferent fiber-mediated or spontaneous excitatory synaptic transmission using transverse spinal cord slices with attached dorsal root. Bath-application of orexinA (100 nM) reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of A- or C-primary afferent fibers. The magnitude of reduction was much larger for EPSCs evoked by polysynaptic C-fibers than polysynaptic A-fibers, whereas it was similar in EPSCs evoked by monosynaptic A- or C-fibers. SB674042, an orexin-1 receptor antagonist, but not EMPA, an orexin-2 receptor antagonist, significantly inhibited the orexin-A-induced reduction in EPSC amplitude from mono- or polysynaptic A-fibers, as well as from mono- or polysynaptic C-fibers. Furthermore, orexin-A significantly increased the frequency of spontaneous EPSCs but not the amplitude. This increase was almost completely blocked by both SB674042 and EMPA. On the other hand, orexin-A produced membrane oscillations and inward currents in the SG neurons that were partially or completely inhibited by SB674042 or EMPA, respectively. Thus, this study suggests that the spinal actions of orexin-A underlie orexin-A-induced antinociceptive effects via different subtypes of orexin receptors.

Keywords: orexin-A; spinal substantia gelatinosa; excitatory synaptic transmission; orexin receptor; oscillation

1. Introduction

The neuropeptides orexin-A and orexin-B (also named hypocretin-1 and hypocretin-2, respectively) are known to regulate sleep/wakefulness states and feeding behavior [14]. Although orexin-A-expressing neurons are located exclusively in the perifornical area of the hypothalamus and the lateral hypothalamus [5, 15], they send axons to many regions of the brain, including the spinal cord [4, 17]. Orexin-A immunoreactivity is seen throughout the spinal cord [4]; however, it is most abundant in the superficial layer (laminae I and II; II, called substantia gelatinosa, SG) of the spinal dorsal horn (DH) [1, 4, 8]. This layer is critical for the transmission and integration of sensory and nociceptive signals from the periphery [24], suggesting a potential role for orexin-A in sensory and pain modulation. Orexin-A and -B bind to two orexin receptors, the orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), which belong to the G-protein-coupled receptors family [5, 15]. While OX1R has a higher affinity for orexin-A than -B by an order of magnitude, the OX2R has similar affinity for both orexin-A and -B. The OX1R has been found in all regions of spinal cord gray matter [1, 9] and in dorsal root ganglion (DRG) neurons immunopositive to a C-fiber marker [9]. In addition, an electron microscopic study showed that OX1Rs are expressed on perikarya (cell bodies) and dendrites associated with OX1R-negative axon terminals, and on axon terminals at asymmetric (i.e., excitatory) synapses in the spinal DH [7]. Based on these reports from the spinal DH, it could be conceived that the OX1R modulates excitatory synaptic transmission and neuronal activity by both pre- and postsynaptic actions, thereby contributing to the control of sensation and nociception. The regulation of sensory/nociceptive transmission involves modulation and plasticity of excitatory synaptic transmission, which is largely glutamatergic, in the spinal DH, as well as a change in the neuronal excitability of DH neurons [16, 24]. Although the synaptic and neuronal actions of orexin-B on superficial DH neurons have been shown [6], the influence of orexin-A on the spinal SG

neurons remains unknown. In addition, the orexin receptors mediating the action of orexins have not been characterized in the SG. Therefore, we determined the effects of orexin-A on excitatory synaptic transmission and neuronal excitability in the spinal SG as well as the receptors mediating these effects.

2. Materials and Methods

2.1. Preparation of spinal cord slices

All the experimental procedures were approved by Institutional Animal Care and Use Committee of Kyungpook National University. Spinal cord slices were prepared from Sprague Dawley rats (15-21 day-old; M/F). After laminectomy under deep urethane anesthesia (1.5 g/kg, i.p.), the spinal cord was dissected out and quickly moved to a petri dish filled with ice-cold Krebs’ solution (composition in mM: NaCl, 117; KCl, 3.6; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 25; and glucose, 11) that was continuously oxygenated with 95% O2/5% CO2 mixed gas. After removing spinal cord membranes (dura, arachnoid and pia) and unnecessary roots and segments, transverse slices (400–500 µm thickness) with an attached dorsal root were cut from the 4th–6th lumbar segments of spinal cord in oxygenated ice-cold Krebs’ solution on a vibratome (Vibratome 1000+; Vibratome, St. Louis, MO, USA), and were placed for at least 1 h at room temperature (24-25 oC) with oxygenated Krebs’ solution to recover. A single slice was then transferred into a recording chamber, and the tip of the dorsal root attached to the slice was fixed into the hole of suction electrode. The recording chamber was continuously perfused (3 ml/min) with preoxygenated Krebs’ solution at room temperature.

2.2. Blind whole-cell patch clamp recordings

Blind whole-cell patch clamp recordings were performed in the SG (lamina II), which was identified as a translucent band in the transverse spinal cord slice through an upright microscope

(BX51WI, Olympus, Tokyo, Japan) with a 4 objective lens. Patch pipettes were made from borosilicate glass (TW150F; WPI, Sarasota, FL, USA), and a tip resistance of patch pipettes was 4-7 MΩ after being filled with internal solution (composition in mM: K-gluconate, 145; NaCl, 5; MgCl2, 1; EGTA, 0.2; HEPES, 10; Tris-ATP, 2; and Tris-GTP, 0.1; pH adjusted to 7.3 with KOH). Upon whole-cell seal formation, the membrane potential was held at −70 mV, which was the reversal potential for inhibitory postsynaptic currents (IPSCs) mediated by GABAA and glycine receptors. Under these recording conditions, inward excitatory postsynaptic currents (EPSCs) appeared spontaneously and could be evoked by electrical stimulation of the dorsal root with a current stimulator (SEN-3301, Nihon Cohden, Tokyo, Japan) connected to the suction electrode. Currents were amplified with a Multiclamp 700A (Molecular Devices, Sunnyvale, CA, USA), filtered at 1-2 kHz, digitized at 5 kHz and collected using pClamp 10 software. Both types of EPSCs could be blocked by the NMDA receptor antagonist D-AP5 (50 μM) and the AMPA/kainate antagonist CNQX (10 μM), indicating that they were mediated by ionotropic glutamate receptors and that excitatory synaptic transmission in the DH is largely glutamatergic. In all experiments, each neuron was recorded in a different slice.

2.3. Drugs and data analysis

Orexin-A,

SB674042

([5-(2-Fluorophenyl)-2-methyl-4-thiazolyl][2(S)-2-[(5-phenyl-1,3,4-

oxadiazol-2-yl)methyl-1-pyrrolidinyl]methanone])

and

EMPA

(N-Ethyl-2-[(6-methoxy-3-

pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide) were purchased from Tocris (Bristol, UK). Drug solutions were applied by exchanging the perfusing solution. Evoked EPSCs were determined as ‘monosynaptic’ inputs if they have constant latencies and no failures during repetitive 10 - 20 Hz stimulation; otherwise, they were considered to be ‘polysynaptic’ inputs. The fiber types producing EPSCs were determined by conduction velocity and stimulus intensity; i.e., ‘Aδ-fiber-mediated’ EPSCs in the case of faster conduction velocities (>1.5 m/s) and lower stimulus intensities (<100 μA/0.1 ms), and ‘C-fiber-mediated’ EPSCs in the case of slower

conduction velocities (< 1.5 m/s) and higher stimulus intensities (>100 μA/0.1 ms). EPSCs were evoked at 0.033 Hz, and the peak amplitude and holding current were measured using pClamp. Peak amplitudes of EPSCs were expressed as the percent of baseline (~5 min). sEPSC were measured in their frequency and amplitude using a template-matching method followed by cut-off filtering of the amplitude threshold (typically 2-3 pA). The analyzed data (30 s bins) were presented as the percent change against the baseline period. Oscillations in membrane holding current were analyzed by power spectrum analysis which is a frequency domain representation of the time domain data. Consequently, this analysis reveals the power levels of different frequency components of the recorded signal. In this analysis, current traces was low-pass filtered at 100 Hz, and the power density (pA2) was expressed as a function of wave frequency. Statistical significance of the data was assessed between two groups using the Student's t-test. Probability of membrane oscillation in different conditions was statistically compared by Fisher’s exact test. Statistical significance was determined as P < 0.05 (*) or P < 0.01 (**). Data are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Orexin-A depresses primary afferent-evoked excitatory synaptic transmission through predominantly OX1R

To examine the effects of orexin-A on primary afferent-evoked excitatory synaptic transmission in lamina II of the SG, SG neurons were voltage-clamped at –70 mV, and the dorsal root attached to the slice was electrically stimulated. Upon bath application of orexin-A (100 nM, 10 min) after a stable baseline period (at least 5 min), we observed a long-lasting reduction of EPSC peak amplitude for all types of synaptic inputs tested (Fig. 1). The magnitude of reduction was similar between A-fiber- and

C-fiber-mediated EPSCs that were defined as monosynaptic inputs (monosynaptic A-fiber, 69.2  5.1 % of baseline at 8-10 min, n = 8 SG neurons; monosynaptic C-fiber, 69.5  3.9 % of baseline at 810 min, n = 8 SG neurons; Fig. 1A). However, the magnitude of reduction was significantly larger in C-fiber-mediated polysynaptic EPSCs than in A-fiber-mediated polysynaptic EPSCs (polysynaptic A-fiber, 88.1  6.6 % of baseline at 8-10 min, n = 8 SG neurons; polysynaptic C-fiber, 57.1  9.9 % of baseline at 8-10 min, n = 5 SG neurons, P < 0.05 vs. polysynaptic A-fiber, Student’s t-test; Fig. 1B). To discern the identity of the receptors mediating the depressant effects of orexin-A on EPSCs, we compared the effects of orexin-A at 8-10 min from the start of application in the presence of the OX1R antagonist SB674042 or the OX2R antagonist EMPA. The depression of orexin-A (100 nM) was significantly reduced, or even completely blocked, in mono- or polysynaptic A-fiber- or C-fibermediated EPSCs by SB674042 (1 μM), but only partially by EMPA (1 μM) (Fig. 1C and E). This significant reduction of orexin-A-induced depression by SB674042 was also apparent when the effects from all types of fibers and synaptic inputs were combined (Fig. 1E). Application of SB674042 and EMPA alone (15 min) had no significant effect on primary afferent-evoked EPSCs (Fig. 1E). Thus, these results reveal a predominant role of OX1R in the modulation of primary afferent-mediated excitatory synaptic transmission by the neuropeptide orexin-A. On the other hand, the depressant effect of orexin-A on EPSC peak amplitude was not observed at a low concentration (3 nM, 98.5  7.8 % of baseline, n = 5 SG neurons; Fig. 1E), while a similar degree of effect was obvious at a higher concentration of orexin-A (300 nM, 74.0  3.5 % of baseline, n = 6 SG neurons, P>0.05 vs. 100 nM, 75.2  5.1 % of baseline, n = 29 SG neurons; Fig. 1E). In addition, orexin-B did not significantly change peak amplitude of EPSCs at a concentration of 100 nM (107.8  3.2 % of baseline at 8-10 min, n = 5 SG neurons), the effect being in agreement with a previous study [6].

3.2. Orexin-A reversibly induces increase of spontaneous EPSC frequency, membrane oscillation and

inward current through both OX1R and OX2R

To determine the effect of orexin-A on spontaneous glutamate release in the DH, as well as the locus of orexin-A-induced depression of primary afferent fiber-mediated excitatory synaptic transmission, we examined its effect on the frequency and amplitude of sEPSCs. Bath application of orexin-A (100 nM, 10 min) reversibly increased the frequency of sEPSCs (Fig. 2A-a and A-b) without altering sEPSC amplitude (Fig. 2Ac). The average changes of sEPSC frequency and amplitude were 65.7  23.6 % (n = 18, **P < 0.01 vs. baseline) and -6.2  12.5 (n = 18, P > 0.05 vs. baseline), respectively (Fig. 2B and C). The increase in sEPSC frequency was significantly reduced by both the OX1R antagonist SB674042 (n = 9 SG neurons; frequency, -5.3  4.5 % change, **P<0.01 vs. control) and the OX2R antagonist EMPA (n = 11 SG neurons; frequency, 2.2  9.3 % change, *P < 0.05 vs. control; Fig. 2C). SB674042 and EMPA alone had no effect on the frequency and amplitude of sEPSC (data not shown). This result indicates that both OX1R and OX2R mediate the orexin-A-induced increase of spontaneous glutamate release in the spinal DH. In this study, we also examined whether bath application of orexin-A could affect the intrinsic membrane properties of SG neurons. As shown in Fig. 2D, a steady membrane holding current in a SG neuron began oscillating upon orexin-A (100 nM) application. The oscillation was typically below 20 Hz in frequency, and was observed in half of recorded neurons (n = 9 out of 18; Fig. 2E). Interestingly, the probability of orexin-A-induced oscillation of membrane current was lower in the presence of SB674042 and completely disappeared in the presence of EMPA (Fig. 2E). In addition to the oscillation, we observed an inward membrane current at rest when applying orexin-A at 100 nM, but not at 3 nM (Fig. 2F). The orexin-A-induced inward membrane current was significantly reduced by both SB674042 (P < 0.05) and EMPA (P < 0.01). Together, these results suggest that the effects of orexin-A on postsynaptic neurons involve OX1R and, to a great extent, OX2R.

4. Discussion

In this study, we found that orexin-A greatly depresses A-fiber-mediated monosynaptic and Cfiber-mediated monosynaptic/polysynaptic excitatory synaptic transmission in the SG of spinal cord. This substantial depression was exclusively mediated by OX1R, regardless of the fiber type and connectivity. In addition, we found that orexin-A facilitates spontaneous glutamate release from presynaptic terminals through both OX1R and OX2R, indicating a differential involvement of orexin receptors in the actions of orexin-A on spontaneous versus evoked glutamate release from primary afferent fibers. Furthermore, we observed that orexin-A induces rhythmic oscillation and an inward membrane current in SG neurons. Interestingly, these postsynaptic effects were mediated by partially OX1R and mostly OX2R. Together, these results demonstrate a variety of pre- and postsynaptic actions of orexin-A on excitatory synaptic transmission and neuronal excitability that differentially involve OX1R and OX2R. A- and C-primary afferent fibers are the main fibers that convey mechanical and thermal nociceptive signals from the periphery to the spinal DH, and are therefore termed ‘pain’ fibers, and their synapses with the spinal DH neurons, particularly SG neurons, are a major site for modulation of pain signals [24]. Therefore, the prominent depressive effect of orexin-A on excitatory synaptic transmission mediated by A- or C-primary afferent fibers via the OX1R (Figs. 1 and 2) could represent the spinal mechanism for orexin-A’s pain-reducing effects that have been identified in the numerous animal models for pain, including inflammatory [1, 23] and peripheral nerve injury-induced neuropathic [10] pain. Furthermore, facilitatory actions of orexin-A on spontaneous glutamate release through both OX1R and OX2R are shown in this study. In the spinal DH, EPSCs ‘evoked’ by electrical stimulation of dorsal roots represent synchronized release of glutamate, whereas ‘spontaneous’ EPSCs represent asynchronous release of glutamate at axon terminals of primary afferent fibers, local interneurons in the DH, and of descending fibers originating in the higher brain regions. Therefore, the facilitatory effect of orexin-A on spontaneous glutamate release reflects differences in receptor

expression or glutamate release machinery at various axon terminals existed in the spinal DH, although it supports a clear presynaptic mechanism for orexin-A’s actions. The neurons expressing orexin-A are concentrated in the perifornical area and the lateral hypothalamus [15], and their axons project widely to many regions in the brain and spinal cord [4, 17]. Therefore, the hypothalamus could be the main source for the orexin-A released into the spinal DH, and the activation of hypothalamic areas may exert pain-modulating effects through orexin actions in the spinal cord, although the orexins produced in the DRG cannot be entirely excluded [1]. Three reports support this assumption. First, cholinergic activation of orexinergic neurons in the posterior hypothalamus produced antinociceptive effects in a neuropathic pain model with chronic constriction injury of the sciatic nerve [10]; second, stress-induced analgesia is hindered in orexin/ataxin-3 mice, in which the orexinergic neurons degenerate, and when orexinergic neuron firing is inhibited by nociceptin/orphan FQ [21]; third, the activation of orexinergic neurons in the lateral hypothalamus by inflammation and stress inhibits pain transmission [20]. Therefore, activation of the orexinergic system in the hypothalamus could also modulate excitatory synaptic transmission in the spinal DH, exerting the modulation of spinal nociceptive signals. In this study, we observed that orexin-A (100 nM) could induce an oscillation of membrane current in a subpopulation of SG neurons, which presumably occurs via postsynaptic actions. To date, rhythmic oscillatory change in membrane current or potential by orexins has been shown in a subpopulation of neurons in the locus coeruleus [18], sympathetic preganglion [19], and superficial DH [6]. Because the orexin-induced oscillation was found only in the sympathetic preganglionic neurons that are electrically coupled [19], synchronized activity of neurons connected by electrical synapses may be a critical factor to generate the oscillation. Alternatively, the oscillation by orexin-B in the superficial DH seems to involve the extracellular signal ATP [6]. In the brain, it has been suggested recently that membrane oscillation potentially contributes to the integration of inputs from multiple sources [2], presumably via the modulation of specific types of synaptic inputs [13]. Therefore, it could be interesting if the membrane oscillation induced by orexin-A influences excitatory or inhibitory synaptic transmission in the SG.

We also observed an inward change in membrane current with bath application of orexin-A, an effect typically regarded as a postsynaptic, mediated by both OX1R and, to a great extent, OX2R. Orexin-A-induced inward currents have been shown to involve an influx of extracellular Na+ [18] and a reduction in K+ conductance [19]. In addition, G protein-cAMP and calcium signals have also been proposed as mechanisms underlying orexin-A-mediated inward current in the sympathetic preganglionic neurons [19] and DH neurons [17], respectively. Thus, although the downstream ionic and signaling mechanisms need to be further characterized in the SG, this result suggests that orexinA affects the excitability of SG neurons. In addition to functional roles of orexins in the short-term food intake [5, 15] and the regulation of sleep and wakefulness [14], the orexins have been known to reduce nociceptive behaviors when they are administered through intra-cerebroventricular or intrathecal routes [12]. Furthermore, recent reports have consistently demonstrated the involvement of orexins, especially orexin-A, in the various types of pain. Particularly, orexin-A is antinociceptive in animal models of neuropathic pain with streptozotocin-induced diabetes [11] or peripheral nerve injury by partial sciatic nerve ligation [22]. Interestingly, the antinociceptive effect on established chronic pain is rapid but short-lasting (~2 h) [3, 11, 22], and is mediated by the high affinity OX1R in the spinal DH, not through the opioidergic system in the brain [1, 3, 11]. In the present study, we provide evidence for the spinal mechanisms underlying the antinociceptive effect of orexin-A. The spinal mechanisms include the modulation of primary afferent-mediated excitatory synaptic transmission and neuronal excitability, i.e., oscillation and inward membrane current in the SG neurons mediated by OX1R and (more so) OX2R.

Acknowledgement

This work was supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2014).

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Figure legends

Fig. 1. Depression of primary afferent-evoked excitatory synaptic transmission by orexin-A in the spinal cord SG is mediated by predominantly OX1R. (A) Bath application of orexin-A (100 nM, 10 min) reduced to a similar degree peak amplitudes of A-fiber- and C-fiber-mediated EPSCs that were defined as monosynaptic (filled circle, A-fiber, conduction velocity of 2.8  0.3 m/sec, n = 8 SG neurons; open circle, C-fiber, conduction velocity of 0.6  0.2 m/sec, n = 8 SG neurons). Representative monosynaptic EPSCs are shown above the time-course graph. Numbers in EPSC traces indicate the time sampled. (B) Bath application of orexin-A (100 nM, 10 min) reduced peak amplitude of polysynaptic EPSCs recorded in SG neurons; however, the magnitude of reduction for C-fiber-mediated EPSCs (open circle, n = 5 SG neurons) was greater than that for A-fiber-mediated EPSCs (filled circle, n = 8 SG neurons). *P < 0.05 or **P < 0.01, Student’s t-test. (C and D) Histograms showing that the OX1 antagonist SB674042 (1 μM) is more effective at inhibiting the orexin-A (100 nM)-induced depression of EPSCs evoked by monosynaptic (C)/polysynaptic (D) Aor C-fibers than the OX2 antagonist EMPA (1 μM). *P < 0.05 vs. control (no antagonist). The numbers indicate number of neurons included. (E) The depressant effect of orexin-A at 100 nM (*P < 0.05 vs. 3 nM) was selectively inhibited by SB674042 (95.2  3.1 % of baseline, n = 19 SG neurons, **P < 0.01 vs. 100 nM orexin-A without antagonist), whereas it was not significantly affected by EMPA (79.7  4.8 % of baseline, n = 13 SG neurons). Bath application of SB674042 and EMPA alone did not significantly change peak amplitude of primary afferent-evoked EPSCs (SB674042, n = 8; EMPA, n = 9).

Fig. 2. Orexin-A-induced increase of sEPSC frequency, membrane oscillation and inward holding current in the spinal SG neurons. (A) Bath application of orexin-A (100 nM, 10 min) increased sEPSCs frequency (A-b; each histogram, every 30 sec), but not amplitude (A-c, cumulative probability). Representative sEPSC traces (A-a) were sampled in the baseline (1) and application (2; a bar on A-b) periods. (B) Scatter diagrams indicate percent changes of sEPSC frequency and amplitude after application of orexin-A for 18 SG neurons recorded. (C) Histograms summarizing the experiment performed in three different conditions: the absence (control) and presence of SB674042 (1 μM) or EMPA (1 μM), for orexin-A-induced changes in frequency or amplitude. *P < 0.05 and **P < 0.01. (D) Upon bath application of orexin-A (100 nM), a membrane oscillation was observed in the example SG neuron. After washout of the peptide, the oscillation was no longer evident. The power spectrum (only up to 50 Hz is shown) shows an increase in the 1 to 20 Hz frequency range after the application of orexin-A compared to the baseline. (E) Histograms compare probabilities of SG neurons with membrane oscillation in the absence and presence of SB674042 or EMPA (**P < 0.01, Fisher’s exact test). Numbers indicate the numbers of neurons with oscillation/total neurons recorded. (F) Averages of whole-cell inward currents induced by the application of orexin-A. The numbers indicate number of SG neurons.

FIG. 1

Fig. 2