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Journal of Physiology (2002), 538.2, pp. 517-525
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013120

Hypocretin-2 (orexin-B) modulation of superficial dorsal horn activity in rat

Timothy J. Grudt *, Anthony N. van den Pol † and Edward R. Perl *

	ABSTRACT

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The hypothalamic peptides hypocretin-1 (orexin A) and hypocretin-2 (Hcrt-2; orexin B) are important in modulating behaviours demanding arousal, including sleep and appetite. Fibres containing hypocretin project from the hypothalamus to the superficial dorsal horn (SDH) of the spinal cord (laminae I and II); however, the effects produced by hypocretins on SDH neurones are unknown. To study the action of Hcrt-2 on individual SDH neurones, tight-seal, whole-cell recordings were made with biocytin-filled electrodes from rat lumbar spinal cord slices. In 19 of 63 neurones, Hcrt-2 (30 nM to 1 µM) evoked an inward (excitatory) current accompanied by an increase in baseline noise. The inward current and noise were unaffected by TTX but were blocked by the P_2X purinergic receptor antagonist suramin (300-500 µM). Hcrt-2 (30 nM to 1 µM) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the majority of neurones. The sIPSC increase was blocked by strychnine (1 µM) and by TTX (1 µM), suggesting that the increased sIPSC frequency was glycine and action potential dependent. Hcrt-2 increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in a few neurones but had no effect on dorsal root-evoked EPSCs in these or in other neurones. Neurones located in outer lamina II, particularly radial and vertical cells, were most likely to respond to Hcrt-2. We conclude that Hcrt-2 has excitatory effects on certain SDH neurones, some of which exert inhibitory influences on other cells of the region, consistent with the perspective that hypocretin has a role in orchestrating reactions related to arousal, including nociception, pain and temperature sense.

(Received 8 August 2001; accepted after revision 24 October 2001)
Corresponding author E. R. Perl: Department of Cell and Molecular Physiology, CB #7545-187 MSRB, University of North Carolina - Chapel Hill, Chapel Hill, NC 27599-7545, USA. Email: erp{at}med.unc.edu

	INTRODUCTION

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Newly discovered hypothalamic peptides, the hypocretins (orexins), have been linked to the regulation of behaviours associated with arousal such as sleep and feeding (de Lecea et al. 1998; Sakurai et al. 1998; van den Pol, 2000). In particular, the sleep disorder narcolepsy has been correlated with either a lowered expression of hypocretin (Chemelli et al. 1999; Nishino et al. 2000; Peyron et al. 2000; Thannickal et al. 2000; van den Pol, 2000) or a defect in a receptor for hypocretin (Lin et al. 1999). Neurones synthesizing the hypocretins are limited to the lateral hypothalamic, perifornical region; however, they send axonal projections to many regions of the central nervous system (Peyron et al. 1998) including a strong distribution to the superficial dorsal horn (SDH, laminae I and II) of the spinal cord (van den Pol, 1999). The SDH is heavily involved in the processing of primary afferent nociceptive and thermoreceptive activity (Perl, 1984; Light, 1992; Han et al. 1998). Our attention was attracted by the functional significance of the fibres containing a hypothalamic peptide that plays a part in maintaining normal wakefulness, for a spinal cord region involved in the transfer of information about pain-causing events.

Only limited information exists on the functional effects produced by the two known forms of hypocretin: hypocretin-1 (Hcrt-1) and hypocretin-2 (Hcrt-2). Hcrt-2 is more likely to activate the hypocretin receptor-2 (Hcrt-R2; Sakurai et al. 1998), the receptor implicated in narcolepsy, than the hypocretin receptor-1 (van den Pol, 2000). Hcrt-2 increases the frequency of glutamate-mediated spontaneous postsynaptic excitatory activity and increases GABA-mediated spontaneous inhibitory synaptic currents in cultured hypothalamic neurones. Hcrt-2 increases intracellular calcium in cultured hypothalamic and spinal cord neurones (van den Pol et al. 1998; van den Pol, 1999). It depolarizes and increases the discharge frequency of locus coeruleus neurones (Horvath et al. 1999). Thus, Hcrt-2 is reported to produce a mix of presynaptic and postsynaptic actions making it difficult to predict possible effects in the SDH.

The SDH contains a number of morphologically and functionally distinguishable classes of neurone (Grudt & Perl, 2002). This, and the variety of its constituent synaptic mediators (Light, 1992; Todd & Spike, 1993), indicates that the SDH represents a complex neural system. Many SDH neurones have axons arborizing locally (Pearson, 1952; Réthelyi & Szentágothai, 1973), further implying integrative circuitry. The hypocretin projection from the hypothalamus presumably contributes a layer of modulation of SDH neurones in addition to that produced by other descending projections. Insight into how the hypocretin input to the SDH fits with its presumptive part in wakefulness and arousal depends upon understanding action of these peptides upon SDH neurones. We undertook to determine the effects of Hcrt-2, the hypocretin most selective in the antagonism of sleep, on a sample of individual SDH neurones. A preliminary report of these observations has been made (Grudt et al. 2000).

	METHODS

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All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill. Young, free-ranging rats (75-125 g), deeply anaesthetized with urethane (1.6 g kg^-1) and cooled to a core temperature of under 25 °C, were killed by exsanguination and cardiac arrest in the process of removal of the spinal cord. Transverse 900-1100 µm slices with attached segmental dorsal roots were prepared from the lumbar spinal cord using a vibrating microtome. Slices were maintained at room temperature (21-24 °C) in artificial cerebral spinal fluid (ACSF) equilibrated with 95 % O₂-5 % CO₂. The ACSF consisted of (mM): NaCl 125, NaHCO₃ 26, NaH₂PO₄ 1.25, KCl 2.5, CaCl₂ 2, MgCl₂ 1 and D-glucose 26.

Tight-seal, whole-cell recordings were made from neurones in both current clamp and voltage clamp modes using pipette electrodes (4-7 M) visually guided into the SDH. The internal pipette solution consisted of (mM): potassium gluconate 130, NaCl 5, CaCl₂ 1, MgCl₂ 1, EGTA 11, Hepes 10 and Na₂ATP 4. The electrodes were back-filled with a solution containing 0.5 % biocytin to label the recorded cell and permit morphological analyses. On completion of recording, the slices were placed in 4 % paraformaldehyde and 4 % sucrose in 0.1 mM phosphate buffer for fixation. After cryoprotective postfixation in 30 % sucrose, sections were usually cut parasagittally at 60 µm in a freezing microtome. Neurones labelled with biocytin were visualized by an immunoperoxidase reaction using avidin-biotin histochemistry (ABC kit, Vector Laboratories).

Signals from the recording micropipette were amplified and conditioned by an Axopatch 200B amplifier (Axon Instruments) and controlled in part by a PC digital computer with pCLAMP 6 software (Axon Instruments). Data were acquired at 4 kHz and filtered at 2 kHz. The firing pattern of neurones was examined in current clamp mode by passing 1 s depolarizing steps from a holding potential of -60 mV. The segmental dorsal root was stimulated with a suction electrode, using graded 0.2 ms pulses. Current-voltage (I-V) relationships were constructed by holding neurones at -60 mV in voltage clamp and stepping every 5 s in 10 mV increments for 500-800 ms, initially to -50 mV and then to -120 mV. To test the effects of Hcrt-2 on I-V relationships, the peptide-containing solution was applied and when the response reached a maximum (noise and depolarization; see below), the I-V protocol was run. Responses to Hcrt-2 and any subsequent desensitization were slow and did not change during the I-V measurement period.

Bicuculline, suramin, strychnine and TTX (tetrodotoxin) were obtained from Sigma (St Louis, MO, USA). Hypocretin-2 was synthesized and purified at the Stanford University Peptide Facility. All agents were bath applied in the concentrations and at the times indicated.

	RESULTS

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Electrophysiological observations

The effects produced by Hcrt-2 applied in the superfusate were studied in stable, whole-cell recordings from 63 SDH neurones. The majority of these recordings were made from cells in lamina I and outer lamina II (II_o).

In 19 of 63 neurones (29 %), in voltage clamp, Hcrt-2 (30 nM to 1 µM) in the superfusate initiated an inward current accompanied by an increase in baseline noise (Fig. 1). The amplitude of the inward current ranged from 5 to 35 pA (mean 13.6 ± 2.4 pA). Such inward currents correspond to depolarization in a voltage recording and would result in increased excitability or neuronal discharge. Repeated applications of Hcrt-2 at nanomolar concentrations (30-100 nM) for 1 min or longer initiated progressively smaller inward currents and noise. On the other hand, short applications at higher Hcrt-2 concentrations (1 µM for 30 s) countered this tachyphylaxis (see below). TTX (1 µM) had no demonstrable effect (n = 2) upon the induced noise and inward currents (Fig. 2), but fast inward currents enlisted by depolarizing steps were completely blocked. In contrast, Fig. 3 shows that suramin (300-500 µM), a P_2X purinergic receptor antagonist, blocked the Hcrt-2-induced noise and inward current (n = 3). Our observations did not permit establishment of whether this suramin action was on presynaptic terminals or on the postsynaptic cell.

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Figure 1. Hcrt-2 in the superfusate evokes an inward current and an increase in baseline noise in certain SDH neurones A, tight-seal, whole-cell voltage clamp recording from a SDH neurone held at -60 mV; Hcrt-2 was superfused at 1 µM. B and C, parts of the trace shown in A on an expanded time base.

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Figure 2. Noise (and accompanying inward current) evoked by Hcrt-2 in SDH neurones is not blocked by TTX TTX (1 µM) was superfused for 5 min prior to application of Hcrt-2 (1 µM).

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Figure 3. The Hcrt-2-evoked increase in noise (and the accompanying inward current) is suppressed by the P2X purinergic receptor antagonist suramin Suramin (300 µM) was superfused for 5 min prior to Hcrt-2 (1 µM) application.

The type of change in I-V relationship during the Hcrt-2-induced inward current is shown in Fig. 4. The Hcrt-2 difference current for the example of Fig. 4A is plotted in Fig. 4B. The average Hcrt-2 current for six neurones is depicted in Fig. 4C. Over the range from -50 to -120 mV, these data show that Hcrt-2 evoked a nearly parallel inward shift of the I-V relationship; the current remained essentially constant for differing clamp voltages. We interpreted this to suggest that changes in the conductance of multiple ions were involved.

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Figure 4. Changes in current-voltage (I-V) relationships produced by Hcrt-2 A, I-V relationships for a neurone showing the change produced by Hcrt-2 (1 µM). The cell was held at -60 mV and stepped to the potentials indicated. B, a plot of the difference between the control I-V and the Hcrt-2 I-V relationships for the observations in A. C, mean ± S.E.M. of Hcrt-2-induced inward currents for six neurones. The current values have been normalized to that induced at -60 mV.

Hcrt-2 increased the frequency of spontaneous outward currents in the majority of neurones (34/55), as illustrated by Fig. 5. These outward currents presumably represented spontaneous inhibitory postsynaptic currents (sIPSCs). In another eight neurones, during Hcrt-2 application baseline noise increased to a level precluding accurate counting of sIPSCs. Robust increases in sIPSC frequency were obtained at Hcrt-2 concentrations of 30 nM for several minutes.

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Figure 5. Hcrt-2 increases the frequency of spontaneous inhibitory (outward) currents Voltage clamp recording with the neurone held at -50 mV; upward deflection is outward current. Hcrt-2 was superfused at 1 µM.

The sIPSCs were largely suppressed by TTX (1 µM). TTX also blocked all of the increase in sIPSC frequency induced by Hcrt-2 (n = 4, Fig. 6A). Suppression of sIPSCs and their increased frequency by TTX was taken to indicate that these outward currents were generated by impulses in presynaptic terminals contacting the recorded neurone. This, in turn, suggested that an action of Hcrt-2 was to increase discharge frequency in a population of presynaptic inhibitory neurones. Strychnine (1 µM) also antagonized the Hcrt-2-induced increase in sIPSC frequency (n = 7; Fig. 6B). Bicuculline (10 µM), a GABA_A antagonist, had little or no effect on sIPSC frequency (n = 2) in cells in which strychnine suppressed the Hcrt-induced increase of sIPSCs. The latter suggested that the neurones which Hcrt-2 activates to increase IPSCs utilize glycine as an inhibitory mediator.

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Figure 6. Characterization of Hcrt-2-induced increased frequency of sIPSCs A, histogram showing that the increase in sIPSC frequency elicited by Hcrt-2 (1 µM) is blocked by TTX (1 µM). Each bin is the sIPSC count for 1 min. The durations of the application of Hcrt-2 and TTX are indicated by bars above the histogram. B, the glycine receptor antagonist strychnine (1 µM) also suppresses the increase in sIPSC frequency. Plotting conditions are the same as for A.

In a small proportion of neurones (6/51), Hcrt-2 provoked a transient increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), as depicted in Fig. 7. This sEPSC action may have been deceptively rare in our accounting because in an additional 12 neurones, the baseline noise after Hcrt-2 application was too great to permit counting of sEPSC frequency. The induced increase of sEPSC frequency was shorter in duration than the Hcrt-2-evoked increase in sIPSC frequency, lasting a minute or less in contrast to several minutes for the sIPSCs (compare Fig. 7A with Fig. 6A). With one exception, a significant increase in EPSC frequency was only seen on the initial Hcrt-2 application. The inability to reproduce the sEPSC frequency increase in successive trials prevented study of its origins and characteristics.

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Figure 7. Hcrt-2 enhances spontaneous excitatory synaptic activity in some SDH neurones A, histogram showing a representative increase in the frequency of sEPSCs in response to Hcrt-2 (1 µM). Each bin is the EPSC count for 12 s. B, Hcrt-2 does not affect dorsal root-evoked EPSCs. Hcrt-2 at 100 nM applied for 5 min increased the frequency of spontaneous synaptic currents without altering the evoked EPSC in the same neurone.

In contrast to effects upon spontaneous synaptic currents, dorsal root (DR)-evoked EPSCs were not affected by Hcrt-2, even though other actions were evident in the same cells (n = 4, e.g. Fig. 7B). Therefore, Hcrt-2 appears to have little effect upon the fast linkage between primary afferent fibres and the neurones from which we recorded.

Given the complexity of the SDH, it is not surprising that some neurones responded to Hcrt-2 in more than one way. All six neurones in which Hcrt-2 increased sEPSC frequency also showed increased sIPSC frequencies. Interestingly, eight of the 19 neurones that responded to Hcrt-2 with an inward current and increase in noise, showed an increase in frequency of IPSCs.

Location and identification of neurones responsive to Hcrt-2

The somata of 56 of the 63 neurones studied were located by the presence of the intracellular biocytin label. Their locations are diagramatically plotted in Fig. 8, which documents that most neurones responsive to Hcrt-2 were located in the outer third of the SDH. Neurones responding with an inward current and increase of baseline noise were primarily located in outer lamina II (II_o) near the border between it and inner lamina II (II_i). With few exceptions, neurones with somata in laminae I or II_i gave no response to Hcrt-2. Neurones with somata located superficial to the laminae II_o-II_i interface generally did not exhibit the inward current response.

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Figure 8. Schematic depiction of the relative locations of neuronal somata according to effects produced by Hcrt-2 Neurones were labelled by biocytin in the recording pipette. The borders of the lamina were determined using darkfield microscopy of tissue sections. Filled circles mark locations of cells that responded to Hcrt-2. Open circles indicate unresponsive cells. In some cells, Hcrt-2 increased noise to an extent that accurate counting of spontaneous synaptic currents was not possible. These cases are omitted from the relevant schematic diagram. Note that some neurones exhibited more than one type of response.

A combination of morphological and functional criteria have been suggested as a means to characterize rodent SDH neurones (Grudt & Perl, 2002). According to this scheme, our studied population includes one 'projection' (prominent axon directed ventrally and contralaterally) and several 'non-projection' (major axon distribution in the SDH) lamina I neurones, 20 lamina II vertical neurones, nine lamina II radial neurones and six lamina II central neurones (see Grudt & Perl, 2002, for information on classification). Examples of the configuration of responsive and unresponsive neurones appear in Fig. 9. Seventeen cells could not be classified. It was noteworthy that this sample from rat did not contain islet cells, a relatively common type in other reports (Gobel, 1978; Todd & Spike, 1993; Grudt & Perl, 2002).

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Figure 9. Examples of neuronal types responsive and unresponsive to Hcrt-2 A, radial cell in which Hcrt-2 increased IPSC frequency and induced an inward current plus an increase in baseline noise. B, vertical cell in which both spontaneous EPSC and IPSC frequency increased. C, vertical cell showing only an increase in sIPSC frequency in response to hypocretin in the superfusate. D, central cell which did not respond to superfusion with Hcrt-2. Note the extensive local distribution of axonic branches in A and C.

Eighteen of the 19 neurones responding to Hcrt-2 with inward current and an increase in noise were labelled with biocytin. As shown in Table 1, two-thirds (6/9) of the radial cells and 30 % (6/20) of the vertical cells responded in this fashion. One neurone exhibiting inward current was morphologically of the vertical cell type but was exceptionally deep in lamina II; there is the possibility that it was a lamina III cell. The one central neurone showing an Hcrt-2-produced depolarizing current was situated very lateral in the SDH; it was the one cell of this type also showing an increase in sEPSC frequency. Table 1 further shows that a variety of cell types exhibited an increased frequency of sIPSCs, including both vertical and radial neurones.

	DISCUSSION

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A unifying and economical explanation for the Hcrt-2 effects on laminae I and II neurones is made difficult by the varied nature of the effects and the present limited insight on details of the region's functional organization. The inward current with the associated increase of synaptic noise and/or the increase in frequency of sEPSCs represent actions that would tend to make a neurone more excitable. In contrast, the increase in frequency of sIPSCs represents an inhibitory action on the recorded neurone, albeit one generated by action potentials in presynaptic neurones. The seemingly contradictory nature of these effects needs to be viewed in the light of the multiple possible functions served by neurones of the region. These functions include the reception and integration of signals associated with (a) nociception and pain, (b) temperature sense, (c) itch and (d) possibly other somatic afferent functions (Perl, 1984; Light, 1992; Han et al. 1998; Andrew & Craig, 2001). Bath-applied Hcrt-2 would simultaneously affect neurones with hypocretin receptors possibly serving more than one of these functions. Two clues may be important in sorting out the underlying processes. One is that while the induction of inward current and the increase in TTX-sensitive sIPSCs could be reproduced in a given cell on repeated tests, a significant increase in sEPSCs almost always was not. This distinction implies some underlying differences in the production of these events. Furthermore, suppression by suramin, a P_2X purinergic receptor blocker, of both the Hcrt-2-induced inward current and the increase of baseline noise, implicates P_2X receptors in these actions. This, in turn, suggests a P_2X receptor agonist, presumably adenosine triphosphate (ATP), is involved in the mediation of the Hcrt-2 action (Li & Perl, 1995; Bardoni et al. 1997).

Hypocretin peptides have excitatory effects on both presynaptic terminals and postsynaptic neurones in other parts of the nervous system (van den Pol et al. 1998; Horvath et al. 1999). In particular, application of hypocretin increases cytosolic Ca²⁺ (van den Pol et al. 1998), which, if it occurred presynaptically, could be related to an increased release of transmitter from presynaptic terminals (Kuno, 1995). Therefore, one possible explanation for certain of the Hcrt-2 actions on SDH neurones would be the activation of Hcrt receptors on presynaptic neurones, triggering entry of Ca²⁺ into presynaptic terminals. The increased cytosolic Ca²⁺ could be hypothesized to cause the presynaptic release of ATP to activate P_2X receptors on the postsynaptic cell. This, in sequence, could open non-selective cation channels inducing inward current (and synaptically generated noise), enhancing excitability of the cell. Extending this postulate further, excited cells may be presumed to be presynaptic to neurones whose activity releases glycine, thereby producing the increased frequency of TTX-sensitive sIPSCs. Such a rationalization could account for the two most commonly observed effects of Hcrt-2 on our spinal cord slices. It could also explain the less frequently observed increase in sEPSCs by presuming that these took place in neurones subserving other functions in which the basic action of Hcrt-2 is to enhance the presynaptic release of transmitter secondary to an evoked influx of Ca²⁺. Unfortunately, we lack evidence on the synaptic mediator involved in the sEPSCs and are left at a loss to explain the striking habituation of the increase in sEPSCs.

In keeping with the above rationalization, activation of P_2X receptors by ATP induces an inward current in a subset of SDH neurones (Li & Perl, 1995). In a small proportion of SDH neurones, synaptic currents are generated by a purine acting on P_2X receptors (Bardoni et al. 1997). Nevertheless, it seems likely that a mediator other than purines was involved. Suramin suppressed the inward current and noise produced by Hcrt-2 but it failed to totally block them. Moreover, our current-voltage observations appear inconsistent with simply opening P_2X-gated channels. The non-selective cation channels that are activated by ATP show inward rectification at negative potentials (Li & Perl, 1995; Bardoni et al. 1997). The current activated by Hcrt-2 in the present work exhibits a near parallel shift from -60 to -120 mV which, as suggested earlier, could reflect the involvement of multiple conductances.

Block of the increase of spontaneous IPSCs by strychnine strongly implicates glycine and glycine receptors in the mediation of the outward currents. Markers for glycine and glycine receptors are present on neurites in the SDH (van den Pol & Gorcs, 1988); however, both spontaneous and evoked IPSCs in SDH neurones are also mediated in part by -aminobutyrate (GABA) acting on GABA_A receptors (Yoshimura & Nishi, 1995; Grudt & Henderson, 1998). The GABA_A receptor antagonist bicuculline had little effect on the increase of IPSCs evoked by Hcrt-2. Therefore, the action of Hcrt-2 appears to be highly selective in evoking glycine-mediated IPSCs. A similar selective activation of glycine IPSCs, in a region also containing GABA-releasing neurones, has been documented in the trigeminal complex (Grudt et al. 1995).

The SDH is composed of numerous morphological and functionally distinguishable cell types (Grudt & Perl, 2002). Of the categories identifiable in the present recordings, radial and vertical cells were most commonly affected by Hcrt-2. These types of cell are common in lamina II_o and at the border between lamina II_o and lamina II_i, the part of the SDH that receives the densest innervation of hypocretin-containing axons (van den Pol, 1999).

The potent effects of the hypocretins upon sleep and appetite suggests that their actions on the SDH probably relate to the regulation of these functions or the arousal associated with them. Since hypocretin absence is associated with narcolepsy (Nishino et al. 2000; Peyron et al. 2000; Thannickal et al. 2000), presumably its presence is related to alertness. Hypocretin's actions on SDH neurones may be to alter responsiveness in relation to the change from sleep to wakefulness. One can speculate that the depolarization and increased baseline noise leads to enhancement of the activity of cells directly or indirectly involved in suppressing nociceptive reflexes or rostral transmission of nociceptive activity. That possibility would be consistent with a recent report indicating orexin-A (Hcrt-1) to be potently analgesic in behavioural tests (Bingham et al. 2001). Clarification and further understanding of the processes involved in hypocretin actions will depend upon a better knowledge of the circuitry of the SDH and a fuller appreciation of the functional part played by the various cell types. At the present level of understanding, it is possible to affirm that the descending connections from the hypothalamus of Hcrt-2-containing neurones has significant functional consequences in terms of regulation of excitability of a substantial number of neurones in part of the SDH. These effects can be rationalized in terms of hypocretin's enhancement of arousal and support of mechanisms associated with active feeding.

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Acknowledgements

This work was supported by grant NS10321 to E.R.P. from the National Institute of Neurological Diseases and Stroke of the National Institutes of Health, USA. Grant NS 41454 provided funds for production of Hcrt-2. We are grateful to Ms Sherry Joseph and Ms Helen Willcockson for their assistance in the preparation of this manuscript and to Mr Kirk McNaughton for excellent histological assistance.

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