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

Abstract

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 microM) 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 microM). Hcrt-2 (30 nM to 1 microM) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the majority of neurones. The sIPSC increase was blocked by strychnine (1 microM) and by TTX (1 microM), 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.

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.

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).

Figure 3. The Hcrt-2-evoked increase in noise (and the accompanying inward current) is suppressed by the P_2X purinergic receptor antagonist suramin

Suramin (300 μm) was superfused for 5 min prior to Hcrt-2 (1 μm) application.

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.

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.

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.

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.

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.

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.