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.2002 Apr 1;22(7):2862-72.
doi: 10.1523/JNEUROSCI.22-07-02862.2002.

Direct and indirect excitation of laterodorsal tegmental neurons by Hypocretin/Orexin peptides: implications for wakefulness and narcolepsy

Affiliations

Direct and indirect excitation of laterodorsal tegmental neurons by Hypocretin/Orexin peptides: implications for wakefulness and narcolepsy

Sophie Burlet et al. J Neurosci..

Abstract

Compelling evidence links the recently discovered hypothalamic peptides Hypocretin/Orexin (Hcrt/Orx) to rapid eye movement sleep (REM) control and the sleep disorder narcolepsy, yet how they influence sleep-related systems is not well understood. We investigated the action of Hcrt/Orx on mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT), a target group whose function is altered in canine narcolepsy and appears pivotal for normal REM and wakefulness. Extracellular recordings from mouse brainstem slices revealed that Hcrt/Orx evoked prolonged firing of LDT neurons. Whole-cell recordings revealed that Hcrt/Orx had actions on both presynaptic neurons and at postsynaptic sites. Hcrt/Orx produced an increase in frequency and amplitude of spontaneous EPSCs without equivalent effect on IPSCs, by triggering action potentials and enhancing spike-evoked synaptic transmission in glutamatergic afferents. Postsynaptically, Hcrt/Orx produced an inward current and an increase in membrane current noise, which were accompanied by a conductance increase. These persisted in TTX, ionotropic glutamate receptor antagonists, and low extracellular calcium. Both presynaptic and postsynaptic actions were specific because they were not mimicked by an Hcrt/Orx fragment, and both actions were observed for cholinergic and noncholinergic LDT neurons. Finally, extracellular recordings during postsynaptic potential blockade demonstrated that postsynaptic actions of Hcrt/Orx alone could evoke prolonged firing. In the context of other recent work, our findings suggest that Hcrt/Orx neurons may coordinate the activity of the entire reticular activating system during waking. Moreover, these findings address specific hypotheses regarding the cellular mechanisms underlying REM disregulation in narcolepsy.

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Figures

Fig. 1.
Fig. 1.
Hcrt/Orx stimulated a long-lasting increase in firing of LDT neuronsin vitro.A1, Extracellular loose-patch voltage recordings indicated that Hcrt/Orx-A (300 nm) stimulated even quiescent neurons to fire repetitively.A2, Instantaneous spike rate of the neuron inA1 shows the long-lasting Hcrt/Orx response. Thebar over the histogram indicates the duration of the superfusion in this and subsequent figures.B, Instantaneous firing rate from another neuron before and after two Hcrt/Orx applications (1 μm) shows that the second response was greatly attenuated. Note that the breaks in the time axis correspond to the time between data files in this and subsequent figures.
Fig. 2.
Fig. 2.
Hcrt/Orx produced an increase in glutamatergic synaptic activity, an inward current, and an increase in membrane current noise.A1, Whole-cell recordings of membrane current at −60 mV in the presence of bicuculline (10 μm) and strychnine (2.5 μm) indicate that Hcrt/Orx-A (1 μm) application produced a large increase in sEPSC activity and an inward shift of the baseline current that reversed after a washout of ∼20 min.A2,Arrow indicates the neuron whose currents are shown inA–C. The neuron was filled with biocytin during the recording and visualized with avidin–Texas Red epifluorescence.A3, The same neuron was also visible with FITC immunofluorescence for bNOS. Scale bar (inA2):A2,A3, 25 μm.B1, An expanded segment of thetrace inA1 before Hcrt/Orx application (labeledB1 inA1).B2, Expanded segment fromA1 after Hcrt/Orx-A application illustrates the greater number of sEPSCs and the inward current shift compared with baseline (horizontal dashed line).B3, These effects recovered by 20 min after the end of the Hcrt/Orx application.C, Cumulative distributions of sEPSC amplitudes (left;Amp) and intervals (right;Int) from before (thin line) and after (thick line) Hcrt/Orx-A application. Kolmogorov–Smirnov statistics demonstrated that Hcrt/Orx-A significantly increased both amplitude and frequency of sEPSCs.D, Population means of sEPSC amplitudes (black bars) and inter-event intervals (white bars) from 15 LDT neurons expressed as percentage of control values. Each neuron showed a significant reduction in the inter-event interval distribution after 1 μm Hcrt/Orx-A. For six of these cells, sufficient recording time was available to observe a recovery after washout of the peptide.E, EPSCs recorded in the presence of bicuculline and strychnine (left;BS) were entirely abolished by the addition of DNQX and APV (middle;DABS). Subsequent application of Hcrt/Orx (300 nm) produced an inward current but no additional PSCs. A substantial increase in current noise accompanied the inward current.Arrows point to expanded sections of thetrace before and after Hcrt/Orx to illustrate the noise at higher time resolution. The time scale in themiddle also applies to theleft. Note the different time scales in theright.
Fig. 3.
Fig. 3.
Hcrt/Orx effects were specific for the full-length peptide. Whole-cell voltage-clamp recordings (−60 mV) were obtained using a K-gluconate internal solution and an extracellular solution containing bicuculline (10 μm) and strychnine (2.5 μm).A, Application of the amide fragment (16–33) of Hcrt/Orx-A did not increase the frequency or amplitude of sEPSCs.B, In the same neuron recorded inA, subsequent application of Hcrt/Orx-A produced the characteristic increase in both sEPSC amplitude and frequency, as well as an inward shift in baseline current.C, Average of mean inter-event intervals and amplitudes are displayed for a population of six LDT neurons exposed first to the peptide fragment and then Hcrt/Orx-A. Means are expressed as percentage of control values.
Fig. 4.
Fig. 4.
TTX blocked the Hcrt/Orx-evoked increase in EPSC frequency and amplitude. Frequency and amplitude distributions of mEPSCs were measured in the presence of extracellular TTX (0.5 μm), bicuculline (10 μm), and strychnine (2.5 μm) from recordings obtained using a K-gluconate (n = 4) or a KCl (n = 5) internal solution.A, Raster display of five consecutive samples of membrane current [K-gluconate (KGlu)] from before (left) and after (right) application of 1 μmHcrt/Orx. Hcrt/Orx evoked an inward shift in membrane current and a large increase in membrane noise but failed to alter mEPSC activity, as indicated by the cumulative distributions of amplitude (left) and interval (right). Control (thin line) and Hcrt/Orx (thick line) distributions were not different, as indicated by thepvalues from the Kolmogorov–Smirnov test.B, Similar data set from another LDT neuron recorded using the internal KCl solution. Hcrt/Orx at 1 μm had no effect on membrane current or noise, allowing unimpeded detection of small EPSCs. Under these conditions, Hcrt/Orx also had no effect on mEPSC activity, as indicated by the cumulative distributions of amplitude (left) and interval (right).C, Summary data indicating that TTX blocked both the change in mean mEPSC amplitude (black bars) and interval (white bars) produced by Hcrt/Orx (1 μm) when neurons were recorded with either internal solution.
Fig. 5.
Fig. 5.
Hcrt/Orx increased the amplitude of evoked EPSCs. Neurons were recorded with a K-gluconate internal solution and an extracellular solution containing bicuculline (10 μm) and strychnine (2.5 μm). EPSCs in LDT neurons were evoked by local electrical stimulation at 0.02 Hz. An Hcrt/Orx superfusion produced the characteristic inward shift in holding current. Epochs markeda,b, andcindicate the control, Hcrt/Orx, and recovery conditions used to compare EPSCs.Broken line indicates time between data files.B, Averages of 50 consecutive EPSCs from the epochsa–c indicated inA. The average amplitude increased from 31 (a) to 41 (b) pA after the application of Hcrt/Orx and recovered as the Hcrt/Orx effect on membrane current subsided (c).
Fig. 6.
Fig. 6.
Hcrt/Orx-evoked inward current and noise were associated with an increase in membrane conductance.A, Membrane current noise (filled triangles) and average membrane current (filled circles) were monitored at a holding potential of −60 mV, and input conductance (filled squares) was monitored with voltage jumps to −90 mV in the presence of a solution (TTX,DABS) containing TTX (0.5 μm), DNQX (15 μm), APV (50 μm), bicuculline (10 μm), and strychnine (2.5 μm) to eliminate action potentials and fast synaptic activity. Under these conditions, 1 μm Hcrt/Orx-A still evoked an increase in noise and an inward current. These changes were accompanied by a slow increase in membrane conductance. Theinset illustrates membrane voltage (top) and current (bottom) during a voltage jump from before and after Hcrt/Orx application as indicated by thearrows. Voltage and current calibration bars are 30 mV and 50 pA, respectively, and the time calibration is 1 sec.B, Time course of mean ± SEM membrane noise (Irms;triangles), current (Ibase;circles), and conductance (Gm;squares) around the time of Hcrt/Orx application (filled symbols;n = 9) or control solution application (open symbols;n = 4). Hcrt/Orx, but not the control solution, produced changes in these measures. *p < 0.001 and #p < 0.05 indicate significantly different from control as determined by a repeated-measures ANOVA andpost hoc testing (Bonferroni).
Fig. 7.
Fig. 7.
Hcrt/Orx-evoked inward current and membrane noise were insensitive to lowering extracellular calcium.A, The increase in membrane noise (triangles) and inward current (circles) evoked by Hcrt/Orx-A were resistant to solutions containing low extracellular calcium (Low Ca2+,TTX, andDABS). Similar low Ca2+ ASCF without TTX, DNQX, and APV abolished evoked EPSCs (seeinset). Calibration: 20 pA and 10 msec.Arrows point to membrane current traces from the indicated time points before and after Hcrt/Orx application and illustrate the large noise increase produced by Hcrt/Orx under these conditions.B, Summary of mean inward current and noise evoked by 1 μm Hcrt/Orx in calcium-containing ACSF and by 300 nm Hcrt/Orx in low-calcium ACSF. The responses were not statistically different. These data strongly indicate that the inward current and membrane noise result from activation of Hcrt/Orx receptors on LDT neurons.
Fig. 8.
Fig. 8.
The Hcrt/Orx-A-mediated current is sufficient to induce spiking in LDT neurons in the absence of fast PSPs.A, Hcrt/Orx-A at 300 nm evoked repetitive firing in LDT neurons in the presence of a solution (DABS) containing DNQX (15 μm), APV (50 μm), bicuculline (10 μm), and strychnine (2.5 μm) to block fast glutamatergic, GABAergic, and glycinergic transmission. This demonstrated that the relatively small current observed under whole-cell conditions is functionally sufficient to produce suprathreshold depolarization in intact LDT neurons.B, Hcrt/Orx-A-mediated excitation of LDT neurons was dose dependent. Hcrt/Orx was applied during each 10 min epoch. Epochs were separated by the indicated times. With sufficient time between doses, a concentration-dependent action was observed.
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