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Department of Physiology, New York Medical College, Valhalla, New York
Submitted 21 December 2007; accepted in final form 23 July 2008
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ABSTRACT |
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30% even in neurons lacking an inward current. This effect was abolished by nifedipine, augmented by Bay-K and abolished by bisindolylmaleimide I. Thus Hcrt/Orx has two independent actions: activation of noisy cation channels that generate depolarization and activation of a protein kinase C (PKC)-dependent enhancement of Ca2+ transients mediated by L-type Ca2+ channels. Immunocytochemistry verified that both these actions occurred in serotonergic and cholinergic neurons, indicating that Hcrt/Orx can function as a neuromodulator in these key neurons of the reticular activating system. Because regulation of Ca2+ transients mediated by L-channels is often linked to the control of transcriptional signaling, our findings imply that Hcrt/Orxs may also function in the regulation of long-term homeostatic or trophic processes. |
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INTRODUCTION |
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; Sakurai et al. 1998) and function in the control of arousal, feeding, metabolism, reward, and stress responses (for reviews, see de Lecea and Sutcliffe 2005; Sakurai 2007). These neurons have projections that notably target brain regions involved with these functions (Nambu et al. 1999; Peyron et al. 1998) including elements of the reticular activating system such as the dorsal raphe (DR) and laterodorsal tegmental nucleus (LDT). The DR and LDT (and the closely related pedunculopontine tegmental nucleus), respectively, provide widespread serotonergic and cholinergic innervation of the brain and are thought to play overlapping, but distinct, roles in the control of arousal states (for reviews, see Jones 2005; McCarley 2007) and other global functions (for reviews, see Jacobs and Fornal 1993; Maskos 2008; Mena-Segovia et al. 2008; Michelsen et al. 2007).
Actions of Hcrt/Orx peptides are mediated by two Gq-protein–coupled receptors (Sakurai et al. 1998) that are expressed throughout the CNS, including within the DR and LDT (Hervieu et al. 2001; Marcus et al. 2001; Trivedi et al. 1998). Such receptors can mediate numerous short- and long-term actions; however, the scope and consequences of Hcrt/Orx receptor signaling on CNS neurons are not yet fully understood. Electrophysiological studies indicate these peptides have both pre-and postsynaptic excitatory effects (van den Pol et al. 1998) and produce membrane depolarization when direct responses are observed. This depolarization is mediated by a variety, and probably a mixture, of mechanisms including the closure of K+ channels (Bayer et al. 2002; Bisetti et al. 2006; Grabauskas and Moises 2003; Hoang et al. 2003, 2004; Hwang et al. 2001; Ishibashi et al. 2005; Ivanov and Aston-Jones 2000), forward activation of the electrogenic Na+/Ca2+ exchanger (NCE) (Burdakov et al. 2003; Eriksson et al. 2001; Wu et al. 2002, 2004), and activation of a cation current (Murai and Akaike 2005; Yang and Ferguson 2002, 2003).
In vivo extracellular recording studies indicate Hcrt/Orx excites both DR neurons (Takahashi et al. 2005) and LDT neurons (Takahashi et al. 2002). In vitro slice studies indicate this excitation is mediated mainly by a cation current in serotonergic and nonserotonergic DR neurons (Brown et al. 2002; Liu et al. 2002) and that a similar current is likely activated in cholinergic and noncholinergic LDT neurons (Burlet et al. 2002; Leonard et al. 2005), although neither the dependence on extracellular Na+ nor the I-V relation has been published or compared with the current reported in DR neurons.
Hcrt/Orx also produces Ca2+ transients in a large fraction of DR and LDT neurons as studied by bulk-loading with the Ca2+ indicator dye fura-2AM under conditions where neither membrane potential nor transmitter phenotype could be determined (Kohlmeier et al. 2004). These Ca2+ transients displayed distinct temporal profiles ranging from "plateau" to "spiker" responses. They were not mediated by Ca2+ release from intracellular stores, as has been described in cells transfected with Hcrt/Orx receptors, nor were they mediated by reverse mode operation of the NCE, but rather were dependent on Ca2+ influx across the plasma membrane. Because an antagonist and agonist of L-type Ca2+ channels, respectively, reduced and promoted these transients, L-type Ca2+ channels seem to be involved in Ca2+ transients elicited by Hcrt/Orx in the DR and LDT (Kohlmeier et al. 2004) as has been described for some other cultured and dissociated central neurons (Uramura et al. 2001; van den Pol 1999; van den Pol et al. 1998). However, membrane potential was not simultaneously recorded in these previous studies, and it was therefore not clear whether Ca2+ channels were simply activated by depolarization or were also modulated by Hcrt/Orx. Moreover, many nonselective cation channels (NSCCs) are quite Ca2+ permeable, and it is not clear whether Ca2+ influx through such channels contribute to the Hcrt/Orx-stimulated Ca2+ influx.
To address these issues, we used whole cell recording and simultaneous Ca2+ imaging in brain slices to compare the currents activated by Hcrt/Orx in DR and LDT neurons and to determine the relative roles played by the cation current and membrane depolarization in producing Hcrt/Orx stimulated Ca2+ transients. We verified that Hcrt/Orx activates a similar noisy, nonselective cation current in both DR and LDT neurons and, contrary to expectations from findings in transfected cells (Kukkonen and Akerman 2001; Lund et al. 2000), found that this current does not make a detectable contribution to Hcrt/Orx-mediated Ca2+ transients in these neurons. Instead, we found that the Hcrt/Orx-mediated somatic Ca2+ transients are voltage-dependent and that Hcrt/Orx enhances the Ca2+ transient resulting from activation of L-type Ca2+ channels, even in the absence of cation current activation. Collectively, these findings offer a simple explanation for the diverse temporal patterns of Ca2+ transients previously observed in these neurons and indicate that Hcrt/Orx can act as a neuromodulator to enhance L-type Ca2+ channel signaling, even in the absence of Hcrt/Orx-evoked depolarization in cholinergic and serotonergic neurons of the reticular activating system.
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METHODS |
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All procedures complied with National Institutes of Health and institutional guidelines (New York Medical College) for ethical use of animals and were approved by the New York Medical College Institutional Animal Care and Use Committee (IACUC).
Slice preparation and artificial cerebrospinal fluid
Brain slices were prepared from 14- to 30-day-old C57/Bl6 mice (Charles River Laboratories). Animals were decapitated following induction of deep anesthesia with isoflurane (Aerrane, Baxter Pharmaceuticals, inhalation). A block of the brain containing the DR or the LDT was rapidly removed and incubated in ice-cold artificial cerebrospinal fluid (ACSF, osmolarity = 295) that contained (in mM) 121 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 1.2 MgSO4, 26 NaHCO3, 20 dextrose, 4. 2 lactic acid, and oxygenated by bubbling with carbogen (95% O2–5% CO2). The brain stem was further sectioned at 250–350 µm in a coronal plane on a Leica vibrotome (VT1000S). Slices containing the DR or LDT were incubated at 35°C for 15 min and stored at room temperature. Recordings were obtained from slices submerged in a recording chamber that was perfused at 2–5 ml/min with the continuously oxygenated ACSF at room temperature (21°C). DR recordings were obtained in slices just rostral to the level of the LDT from cells in the central gray within 50 µm of the midline. LDT recordings were obtained from the LDT cholinergic cell group, beginning rostral to the main locus coeruleus, in slices in which the dorsal tegmental nucleus was also present. Relatively large cells appearing in clusters were targeted in both nuclei, with large cells apposing the blood vessel within the LDT preferred (estimated long axis DR: 17.3 ± 0.7 µm, n = 19; LDT: 18.6 ± 1.0 µm, n = 19 from fura2 fluorescence in the slice). However, histochemical methods were used for final confirmation of neurotransmitter phenotype.
Drugs and experimental solutions
Orexin-A (Sigma; Phoenix Pharmaceuticals; American Peptides) was dissolved in water in 10 µM aliquots and frozen (–80°C). Aliquots were dissolved in ACSF to a final concentration of 300 nM or 1 µM immediately before use. Orexin was either bath applied or delivered more locally via a "Y" tube (modified from Wu et al. 2002). A "Y" tube was made by forming silicone tubing into a U and converting this construction to a Y tube by inserting a microfil tubing (ID, 100 µm; OD, 164 µm; CMF34GxxL, World Precision Instruments, Sarasota, FL) into the bend of the U with cyanoacrylic glue. During recordings, the microfil tubing was placed within 50 µm of the cell. The 15 s application time was controlled by a ALA-VM4 valve manifold (Scientific Instruments). Latency from closing of the valve (which started drug solution flow into tissue) and response of the neuron was typically <15 s. Rapid wash of orexin was obtained by switching superfusion solutions to control ACSF. Leakage of agonist was monitored and found to not be an issue. TTX (Alomone) was dissolved in ACSF to a final concentration of 500 nM to block voltage-gated sodium channels. In most of these experiments, DR or LDT neurons were recorded in an ACSF solution (DABST) containing the ionotropic receptor antagonists DNQX (15 µM, Sigma), APV (50 µM, Sigma), bicuculline (10 µM, Sigma), and strychnine (2.5 µM, Sigma), in addition to TTX (0.5 µM, Alamone). In some recordings, CsCl (2 or 3 mM) was added to the DABST to block H-current. In some experiments, the [Ca2+] of the ACSF was buffered to <20 µM by the addition of 2.7 mM EGTA (calculated with Patcher's Power Tools XOP for Igor Pro). In others, a low-Na+ ACSF was used in which NaCl in the ACSF was replaced with an equivalent concentration of N-methyl-D-glucamine Cl resulting in a solution containing 26 mM Na+. The L-type Ca2+ channel antagonist nifedipine and agonist, Bay-K-8644 (Bay-K, Sigma) were dissolved in DMSO to a stock concentration of 10 mM and delivered at the final concentration of 10 µM in ACSF. Bisindolylmaleimide I, HCl (Calbiochem, EMD Biosciences) was dissolved in DMSO to a stock concentration of 5 mM. On the day of experiments, it was diluted in TTX-ACSF to a final concentration of 1 µM and applied for 5 min before application of orexin. Final dilutions of these drugs were made immediately before application, and light exposure was minimized during all phases of their preparation and application.
Whole cell electrophysiological recording and imaging
Micropipettes (2–4 MOhms) used for patch-clamp recordings (Borosilicate, cat number 8050, AM systems) were pulled on a horizontal puller (Sutter Instruments, P87). Pipettes were filled with a recording solution of either the potassium salt of Fura-2 (bis-fura 2, 50 µM, Molecular Probes) dissolved in the recording solution (in mM) 144 K-gluconate, 3 MgCl2, 10 HEPES, 0.3 NaGTP, and 4 Na2ATP or, in those cases where calcium imaging was not being conducted, pipettes were filled with a recording solution containing (in mM) 144 K-gluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 0.3 NaGTP, and 4 Na2ATP (osmolarity = 310). In some experiments, a Cs+-based patch solution containing 143 Cs+-gluconate, 0.2 EGTA, 5 KCl, 4.3 NaCl, 2 MgCl2, and 10 HEPES was used. In most cases, biotinylated Alexa-594 (25 µM) was also included in the patch solution so cells could be histochemically identified. Neurons were visualized for whole cell recordings at x160 magnification with visible-light, differential interference contrast optics, using a Nuvicon tube camera (Dage VE-1000) mounted on a fixed stage microscope (Olympus BX50WI). Cells for recording were chosen within the boundary of the DR or LDT nuclei which were identified using a x4 objective.
Gigaseals were obtained under visual control using an Axopatch 200B amplifier (Axon Instruments) operated in voltage-clamp mode and filtered at 2 or 5 KHz with a four-pole Bessel filter at the amplifier output and sampled at 4, 10, or 20 KHz. In those cases where calcium imaging was performed, after establishing the whole cell recording configuration, cells were filled with both the calcium indicator and biotinylated Alexa-594 by either passive diffusion or brief hyperpolarizing pulses. Data were not collected until 
20 min had passed following break-in to allow dye equilibration. Neurons were imaged through a x40 objective using a cooled, CCD camera equipped with a back-illuminated EEV 57 frame transfer chip having an imaging area of 512 x 512 pixels (field size = 160 µm/side; MicroMax, Roper Scientific). Fura 2 was excited at 380 nm with light from a 75-W Xenon lamp that was shuttered to reduce light exposure to the tissue in between data acquisition. Recordings were either conducted in voltage clamp or "I-clamp fast" current-clamp mode, following appropriate compensation for the pipette capacitance; quality of the cells was assayed by monitoring the holding current and the input resistance as determined by the voltage or current response to a brief, negative going step. Recordings were uncorrected for liquid junction potentials, which were calculated to be 12 mV. Current and voltage traces were digitized, and command pulses were generated by custom software (TIWB; Inoue et al. 1998) run on a Mac OS computer that controlled an ITC-18 interface (Instrutech). TIWB software also controlled the camera and shutter that allowed precise synchronization between electrophysiological and optical signals. The camera was read out through a 1-MHz, 14-bit A/D converter. Images were binned on the chip at 4 x 4 and acquired in two different modes: 1) "discontinuous," a low-speed imaging mode used to monitor changes in calcium associated with depolarizations or inward currents in which an image was collected every 4 s or 2) "continuous," a high-speed imaging mode in which an image was collected every 50 ms, a rate fast enough to monitor changes in calcium accompanying rapid alterations of the membrane potential. Changes in fluorescence (dF/F) were quantified by the average pixel values within regions of interest (ROIs) that were positioned on background- and baseline-subtracted fluorescent images. The background was determined from a ROI that was positioned at a location remote from the filled cell or its processes. Baseline fluorescence was determined as the fluorescence measured in the first few frames of each sequence before stimulation. bis-Fura 2 fluorescence decreases when calcium increases and is bound by the dye; however, for purposes of clarity, dF/F responses have been inverted in figures such that upward dF/F deflections indicate rises in calcium.
In those cases where calcium imaging was not being performed, membrane currents and voltages were controlled and recorded with PCLAMP8 software (Axon Instruments). Access resistance (Racc) was estimated on-line by exponential fitting of the uncompensated capacitative transients resulting from ±2.5-mV voltage steps delivered at 3.3 Hz using the software's membrane test routine. The quality of recorded cells was assessed with this routine by simultaneously monitoring input resistance, holding current, and capacitance. These parameters were monitored continuously over the initial several minutes after establishing the whole cell configuration, as well as before and after each experimental protocol. In all cases, recordings were terminated if the estimated Racc became unstable or changed by >20% between measurements. Recordings were also terminated if cell parameters became unstable.
Electrophysiological analysis
Current and voltage waveforms were analyzed using Igor Pro Software (Wavemetrics). Holding current was measured from 1 s averages of the holding current at –60 mV. I-V relations were obtained from voltage ramps from –100 to –30 mV or to +10 mV (Cs+-rich internal solution). Ramps (14 mV/s) began after holding Vm at –100 mV for 1 s. Reversal potentials were measured by a linear fit to the I-V curve from just negative to just positive to the zero current potential. In some experiments, a previously recorded Hcrt/Orx current (Vm = –60) was used as a command current while monitoring membrane potential. To examine the effect of Hcrt/Orx on the Ca2+ influx mediated by voltage-gated Ca2+ channels, Hcrt/Orx (300 nM) was applied while the voltage of the cell was periodically stepped from a holding potential of –60 to –30 or –40 mV for 5 s. To determine the contribution of L-type Ca2+ channels and the involvement of protein kinase C (PKC) on Hcrt/Orx-induced calcium increases, this protocol was also performed in the presence of nifedipine, a blocker, or Bay-K, an agonist, of this channel or bisindolylmaleimide I, HCl, a PKC inhibitor, respectively. Results were compared with Hcrt/Orx effects in the absence of these agents. Results are reported as means ± SE. Differences between means were compared using either a paired or unpaired two-tailed Student's t-test with 
set at 0.05, as appropriate.
Histochemistry
For histochemical analysis of recorded cells, slices were fixed in 4% paraformaldehyde at 4°C and stored in 0.01 PBS and 30% sucrose for cryoprotection. Slices were resectioned at 40 µm and incubated with gentle shaking at 4°C for 24 h in the dark with the tryptophan hydroxylase (TpH) antibody (Abcam; catalog 3907 sheep 1:1,000) or a bNOS antibody (Sigma, catalog N7280, rabbit polyclonal 1:400), which is a specific marker for cholinergic neurons in the LDT and pedunculopontine tegmentum (Vincent and Kimura 1992). This was followed by incubation with the FITC-conjugated secondary antibody (TpH: A11015 donkey, anti-sheep; bNOS: A11008
[GenBank]
, goat, anti-rabbit; Molecular Probes). Further histochemical processing was not necessary to visualize the Alexa-594–filled cell, which was viewed under a Texas-red filter-cube set.
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RESULTS |
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Hcrt/Orx depolarizes DR and LDT neurons and evokes a coordinated elevation of [Ca2+]i
Application of 300 nM Hcrt/Orx produced membrane depolarization and action potentials in a majority of DR and LDT neurons (
70%) as expected from previous reports (Brown et al. 2002; Burlet et al. 2002; Leonard et al. 2005; Liu et al. 2002). Correlated with these depolarizations, in many cells, were changes in dF/F measured at the soma and proximal dendrites, indicating a rise in intracellular calcium (Fig. 1, A and B). These findings confirm our prior observation in fura-2AM–loaded neurons that Hcrt/Orx elicits Ca2+ transients in both the soma and dendrites (Kohlmeier et al. 2004). Changes in dF/F were never elicited in the absence of depolarization; however, Hcrt/Orx could produce depolarization without detectable change in dF/F in cells from both nuclei—a point we examined in greater detail later. In the DR, the average depolarization was 3.8 ± 1.2 mV and the maximal dF/F increase at the soma was 5.4 ± 1.3% (n = 5). In the LDT, the average depolarization was 3.5 ± 0.8 mV and the maximal dF/F increase at the soma was 5.2 ± 1.8% (n = 10). To determine whether Hcrt/Orx induced these changes in the principal cells of the DR and LDT, we immunolabeled for TpH or bNOS, respectively, in slices containing recorded Alexa-594–injected neurons. Immunohistochemistry from a population of recovered cells showed that Hcrt/Orx-induced effects were present in serotonergic DR cells and cholinergic LDT neurons (Fig. 1C).
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). The responses shown in Fig. 1 are reminiscent of the smooth/spiker responses described previously and suggest that "plateau" responses might be mediated by subthreshold depolarizations, whereas "spiker" responses might be mediated by action potentials. The Ca2+ influx during the subthreshold depolarization might be mediated by activation of voltage-gated Ca2+ channels during the depolarization and/or by Ca2+ influx though any Ca2+-permeable cation channels contributing to the depolarization. Hcrt/Orx depolarizes DR and LDT neurons by activating a noisy inward current that is Na+ dependent
Previous studies have implicated a cation current in mediating the Hcrt/Orx-evoked depolarization of both DR and LDT neurons, although no I-V data have been published for the orexin currents in LDT neurons. To extend these observations and compare the currents under identical recording conditions, we examined the Hcrt/Orx current from the somata of 45 DR neurons and 58 LDT neurons using whole cell voltage-clamp methods. DR neurons recorded with a K+-rich internal solution had an average input resistance of 511 ± 31 M
(range: 265–990 M, n = 33) and LDT neurons recorded with a K+-rich internal solution had an average input resistance of 310 ± 25 M (range: 106–771 M, n = 36). Orexin-A (300 nM; 1.5-min superfusion; holding potential = –60 mV) produced a long-lasting inward current (–35.3 ± 5.9 pA, n = 5, DR and –22.7 ± 2.7 pA, n = 11, LDT; Fig. 2, A and H) that was accompanied by an increase in membrane current noise (Fig. 2, A and H) in neurons recorded with ACSF containing DABST to block action potentials and fast synaptic potentials and 2 mM Cs+ to block Ih. In this sample of neurons, the average membrane noise at –60 mV increased over control by 108.5 ± 15.9% (n = 5) in the DR and 55.6 ± 9.0% (n = 11) in the LDT. These effects were long-lasting and were typically only partly reversed by the end of the recording period (10–20 min). As previously reported, for both DR and LDT neurons, these effects resulted from a direct action on the recorded neurons because neither the inward current nor the membrane current noise increase were attenuated in low Ca2+ ACSF, which was shown previously to block evoked excitatory postsynaptic currents (EPSCs) in the LDT (Burlet et al. 2002). In DR neurons, the orexin-A current (at –60 mV) was larger, on average (–65.0 ± 9.6 pA, n = 7, P < 0.05) in low Ca2+ ACSF, whereas it was not significantly larger in LDT neurons (–27.9 ± 3.7 pA, n = 4, P > 0.05).
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In some neurons, the Hcrt/Orx current seems to be sensitive to KB-R7943, an antagonist of the electrogenic Na+/Ca2+ exchanger (Burdakov et al. 2003; Eriksson et al. 2001; Wu et al. 2002, 2004). Because we previously found that the calcium influx evoked by Hcrt/Orx in both DR and LDT neurons was insensitive to KB-R7943 (Kohlmeier et al. 2004), a role for the electrogenic Na+/Ca2+ exchanger seemed unlikely. Nevertheless, we verified that KB-R7943 did not attenuate the current activated by orexin-A in DR neurons (data not shown). As expected, a low concentration of KB-R7943 (10 µM) had no effect on the inward current evoked by orexin-A (95.8 ± 21.9% of control, n = 2), whereas a higher concentration (80 µM), which is reported to have nonspecific effects (Iwamoto et al. 1996), trended toward only a modest attenuation (76.5 ± 6.9% of control, n = 2). These findings are in concordance with prior findings (Kohlmeier et al. 2004; Liu et al. 2002) and indicate that the Na+/Ca2+ exchanger does not make a significant contribution to the Hcrt/Orx-mediated depolarization and calcium influx in DR and LDT neurons.
Noisy inward current in DR and LDT neurons is a nonselective cation current
Using voltage-clamp ramps from –100 to –30 mV, we examined the current-voltage relationship of the orexin-A–induced current in DR (Fig. 2E) and LDT (Fig. 2J) neurons using a K+-rich pipette solution. These data showed that Hcrt/Orx produced an inward shift in the membrane current and an increase in noise that were both larger at –100 than –30 mV. Subtracting these curves and plotting them against Vm shows the I-V relation of the Hcrt/Orx-evoked current (Iorx), which was approximately linear over this range of voltages in DR (Fig. 2F) and LDT (Fig. 2K) neurons. By plotting the rms current versus Vm (computed every 5 mV), it was also evident that the current noise was largest at negative potentials and decreased as membrane potential moved more positively, as expected for channel noise associated with Iorx. We estimated a slope conductance by fitting a line to the I-V relation between –100 and –40 mV, yielding an average value of 1.65 ± 0.42 nS in DR neurons (n = 6) and 0.50 ± 0.10 nS in LDT neurons (n = 11). These data suggest that the current may reverse near 0 mV and are consistent with Hcrt/Orx activating a nonselective cation current in both DR and LDT neurons.
To further test this idea, we made recordings using a cesium-based internal solution and low calcium ACSF containing DABST to improve the space clamp by blocking K+ currents and to limit current from voltage-gated Ca2+ currents, which produced Ca2+ spikes otherwise. Under these conditions, orexin-A also induced an inward current at –60 mV in both the DR (–74.0 ± 22.6 pA, n = 6) and LDT (–21.5 ± 5.0 pA, n = 8) that was accompanied by increased membrane current noise (DR: 223.13 ± 51.71%; LDT: 62.0 ± 20.7%).
This current was studied with slow voltage ramps between –100 and +10 mV, and its I-V relation was constructed from the difference between ramp currents measured at the peak of the Hcrt/Orx effect and from before Hcrt/Orx application. The resulting I-V curves were approximately linear below –40 mV, as observed with the K+-rich internal solution, with a slope conductance of 0.97 ± 0.25 nS for DR (Fig. 2G) and 0.25 ± 0.09 nS for LDT (Fig. 2L) neurons. These I-V curves flattened at more positive potentials and reversed near 0 mV for both DR (–10.2 ± 1.8 mV, n = 4) and LDT (–11.4 ± 2.5mV, n = 8) neurons. Moreover, the membrane current noise behaved similarly to that observed with the K+-rich intracellular solution, was largest at –100 mV, and decreased as the reversal potential was approached. These data suggest that channels mediating Iorx have similar permeabilities to Cs+ and K+. To better evaluate this point, we recorded from four DR neurons with the K+-rich internal solution and measured Iorx using voltage ramps to +10 mV. Under these conditions, the measured reversal potential was –3.6 ± 3.4 mV (n = 4), which was not different from that measured using the Cs+-rich internal solution (P > 0.05), consistent with equal Cs+ and K+ permeabilities and indicating that Iorx is mediated by nonselective cation channels.
In addition to Hcrt/Orx activating cation channels in LDT neurons (n = 11/16 responders recorded with the K+ internal solution, 69%), a subpopulation had orexin currents with I-V curves that were flat or inwardly sloping between –100 and –40 mV in recordings with a K+-rich internal solution and ACSF containing DABST (n = 5/16 responders, 31%). For these cells, the peak current evoked by 300 nM orexin-A at –60 mV was –10.0 ± 2.3pA (n = 5), and the membrane current noise only increased by 9.7 ± 8.4%. The Hcrt/Orx-evoked current and membrane noise in these cells were significantly smaller than those observed in LDT neurons with cationic currents (P < 0.01).
Collectively, these data indicate that Hcrt/Orx-induced depolarizations are mediated by the activation of a noisy NSCC in DR neurons and a majority of LDT neurons. A minority of LDT neurons responded to Hcrt/Orx with smaller inward currents that, based on the I–V curves and current noise, were mediated by one or more additional mechanisms that we did not further study.
Hcrt/Orx-evoked elevation of [Ca2+]i requires depolarization and is not mediated by Ca2+ influx through Hcrt/Orx activated nonspecific cation channels
Given the potential space-clamp issues associated with clamping neurons with extended processes and the fact that Ca2+-spiking could occur at positive membrane potentials during voltage ramps in normal extracellular calcium, we did not attempt to estimate the Ca2+ permeability of the NSCCs by measuring the reversal potential shifts produced by changing extracellular Ca2+ concentration. Instead, we measured somatic intracellular Ca2+ transients while manipulating membrane potential. We first examined the Ca2+ transients produced by orexin-A (300 nM) depolarizations in neurons recorded in current clamp following blockade of Na+ spikes and ionotropic synaptic receptors. Under these conditions, the average change in dF/F was 5.1 ± 1.4% in the DR (n = 8) and 4.5 ± 1.8% in the LDT (n = 5). Interestingly, Hcrt/Orx sometimes induced small amplitude spikes in both DR (n = 3) and LDT (n = 4) neurons that were similar to the calcium spikes evoked by somatic current injection in the presence of TTX (Fig. 3, A and inset). However, changes in dF/F were also evident when these presumed Ca2+ spikes were not elicited (DR: n = 5; LDT: n = 1, Fig. 3B). Conversely, in some neurons, Hcrt/Orx elicited changes in membrane potential without corresponding changes in dF/F (Fig. 3C). Indeed, we found that the magnitude of the elicited Ca2+ transient was correlated with the membrane potential before orexin-A application. On average, orexin-A produced significantly smaller Ca2+ transients in neurons with membrane potentials negative to –46 mV (n = 16) than in neurons with membrane potentials positive to –45 mV (n = 22; Fig. 3, histogram). These data suggest that the Hcrt/Orx depolarization, per se, might be responsible for triggering the majority of the Ca2+ influx.
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; Fig. 3D1, left), but when the same current was injected from a membrane potential of –60 mV, little change in dF/F was apparent (LDT; Fig. 3D1, right). Similarly, when the amplitude of the simulated current was increased to produce a greater depolarization, the change in dF/F also increased (Fig. 3D2). Thus simulated Hcrt/Orx depolarizations mimic the Ca2+ transients produced by authentic Hcrt/Orx. These data suggest that depolarization is necessary for Hcrt/Orx to evoke a Ca2+ transient in these neurons. To directly test this possibility, we applied orexin-A (300 nM) and monitored dF/F while voltage clamping the soma to –60 mV. Under these conditions, Hcrt/Orx produced typical inward currents but without detectable changes in dF/F. In our sample of DR neurons, orexin-A (300 nM) produced an inward current of 12.3 ± 2.2 pA (n = 6/6 cells tested) but only an average change in dF/F of 0.75 ± 0.28% (n = 6/6; Fig. 4A, left). In the LDT, orexin-A (300 nM) produced an inward current of 15.4 ± 0.63 pA (n = 6/6 cells tested) but only an average change in dF/F of 0.4 ± 0.33% (Fig, 4A, right). These changes in dF/F were not significantly different from baseline (P > 0.05). Thus depolarization is necessary for Hcrt/Orx to stimulate Ca2+ transients in DR and LDT neurons.
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Hcrt/Orx enhances Ca2+ transients mediated by L-type calcium channels
Presumably, this depolarization is necessary to trigger voltage-operated Ca2+ channels, like the L-type channels that we have already shown contribute to the Hcrt/Orx-mediated Ca2+ transients in DR and LDT neurons (Kohlmeier et al. 2004). However, is depolarization alone sufficient to account for the Hcrt/Orx-mediated Ca2+ transients or does Hcrt/Orx also enhance the Ca2+ transients evoked by a given depolarization? To address this question, we monitored the changes in dF/F resulting from voltage clamp steps from —60 to –30 mV in DR and LDT neurons before and after orexin-A (300 nM) application. We found that the Ca2+ transients produced by these voltage jumps were significantly enhanced by 30% following Hcrt/Orx application (Fig. 5A). Interestingly, on average, this enhancement was the same regardless of whether orexin-A also activated a slow inward current in the recorded neuron (P > 0.05). In DR neurons, the Ca2+ transient was enhanced by 34.4 ± 3.1% in cells with an inward Hcrt/Orx current (n = 3/14 cells tested) and by 26.0 ± 4.84% in cells without inward current (n = 6/14 tested). In LDT neurons, the Ca2+ transient was enhanced by 33.3 ± 12.4% in cells with inward current (n = 4/13 tested) and by 30.0 ± 5.7% in cells without inward current (n = 5/13 tested). In five DR cells and four LDT neurons, step-evoked calcium transients were not enhanced regardless of whether orexin-A elicited an inward current (inward current evoked in 3/5 DR cells and 1/4 LDT cells). These data indicate that, in a large fraction of DR and LDT neurons, Hcrt/Orx enhances Ca2+ transients independently of the inward current that produces direct excitation. Although this enhancement is most likely caused by increased Ca2+ influx through voltage-gated Ca2+ channels, we saw only small and variable changes in the total whole cell current, which was monitored simultaneously during the voltage steps (Fig. 5A, middle trace). This probably reflects the fact that the Ca2+ current makes up only a small fraction of the total current that is dominated by outward currents, some of which are also Ca2+ dependent. Indeed, on average, this current was 11.1 ± 1.1 pA more outward (P < 0.05; n = 8) at –30 mV for the same sweeps in which the Ca2+ transients were enhanced by 30%.
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To further test the role of L-channels, we examined the effect of Bay-K, an L-type Ca2+ channel agonist. Application of Bay-K (10 µm) alone significantly enhanced the step-evoked Ca2+ transient by 87.3 ± 40.3% in the DR (n = 5) and by 126.9 ± 46.0% in the LDT (n = 5). Application of orexin-A (300 nM) in the presence of Bay-K produced a further enhancement of the step-evoked Ca2+ transient in both nuclei (DR: 159.4 ± 42.6%, n = 5; LDT: 303.5 ± 96%, n = 5; compared with the baseline transient before Bay-K and orexin-A), suggesting that the Hcrt/Orx enhancement was augmented by potentiating L-channel activity with Bay-K.
Indeed, the enhancement in the step-evoked Ca2+ transient produced by Hcrt/Orx in the presence of Bay-K was significantly greater than that produced in the control population without Bay-K (Fig. 5E). In the DR, orexin-A application increased the step-evoked Ca2+ transient measured in Bay-K by 45.7 ± 10.8% (n = 5) compared with a 28.6 ± 3.5% increase measured in the absence of Bay-K (n = 9; P < 0.05). In the LDT, a similar relation was seen, with orexin-A increasing the step-evoked Ca2+ transient measured in Bay-K by 71.7 ± 21.5% (n = 5) compared with the 31.6 ± 5.9% increase in the absence of Bay-K (n = 9; P < 0.05; Fig. 5E). This is the outcome expected if Hcrt/Orx is specifically enhancing influx through L-type Ca2+ channels, because a larger fraction of the total Ca2+ transient is mediated by L-channels following Bay-K action. Taken together, these data strongly indicate that Hcrt/Orx enhances the depolarization-evoked Ca2+ transients mediated by L-type Ca2+ channels in DR and LDT neurons.
PKC mediates the Hcrt/Orx enhancement of Ca2+ transients
Previously, we found that the PKC inhibitor Bis I attenuated, but did not abolish, the orexin-induced calcium transients in Fura AM–loaded DR and LDT neurons. Because our findings here indicate that these Ca2+ transients are produced by both depolarization and enhanced L-type Ca2+ channel transients, we determined whether Bis I antagonized one or both of these actions. To do so, we examined the effect of applying Hcrt/Orx on voltage step-evoked Ca2+ transients following pretreatment of the slices with Bis I (1 µM). We found that Bis I application alone sometimes resulted in a slight, slow outward drift in the holding current at –60 mV and a progressive increase in the outward current produced by the voltage step (–60 to –30 mV). Despite these subtle changes in whole cell current, Bis I did not have an effect on the Ca2+ transients evoked by voltage steps. The average dF/F produced by these steps in the DR (6.7 ± 0.8%, n = 7) and LDT (5.5 ± 9.2%, n = 11) after Bis I application was not different from the values before Bis I application (DR: 6.4 ± 1.9%; LDT: 5.8 ± 0 0.9%; P > 0.05) for the same cells. Moreover, in the presence of Bis I, Hcrt/Orx failed to increase the step-evoked Ca2+ transients above control levels (DR: 5.7 ± 07%, n = 7/7; LDT: 5.6 ± 0.9%, n = 11/11; P > 0.05, Fig. 6A). Because Hcrt/Orx enhanced these Ca2+ transients in 70% of recorded cells in the absence of Bis I, these data indicate that activation of PKC is required for the enhancement of L-type Ca2+ channel function by Hcrt/Orx in DR and LDT neurons.
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) and DR and LDT Ca2+ transients (Kohlmeier et al. 2004). Hcrt/Orx enhances the Ca2+ transients in identified serotonergic DR neurons and cholinergic LDT neurons
To determine whether Hcrt/Orx enhances the depolarization-evoked Ca2+ transients in the serotonergic and cholinergic neurons of these nuclei, we immunolabeled for TpH or bNOS in slices containing Alexa-594–positive neurons. In a small sample of recovered neurons, we verified that Hcrt/Orx enhanced voltage step–evoked Ca2+ transients in both serotonergic DR (n = 3) and cholinergic LDT neurons (n = 4). This also occurred in cells that were not TpH+ (n = 2) or bNOS+ (n = 3). We confirmed that the Hcrt/Orx-mediated enhancement was augmented after Bay-K application in both serotonergic (n = 3) and cholinergic neurons (n = 2). Thus Hcrt/Orx enhances Ca2+ transients from L-channels in both DR and LDT principal neurons.
Transmitter phenotype and the temporal pattern of calcium transients evoked by Hcrt/Orx
Using this double-labeling approach, we were also able to determine whether the temporal pattern of Ca2+ transients evoked by Hcrt/Orx was associated with neurons having a particular transmitter phenotype—a point that was not resolvable in our fura-2AM study (Kohlmeier et al. 2004). In this study, we found that all three temporal profiles of Ca2+ signals were elicited in both TpH+ (n = 11) and TpH–(n = 9) DR neurons and both bNOS+ (n = 15) and bNOS–(n = 8) LDT neurons. What did co-vary with the calcium response pattern was whether Hcrt/Orx elicited Ca2+ spikes. This was apparent in DABST-containing ACSF, where both "smooth/spiker" and "spiker" responses were observed when Hcrt/Orx induced calcium spikes (Fig. 2A). In contrast, "Plateau" responses were observed when Hcrt/Orx produced a depolarization without Ca2+ spikes (Fig. 2B). We also found that the propensity to fire Ca2+ spikes in response to depolarization was not strictly related to neurons of a particular transmitter phenotype. In DABST-containing ACSF, Ca2+ spikes were elicited by DC injection in the majority of DR and LDT neurons tested (TpH+ DR, n = 10/13; TpH–DR n = 2/4; bNOS+ LDT, n = 10/14, bNOS–LDT, n = 2/4). Taken together, neither the ability to generate Ca2+ spikes nor the calcium response profile elicited by Hcrt/Orx seemed to correlate with neurotransmitter phenotype in the DR or LDT.
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DISCUSSION |
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). In addition to clarifying the mechanisms underlying Hcrt/Orx-evoked Ca2+ transients in key neurons of the reticular activating system, our findings underscore the role of Hcrt/Orx peptides as neuromodulators capable of enhancing an important Ca2+-signaling pathway mediated by L-type Ca2+ channels. Mechanisms mediating Hcrt/Orx-induced depolarization in DR and LDT neurons
The Hcrt/Orx-mediated depolarization in rat DR neurons was initially ascribed to blockade of a leak potassium current (Brown et al. 2001), but in a later publication (Brown et al. 2002), the same group suggested a more important involvement of a NSCC. The findings of Liu et al. (2002) in rat, our preliminary findings (Leonard et al. 2005; Tyler et al. 2001), and our findings here, in mouse, support this view. Our data also confirmed that this current substantially increases membrane noise, is very attenuated at –60 mV in a low-Na+ extracellular solution, and is insensitive to low concentrations of an antagonist of the electrogenic Na+/Ca2+ exchanger (Brown et al. 2002; Liu et al. 2002). Our new data show that the evoked membrane current noise co-varies with current magnitude between –100 and +10 mV as expected, if it results from channels opened by Hcrt/Orx. We also found that the current is not blocked by extracellular Cs+ at concentrations that block H-current and some K+ channels. Moreover, the Hcrt/Orx current was similar in neurons recorded with a Cs+-rich intracellular patch solution and reversed near 0 mV with both internal solutions.
In LDT neurons, the current activated by Hcrt/Orx was very similar. For example, membrane noise co-varied with current magnitude between –100 and +10 mV, the current was very attenuated at –60 mV in low Na+ ACSF, it was insensitive to extracellular Cs+, it reversed near 0 mV, and it was of a similar magnitude in neurons recorded with intracellular K+ or Cs+. Collectively, these data suggest that Hcrt/Orx activates cation channels that are permeable to Na+, K+, and Cs+, consistent with an NSCC in LDT and DR neurons. It is noteworthy that, on average, the Hcrt/Orx slope conductance measured with the K+ internal solution was 3 times larger in DR neurons than in LDT neurons. This may indicate that DR neurons are more powerfully recruited to fire than are LDT neurons during periods of high Hcrt/Orx release.
It is also of interest to note that, in DR and LDT neurons recorded with the Cs+-based internal solution and low extracellular Ca2+, the I-V curve of the Hcrt/Orx current showed a decrease in slope conductance positive to –40 mV and outward rectification again as the membrane potential passed through the reversal potential. The tendency for the I-V to flatten at potentials positive to –40 mV was also observed in neurons recorded with a K+-rich intracellular solution and suggests that the Hcrt/Orx-activated cation current is slightly voltage dependent in DR and LDT neurons. This similarity further suggests that Hcrt/Orx activates a similar class of cation channels in both types of neurons. Several issues concerning this current remain unresolved. For example, it is not clear what makes this current so noisy and we did not determine the relative permeabilites to Na+ and K+ because we could only make slow changes in extracellular Na+ and did not use voltage ramps over a sufficiently large range of potentials to delineate the I-V and reversal potential shifts. These points should be studied in future experiments using isolated neurons, where rapid solution changes and better space clamp can be achieved.
Hcrt/Orx-activated cation currents have also been reported in area postrema neurons (Yang and Ferguson 2002), nucleus tractus solitarius (Yang and Ferguson 2003), paraventricular hypothalamic neurons (Yang and Ferguson 2002), and locus coruleus (LC) neurons (Murai and Akaike 2005). However, the current reported in those neurons seems different in several respects. First, there was no evidence of increased membrane current noise at negative membrane potentials in the published accounts, and second, the I–V curves where essentially linear and reversed without rectification near –40 mV. Cs+ permeability was not tested, but based on the lack of current noise and the nearly linear I-V relation, those currents are likely mediated by different NSCC channels. Thus it is likely that Hcrt/Orx activates more than one type of NSCC in the CNS.
Mechanisms mediating Hcrt/Orx-induced calcium transients
Based on the findings that most NSCCs carry calcium ions and the results of our previous study, which showed that Hcrt/Orx stimulates a somatic Ca2+ influx in neurons of the DR and LDT, we postulated that the NSCC activated by Hcrt/Orx contributed to the Hcrt/Orx-stimulated Ca2+ influx in these neurons. Contrary to this idea, however, concurrent monitoring of membrane potential/holding current and [Ca2+]i showed that the NSCC activated by Hcrt/Orx was not a measurable source for these transients at –60 mV or even after increasing the Ca2+ driving force by stepping the membrane to –100 mV. These data, along with the observations in unclamped cells that a simulated Hcrt/Orx depolarization mimics the Ca2+ transients evoked by Hcrt/Orx, indicate that the activation of voltage-operated Ca2+-permeable channels is necessary. It is worth noting that the NSCC activated by orexin during our imaging experiments appeared somewhat smaller than we previously observed under standard whole cell recording conditions. Initially, we thought this might result from some rundown of the NSCC because of the longer waiting period before Hcrt/Orx application that was required for dye-filling in the imaging experiments. Indeed, under standard recording conditions, most of our Hcrt/Orx applications started by 11 min after breakthrough. Because we also had numerous recordings where orexin was applied between 20 and 35 min after breakthrough, which is comparable to the times used in the imaging experiments, we compared the average magnitude of orexin current evoked by early (n = 22) and late applications (n = 6). We found they were not significantly different (P > 0.2), suggesting that delayed application of Hcrt/Orx is unlikely to importantly alter the nature of the NSCC current. Another possibility is that the signaling pathways required to activate the NSCC may be somewhat reduced because of increased intracellular Ca2+ buffering expected from bis-Fura2. Although this might account for somewhat smaller currents, this too is unlikely to have altered the channels underlying this current, because a noisy inward current was still clearly present in our imaging experiments. Thus our combined whole cell and imaging data argue strongly against involvement of the Hcrt/Orx-activated NSCC in mediating the Hcrt/Orx-evoked Ca2+ transients in DR and LDT neurons.
These findings may also provide some insight into the molecular identity of the channels underlying this NSCC in DR and LDT. Transient receptor potential canonical (TRPC) channel subunits (TRPC I-VIIs) form classic, nonselective cation channels (for review, see Ramsey et al. 2006) and are expressed throughout the nervous system and in serotonergic DR neurons, with TRPC5 and TRPC6 mRNAs occurring most frequently (Sergeeva et al. 2003). TRPC I-VII mRNAs also appear present within the LDT (Allen Brain Map, www.brain-map.org). TRPCs mediate cation currents activated by Gq-coupled receptors and have recently been shown to mediate a Ca2+ influx activated by Hcrt/Orx in mammalian expression systems. Orexin receptors expressed in CHO cells elevate [Ca2+]i via activation of both a store-independent Ca2+ influx and a store-operated Ca2+ influx triggered by Ca2+ release from thapsigargin-sensitive intracellular stores (Kukkonen and Akerman 2001; Lund et al. 2000). Using a dominant negative approach, TRPC1 and TRPC3 channels were elegantly shown to be involved in the store-independent Ca2+ influx (Larsson et al. 2005). Hcrt/Orx also activates a calcium influx pathway in excitable neuroblastoma cells with involvement of TRPC6 channels (Nasman et al. 2006). It therefore seems reasonable to speculate that native orexin receptors activate TRPCs in DR and LDT neurons because these channels can be operated by orexin receptors and are expressed in DR and LDT neurons. On the other hand, following Hcrt/Orx activation, a Ca2+ influx was undetectable at subthreshold membrane potentials in these neurons. Thus despite the relatively higher abundance of TRPC5 and 6 mRNA in DR, perhaps Hcrt/Orx activates channels formed by TRPC1-4 subunits because they have a lower Ca2+ selectivity (PCa/PNa = 1–3) than do channels formed from TRPC5 and 6 subunits (Pca/PNa = 5–9; see Table 1 of Owsianik et al. 2006). Of course, it is also possible that the Ca2+ influx mediated by these channels is too low, too restricted, or too remote from our measurement sites to be detected in our preparation. Unfortunately, there are currently no pharmacological agents that can distinguish between TRPC channels, so their particular roles in mediating Hcrt/Orx actions in DR and LDT neurons remains a topic for future study.
Hcrt/Orx likely modulates L-type Ca2+ channels in DR and LDT neurons
Our findings indicated that Hcrt/Orx produces a membrane depolarization that activates voltage-operated Ca2+ channels and produces Ca2+ spiking in some DR and LDT neurons. Evidence from Ca2+ imaging studies in dissociated hypothalamic and spinal neurons (van den Pol 1999; van den Pol et al. 1998) and VTA neurons (Uramura et al. 2001) found that Hcrt/Orx-stimulated Ca2+ transients were sensitive to L- or L- and N- type channel antagonists. Because patch-clamp studies of hypothalamic neurons failed to show an Hcrt/Orx-mediated depolarization, a direct modulatory role of Hcrt/Orx on L-type channels was proposed. However, in the VTA, Hcrt/Orx strongly depolarizes dopamine and GABA neurons (Korotkova et al. 2003), so activation of Ca2+ channels might be secondary to this depolarization. Similarly, because Hcrt/Orx depolarizes DR and LDT neurons, it was unclear from our previous fura-AM measurements whether L-type Ca2+ channel involvement was merely a result of activation by depolarization, although indirect evidence suggested more than a depolarization was involved (Kohlmeier et al. 2004). To test whether depolarization was sufficient, in this study, we examined the effect of orexin-A on a voltage-clamp step-evoked Ca2+ transient. This showed that Hcrt/Orx significantly enhances these Ca2+ transients. The enhancement was prevented by pretreating with nifedipine and was augmented by pretreatment with Bay-K, indicating the enhancement was largely, if not exclusively, mediated by enhancement of L-channel Ca2+ transients in both DR and LDT neurons. Because orexin-A enhanced these Ca2+ transients both in cells with and without an orexin-A–evoked inward current, these data indicate Hcrt/Orx can independently enhance the Ca2+ transients generated by L-type Ca2+ channels in these cells. The precise mechanism by which this happens has not been resolved, but our data are consistent with Hcrt/Orx increasing the L-type calcium current, which is present in both DR (Penington et al. 1991) and LDT neurons (Kohlmeier and Leonard 2006). Indeed, Hcrt/Orx has been shown to increase L-current in cultured ovine somatotropes (Xu et al. 2002), where is does so without altering voltage dependence and depends on PKC signaling. PKC inhibition also attenuates Hcrt/Orx-mediated Ca2+ transients in hypothalamic neurons (van den Pol et al. 1998) and VTA neurons (Uramura et al. 2001). Previously, we found PKC inhibition partly blocked these transients in DR and LDT neurons (Kohlmeier et al. 2004). In this study, we extended this observation and showed that the orexin-A–mediated enhancement of the voltage step–evoked Ca2+ transient was completely abolished by pretreatment with Bis I, a widely used inhibitor of PKC. Presumably, the partial block we observed previously results from residual depolarization and the activation of non–L-type Ca2+ channels because some DR and LDT neurons showed a PKC inhibitor–resistant inward current. Thus our data are consistent with a PKC-dependent enhancement of the L-type Ca2+ current in DR and LDT neurons, although the particular pore-forming subunit involved and the nature of the modulation by Hcrt/Orx remains to be determined. It is noteworthy, in this regard, that although the Ca2+ transients produced by three to five spikes in LDT neurons are partly sensitive to L-channel antagonists, these spike-evoked transients were not enhanced by orexin-A (Kohlmeier and Leonard 2006). This implies that the Hcrt/Orx enhancement of L-type Ca2+ transients might require particular stimulus conditions like prolonged depolarization to be manifest.
Ca2+ response profiles and transmitter phenotype of DR and LDT neurons
In our previous study of fura-AM–loaded neurons of the DR and LDT, we found that Hcrt/Orx elicited three types of calcium response profiles: plateau responses that exhibited a slow rise time, a stable plateau, and were slow to recover; spiker responses, which showed rapid, transient increases of calcium from a stable baseline; and smooth/spikers, which were a combination of these other two responses (Kohlmeier et al. 2004). Because of the recording method, it was not possible to determine whether these response profiles co-varied with transmitter phenotype. In this study, transmitter phenotype was identified for recorded cells, and we found no particular relation between Ca2+ response profile and transmitter phenotype. All three response types were observed in transmitter identified cells of the DR and LDT and in other cell types. Rather, the response profile depended on the magnitude of the Hcrt/Orx depolarization and the ability of the recorded neuron to fire action potentials or Ca2+ spikes. Plateau responses were generated by an Hcrt/Orx-mediated depolarization and activation of voltage-gated Ca2+ channels (VGCCs) in neurons with insufficient current density to generate action potentials or Ca2+ spikes. Spiker responses were generated when the Hcrt/Orx-mediated depolarization triggered action potentials or Ca2+ spikes but was insufficient to maintain steady activation of voltage-gated Ca2+ channels. Smooth/spikers were generated by an Hcrt/Orx-mediated depolarization sufficient to activate VGCCs and to generate action potentials or Ca2+ spikes (in TTX). These latter responses were readily observed in current-clamped cells following orexin-A application (Fig. 1). In our previous study, we found that nifedipine reduced, but did not abolish, the Hcrt/Orx-evoked Ca2+ transients. Presumably, this reflects the ability of the Hcrt/Orx mediated depolarization to also activate other types of VGCCs. The effect of Hcrt/Orx to enhance the L-type Ca2+ channel-mediated Ca2+ transient would be expected to facilitate all of these response types. It should also be noted that in slices loaded with fura-AM, it was not possible to determine the resting membrane potential of the imaged neurons, and some cells might have been quite depolarized. Indeed, such neurons might be induced to fire Ca2+ spikes following enhancement of L-type Ca2+ channels by Hcrt/Orx even in the absence of an Hcrt/Orx-mediated depolarization. This is supported by our prior observations that Bay-K alone sometimes induced "spiker" responses and that "spiker" responses were more likely to be abolished by nifedipine than other types of responses (Kohlmeier et al. 2004). Thus the range of Ca2+ responses previously observed by Ca2+ imaging alone in the DR and LDT can be explained by the dual actions of Hcrt/Orx elucidated here.
Functional considerations
Although the Hcrt/Orx peptides influences several central systems, disruption of Hcrt/Orx signaling results in a striking sleep phenotype that includes increased numbers of shorter duration bouts of waking, non-REM, and REM sleep and the appearance of both abrupt- and slow-onset motor arrests similar, respectively, to the cataplexy and sleep attacks of human narcolepsy (Beuckmann et al. 2004; Chemelli et al. 1999; Gerashchenko et al. 2001; Hara et al. 2001; Lin et al. 1999; Mochizuki et al. 2004; Willie et al. 2003). Indeed, reduced Hcrt/Orx levels resulting from the loss of Hcrt/Orx-synthesizing neurons seems to cause human narcolepsy with cataplexy (Nishino et al. 2000; Peyron et al. 2000; Thannickal et al. 2000). How Hcrt/Orx signaling normally promotes arousal, stabilizes behavioral states and limits epochs of motor atonia to REM sleep is not clear but may involve the ability of Hcrt/Orx to influence elements of the reticular activating system, including the DR and LDT, which functions in the initiation and stabilization of waking and REM states (Pace-Schott and Hobson 2002; Saper et al. 2001). Indeed, intracerebroventricular injection of orexin-A (Espana et al. 2001; Hagan et al. 1999; Piper et al. 2000) or microinjection of orexin-A into the LDT (Xi et al. 2001) or the locus coeruleus (Bourgin et al. 2000), another component of the reticular activating system, suppresses REM sleep and promotes waking.
Results from our study have several implications for considering how the DR and LDT might influence these arousal-related functions of Hcrt/Orx. First, our data showed that serotonergic and cholinergic neurons of the DR and LDT are targets for both direct excitation and enhancement of L-type Ca2+ channel function by Hcrt/Orx. Thus the release of Hcrt/Orx during active waking would be expected to excite both types of neurons, thereby promoting waking and suppression of REM sleep. Second, our direct comparison of DR and LDT neurons indicates that Hcrt/Orx produces at least a two-fold larger inward current in DR neurons. Because LDT cholinergic neurons receive input from serotonergic DR neurons (Honda and Semba 1994; Semba and Fibiger 1992; Vertes and Kocsis 1994) and are strongly inhibited by serotonin (Leonard and Llinás 1994; Leonard et al. 2000; Luebke et al. 1992), it suggests that cholinergic neurons will receive competing direct Hcrt/Orx excitation and indirect serotonergic inhibition. This inhibition might dominate the activity of some LDT neurons, including the REM-on neurons, whose activity is strongly inhibited by 5-HT1A receptor activation (Thakkar et al. 1998), a situation that would be expected to promote narcolepsy signs in the absence of Hcrt/Orx drive.
Finally, our study underscores the idea that Hcrt/Orx peptides function as neuromodulators in addition to having a role in direct excitation, because Hcrt/Orx-mediated enhancement of L-type Ca2+ channel transients would only be manifest when these channels are activated during epochs of high activity. The consequences of this modulation remain to be determined but are expected to influence both the short-term excitability of these neurons through the control of Ca2+-dependent channel activity and the longer-term regulation of the homeostatic or trophic properties of these neurons, because elevation of [Ca2+]i mediated by L-type Ca2+ channels has been consistently linked with activity-dependent transcriptional signaling pathways involved with plasticity and development (Dolmetsch et al. 2001; West et al. 2001). Changes in these latter processes could provide a powerful means to adjust the neuronal circuits coordinating expression of sleep-wake–related physiological components during development and throughout life. Impairment of these processes resulting from the loss of orexin signaling might therefore also be expected to factor into the expression of narcolepsy symptoms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. S. Leonard, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: chris_leonard{at}nymc.edu)
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