Dual orexin actions on dorsal raphe and laterodorsal tegmentum neurons: noisy cation current activation and selective enhancement of Ca2+ transients mediated by L-type calcium channels - PubMed (original) (raw)

Dual orexin actions on dorsal raphe and laterodorsal tegmentum neurons: noisy cation current activation and selective enhancement of Ca2+ transients mediated by L-type calcium channels

K A Kohlmeier et al. J Neurophysiol. 2008 Oct.

Abstract

The hypocretin/orexins (Hcrt/Orxs) are hypothalamic neuropeptides that regulate stress, addiction, feeding, and arousal behaviors. They depolarize many types of central neurons and can increase [Ca2+]i in some, including those of the dorsal raphe (DR) and laterodorsal tegmental (LDT) nuclei-two structures likely to contribute to the behavioral actions of Hcrt/Orx. In this study, we used simultaneous whole cell and Ca2+-imaging methods in mouse brain slices to compare the Hcrt/Orx-activated current in DR and LDT neurons and to determine whether it contributes to the Ca2+ influx evoked by Hcrt/Orx. We found Hcrt/Orx activates a similar noisy cation current that reversed near 0 mV in both cell types. Contrary to our expectation, this current did not contribute to the somatic Ca2+ influx evoked by Hcrt/Orx. In contrast, Hcrt/Orx enhanced the Ca2+ transients produced by voltage steps (-60 to -30 mV) by approximately 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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Figures

FIG. 1.

FIG. 1.

Hypocretin/orexin (Hcrt/Orx) depolarizes dorsal raphe (DR) and laterodorsal tegmental (LDT) neurons causing action potentials and rises in somatic and proximal dendritic [Ca2+]i. A and B: (top right) images of bis-fura-2 fluorescence in a DR (A) and LDT (B) neuron during whole cell recordings from which changes in fluorescence and membrane potential were measured (left column). Boxes indicate regions of interest (ROIs) from which dF/F signals were computed. Scale equals 10 μm. In left panels, bottom trace indicates membrane potential, middle trace indicates somatic dF/F, and top trace indicates dendritic dF/F. Orexin-A (300 nM) induced a depolarization, firing, and rises in [Ca2+]i in these DR and LDT neurons. Boxed insets are high-gain traces showing that the Ca2+ signals preceded the induction of spiking (arrows). Scales in insets equal 10% dF/F, 10 mV, and 20 s. C: immunocytochemistry verified that these actions occurred in serotonergic and cholinergic neurons of the DR (C1) and LDT (C2). Left image was taken at low power (×4 objective) and shows TpH-positive cells in the Raphe (C1) and bNOS-positive cells in the LDT (C2) labeled with FITC. Top image in right columns show a higher-power FITC image (×20 objective) of the field containing the recorded neuron. Bottom image in right columns shows the recorded neuron (arrow) in the DR (C1) and the LDT (C2) visualized with alexa-594, which was included in the pipette solution. These neurons showed an orexin-A (300 nM)–mediated depolarization and change in [Ca2+]I and were immunopositive for TpH (C1) and hence serotonergic and bNOS (C2) and hence cholinergic. Scale equals 100 (×4) and 20 μm (×20). Image brightness and contrast were adjusted uniformly for each image to facilitate viewing fluorescent labels. Aq, aqueduct; MnR, median raphe; DTg, dorsal tegmental nucleus.

FIG. 2.

FIG. 2.

Orexin activates an Na+-dependent cation current that is similar in DR (A–G) and LDT neurons (H–L). A: bath application of orexin-A (300 nM) produces a long-lasting inward current (bottom trace) and increase in current noise (top trace) in DR neurons (holding potential = –60 mV). B: following activation of this current by orexin-A, superfusion with low-Na+ artificial cerebrospinal fluid (ACSF) rapidly attenuated the current and the noise increase, consistent with a large reduction in concentration of a permeant ion. Return to ACSF containing normal Na+ reinstated a residual noisy inward current. C: application of low Na+ ACSF also attenuated the baseline holding current and noise (−60 mV), showing that the basal inward current at −60 mV is also Na+ dependent. D: application of orexin-A (300 nM) under low Na+ conditions produced only a small inward current in DR neurons. E: Voltage ramps between –100 and –30 mV obtained before and after orexin application showed an inward current that was larger and noisier at –100 than at –30 mV. F: I-V relation of the orexin-evoked current (bottom) appeared quite linear over this range as indicated by the best fit line. The noise also decreased linearly between –100 and –30 mV (top). G: the orexin current appeared similar in recordings obtained with Cs+-rich intracellular solutions and reversed near 0 mV, suggesting the orexin-activated channels were permeable to both Cs+ and K+. H: in LDT neurons, bath application of orexin-A (300 nM) also produced a long-lasting inward current (bottom trace) and an increase in membrane current noise (top trace). I: both were rapidly attenuated by low-Na+ ACSF. J: voltage ramps (–100 to –30 mV) often showed a noisy current similar to that seen in DR neurons. K: in these LDT neurons, the I-V relation of the orexin-evoked current was linear over this range as indicated by the best-fit line (bottom). The membrane current noise also decreased between –100 and –30 mV (top). L: this orexin current appeared similar in LDT neurons recorded with Cs+-rich intracellular solutions and reversed near 0 mV, suggesting the underlying channels are permeable to Cs+ and K+ as in DR neurons. For experiments A–F and H–K, the internal solution contained K+ gluconate and the ACSF contained DABST and 2 mM Cs+. For experiments G and L, the internal solution contained Cs+ gluconate and the ACSF contained low Ca2+, DABST, and 2 mM Cs+.

FIG. 3.

FIG. 3.

Orexin-A causes rises in [Ca2+]i and Ca2+ spiking in some DR and LDT neurons, if they are sufficiently depolarized. A: following application of TTX and the blockade of ionotropic glutamate, GABA, and glycine receptors, orexin-A often elicited Ca2+ spikes in DR (left traces) and LDT (middle traces) neurons. Right traces show the Ca2+ spikes obtained by depolarization in the DABST-containing ACSF and the spikes obtained in normal ACSF. Note the difference in threshold and amplitude. B: changes in [Ca2+]i were also elicited by orexin-A in some cells in which Ca2+ spikes were not induced. C: in some cells, the orexin-A–induced depolarization was not accompanied by changes in dF/F. Note: the small change in dF/F during the application is artifact, often seen with the “Y-tube” method of drug delivery, and was easily distinguished from specific peptide effects. The bar graph shows the maximal percent change in dF/F produced by orexin-A (300 nM) in cells that were held close to −45 mV and those that were held close to −55 mV. The holding potential influenced the change in [Ca2+]i induced by the Hcrt/Orx depolarization. D: depolarization elicited by a simulated Hcrt/Orx current induced rises in [Ca2+]i. The current command was derived from a previously recorded inward current induced by orexin-A. This current command produced a slow depolarization that was sufficient to induce an increase in dF/F in the absence of Hcrt/Orx in this DR cell at a baseline potential of –43 mV (D1, left). The same current command elicited a negligible change in dF/F when injected from a baseline potential of –60 mV in the same cell (D1, right). Similarly, at a given baseline potential, increasing the injected current, increased the resulting dF/F (D2; LDT neuron).

FIG. 4.

FIG. 4.

The nonselective cation channel (NSCC) activated by Hcrt/Orx is not a Ca2+ influx pathway that contributes to the Ca2+ transients elicited by Hcrt/Orx. A: voltage-clamp recordings (holding = −60 mV) and corresponding changes in dF/F following Y-tube application of ACSF (1) and orexin-A in ACSF (2) to a DR (left traces) and LDT (right traces) neuron. Orexin-A induced an inward current but no rise in somatic or dendritic [Ca2+]i when DR and LDT neurons were voltage clamped near rest (−60 mV). B: voltage jumps to –100 mV increased the driving force for Ca2+ and augmented the dF/F under baseline conditions, indicating a resting Ca2+ permeability. However, this increase in dF/F was not further augmented during activation of the NSCC by orexin-A. B1: membrane current (holding = –60 mV) recorded from a DR neurons while orexin-A (300 nM) was applied. The trace was “blanked” during voltage jumps before (B1a) and after (B1b) orexin-A application. B2a: dF/F (top trace) increased during voltage steps from –60 to –100 mV (bottom traces), indicating a resting Ca2+ permeability (3 sweeps superimposed; middle trace is membrane current). B2b: This slight increase in dF/F was not different when tested during the inward current elicited by orexin-A application (3 traces superimposed). B3: bar graph summarizes these changes in dF/F elicited by voltage jumps to −100 mV from before and after orexin-A application. These changes were not different (P > 0.05, n = 5), indicating that any Ca2+ influx that might occur via the NSCC does not contribute directly to the Hcrt/Orx-mediated Ca2+ transients.

FIG. 5.

FIG. 5.

Hcrt/Orx enhances the Ca2+ transients produced by activation of L-type Ca2+ channels in DR and LDT neurons. A: Ca2+ transients elicited by voltage steps from –60 to –30 mV were reversibly enhanced by ∼30% following application of orexin-A (300 nM) in DR and LDT neurons, even in the absence of an orexin-A–induced inward current. Top traces show the Ca2+ transients measured as dF/F superimposed from before orexin-A application (Con), after orexin-A application (Orx), and after recovery from the orexin-A application (Rec). The bottom and middle traces show the corresponding membrane voltage (bottom) and membrane current traces (middle). B: nifedipine (10 μM) nearly completely attenuated the Ca2+ transient enhancement produced by Hcrt/Orx. Top traces show the Ca2+ transients elicited by voltage steps from –60 to –30 mV in the presence of nifedipine (Nif) after orexin-A application (Orx/Nif) and after the expected orexin effect would have recovered (Rec Orx/Nif). C: bar graph summarizes the effect of nifedipine (10 μM) on the Ca2+ transient enhancement produced by Hcrt/Orx. Nifedipine significantly attenuated this effect in both the DR and LDT (*P < 0.05). D: application of Bay-K 8644 (10 μm; Bay-K), which enhances current through L-type Ca2+ channels, increased the control Ca2+ transients elicited by voltage steps from –60 to –30 mV (Con). Application of orexin-A (300 nM) further enhanced these Ca2+ transients (Orx/Bay-K), which recovered after wash-out of orexin-A (Rec Orx/Bay-K). E: bar graph comparing the enhancement of the voltage step–evoked Ca2+ transient produced by orexin-A in normal ACSF (Orx ACSF) and in ACSF containing Bay K (Orx + Bay). The Ca2+ transient enhancement was significantly larger in the presence of Bay-K than it was in the absence of Bay-K for both DR and LDT neurons (*P < 0.05).

FIG. 6.

FIG. 6.

Inhibition of protein kinase C (PKC) with bisindolylmaleimide I abolishes enhancement of voltage-step evoked Ca2+ transients by Hcrt/Orx but does not completely block the Hcrt/Orx-evoked inward currents. A: Ca2+ transients elicited in DR and LDT neurons by voltage-steps from –60 to –30 mV following preincubation with bisindolylmaleimide I (Bis I; top trace labeled bis) were not enhanced by orexin-A (300 nM; top trace labeled Orx/bis). Corresponding voltage (bottom) and total membrane current (middle) traces are also shown. B: following preincubation with Bis I, orexin-A (300 nM) retained the ability to evoke an inward current in some neurons even though these neurons did not show an enhancement in Ca2+ transients evoked by voltage steps. Trace in B is taken from 1 such neuron recorded in the DR. Note that the vertical deflections are currents associated with test voltage steps and are truncated. C: left bar graph: effect of Hcrt/Orx on Ca2+ transients following preincubation with Bis I. The average Ca2+ transient amplitude was not different following orexin-A application. Right graph: magnitude of the current evoked by orexin-A following preincubation of the slice with Bis I in the same neurons that were imaged. There was no detectable current in 5/7 DR neurons and 9/11 LDT neurons. The average (±SE) Hcrt/Orx current for these neurons is plotted as solid symbols for DR and LDT neurons (_I_Orx). Two neurons from each nucleus, however, retained an _I_Orx of apparently normal magnitude. The amplitudes of these currents are plotted as open symbols for each DR and LDT neuron.

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