Keywords: arousal, narcolepsy, tuberomammillary, voltage clamp, orexin, dorsal raphe

Table 1.

Responses of serotonergic cells to orexins in current clamp

As we have reported previously (Brown et al., 2001a), bath application of low concentrations (5–300 nm) of orexin A depolarized serotonergic cells and elicited sodium-dependent action potentials (n = 36). In the presence of the selective voltage-dependent sodium channel blocker TTX (0.5 μm), orexin A still depolarized serotonin neurons; in some neurons (6 of 13 neurons tested with 100 or 300 nm orexin A), high-amplitude calcium-dependent action potentials were observed. These calcium-dependent action potentials were broader than TTX-sensitive action potentials and could be blocked by the addition of cadmium (100 μm) to the bathing medium. Application of 100 nmorexin A led to a depolarization of 8.7 ± 1.3 mV (n = 8). A higher concentration of orexin A (300 nm) did not lead to a larger effect (9.2 ± 1.4 mV;n = 5), so this concentration was used as standard in subsequent experiments. Washout was slow; complete reversal of the effect often required at least 1 hr.

In three of six cells, when the membrane potential was manually clamped at the pre-drug level during the orexin A-induced depolarization, an increase in the voltage responses to hyperpolarizing current steps could be observed, i.e., the input resistance was increased (to 127 ± 9% of control) (Fig.1A(i)). However, the magnitude of this effect was variable, and large depolarizations could also be observed without significant changes in input resistance (Fig.1A(ii)). On average, in the presence of TTX, 100 nm orexin A caused an increase in the input resistance to 112 ± 8% of control (n = 6).

Although orexin A activates both orexin receptors with a similar affinity, orexin B is a relatively selective agonist for the type II orexin receptor (Sakurai et al., 1998). Bath application of orexin B caused a similar response as orexin A, i.e., it depolarized the serotonin neurons and caused the appearance of action potentials (Fig.1B(i)). In the presence of TTX, 100 nm orexin B caused a depolarization in all cells tested, the magnitude of which (9.3 ± 1.4 mV;n = 8) (Fig.1B(ii)) was not significantly different from that caused by 100 nm orexin A. Changes in input resistance were variable, as was the case with orexin A. On average, 100 nm orexin B increased the input resistance to 119 ± 7% of control (n = 8). In contrast to the effects of orexin A and B, another peptide neurotransmitter produced in the lateral hypothalamus, melanin-concentrating hormone (MCH), had only minor effects on the membrane potential of serotonin neurons (Fig.1B(i)) (1.3 ± 0.7 mV;n = 6).

Responses of serotonergic cells to orexins under voltage clamp

In voltage-clamp experiments, cells were held at −75 mV, and slow voltage ramps from −75 to −130 mV were applied every 30 sec (Fig.2). Bath application of orexin A (100 nm) led to the appearance of an inward current that was associated with a large increase in channel noise (Fig.2A). On average, the inward current at −75 mV amounted to −44.1 ± 4.0 pA (n = 7). Voltage ramps in the absence and presence of orexin A (Fig.2B) were converted to current–voltage (I–V) plots. Because orexin A increased input resistance under current-clamp conditions, we expected to observe that theI–V plots would cross at the potassium equilibrium potential. However, this was not observed in any experiment with orexin A. Three types of responses were observed. In three of seven cases we observed that the curves converged from −75 to −100 mV but remained parallel thereafter (type A response) (Fig.2A(iii)); in two of seven cases the curves remained parallel over the entire voltage range tested (type B response) (Fig.2B); in the remaining two cases the curves converged as the voltages became more positive (type C response) (Fig.2C). In contrast to the results obtained with orexin A, application of the selective 5-HT_1A receptor agonist, 8-OH-DPAT, which is known to activate an inwardly rectifying potassium conductance in these neurons (Aghajanian and Lakoski, 1984;Penington et al., 1993), caused an outward current of 67 ± 19 pA (n = 4).I–V curves in control and in the presence of 8-OH-DPAT crossed at −101.1 ± 1.4 mV (n = 4), which was near the calculated potassium equilibrium potential (−99.5 mV).

Application of orexin B under voltage clamp led to essentially identical responses as seen with orexin A (Fig.3A), i.e., an inward current with increased noise, with a current–voltage relationship that did not cross at the potassium equilibrium potential (Fig.3A(ii)). At 100 nm, orexin B induced an inward current of −57.2 ± 8.1 pA (n = 7), which was not significantly stronger than that of orexin A (p= 0.17). The effect of orexin B was dose dependent, with higher concentrations giving larger inward currents; the EC₅₀ was 22 nm (Fig.3A(iii)). In 2 of 18 neurons tested with high (≥100 nm) concentrations of orexin B, there was no response, although these neurons subsequently responded to orexin A (100 nm) or phenylephrine (3 μm; see below) indicating that some neurons may lack type II receptors. Taking together the current–voltage responses of all experiments with high concentrations of orexin B (100, 300, or 600 nm), there were three type A responses, eight type B responses, and four type C responses.

The fact that orexin B was similarly effective as orexin A in exciting serotonin neurons at low concentrations suggested a role for the orexin type II receptor. Because pharmacological tools for investigating orexin receptors are not available at the current time, we took an alternative approach and determined the expression of orexin receptors in neurons acutely isolated from the dorsal raphe region.

Single-cell PCR for orexin receptors

Serotonin neurons were identified by the presence of signal for Tph. The Tph-positive neurons (14 of 22) had somata 20–30 μm in diameter with polygonal to round shapes and several dendrites. Some neurons from the dorsal raphe region that were collected for the single-cell PCR study turned out to be Tph negative (8 of 22); these neurons were similar in size and shape to the Tph-positive neurons and were not investigated further. We cannot exclude the possibility that these neurons also express Tph, but below our detection level; another possibility is that they are GABAergic interneurons. In the amplifications with orexin receptor-specific primers, the obtained amplimers had the expected sizes of 108 and 156 bp for OX₁ and OX₂, respectively. Genomic DNA amplification products with our primers would have the expected sizes of 605 bp (OX₁) and 653 bp (OX₂), but these products were never seen on the stained gels.

The majority of the Tph-positive neurons, 9 of 14, expressed both types of orexin receptors, whereas 5 cells expressed only OX₁ (Fig.3B). Although we did not do a detailed quantification, we could observe that the signal for OX₁ was generally strong, whereas the OX₂ signal was more variable in strength and usually weaker.

The orexin-induced excitation of serotonergic neurons is occluded by activation of α₁ adrenergic receptors

The tonic firing of serotonergic neurons during waking is thought to be maintained by the activity of locus coeruleus noradrenergic neurons; noradrenaline depolarizes 5-HT neurons via activation of α₁ adrenoceptors (Vandermaelen and Aghajanian, 1983;Pan et al., 1994). Two observations suggested that orexins and noradrenaline might excite serotonin neurons by similar mechanisms. First, both orexin receptors and α₁adrenoreceptors are coupled to G_qG-proteins and activation of phospholipase C (PLC) (Berridge, 1993;Sakurai et al., 1998). Second, the responses to orexins in current and voltage clamp that we have observed bear a strong resemblance to those described previously for α₁ adrenoceptor agonists (Pan et al., 1994). We tested the idea that these two systems act via a common mechanism by performing occlusion experiments. Addition of 3 μmphenylephrine to the bath caused an inward current of −60.3 ± 12.2 pA (n = 8) and an increase of current noise (Fig.4A). Analysis of the current–voltage relationships revealed that the curves obtained in control and in the presence of phenylephrine did not cross; three type A responses and five type B responses were observed. Application of orexin A (100 nm) in the continued presence of phenylephrine did not lead to an additional inward current (1.3 ± 4.3 pA;n = 6), even in cells such as the one illustrated in Figure4 where phenylephrine had a relatively weak effect.

In addition to causing an inward current, noradrenaline modulates the firing of serotonin neurons by inhibition of the voltage-gated potassium current,I_A, and the calcium-activated potassium current responsible for the slow afterhyperpolarization (Aghajanian, 1985;Pan et al., 1994). Similarly, orexins may modulate conductances other than that responsible for the inward current. To investigate interactions between the two systems on spontaneous firing of serotonin neurons under conditions in which all mechanisms are active and the internal milieu of the neurons is undisturbed, we performed single-unit extracellular recordings in the presence of the α₁ adrenoceptor agonist, phenylephrine. A concentration of phenylephrine (3 μm) was chosen that elicits a rate of tonic firing similar to that seenin vivo (Vandermaelen and Aghajanian, 1983). Application of orexin A (100 nm) in the presence of phenylephrine did not increase the firing frequency of 5-HT neurons (n = 8) (Fig.4B(i)). When phenylephrine was washed out of the slice, the firing of the cells slowly diminished and finally ceased. Application of 100 nm Orexin A, 30 min after washout of phenylephrine, caused the reappearance of action potentials and increased the firing rate to levels similar to those seen in the presence of phenylephrine (seven of seven cells) (Fig.4B(ii)).

It has been shown previously that nicotine excites dorsal raphe neurons by depolarizing noradrenergic axons, releasing noradrenaline, which then activates α₁ receptors on serotonin neurons (Li et al., 1998). To determine whether this was a possible explanation for the similar responses seen with orexin and phenylephrine, we performed a further series of experiments in which the α₁ adrenoceptor antagonist prasozin (1 μm) was added to the perfusing solution containing phenylephrine. This led to a complete cessation of firing in three of four cells and a strong reduction in the firing rate of the other cell. Subsequent application of orexin A (100 nm) under these conditions still led to an increase in firing similar to that seen in the absence of prasozin (Fig.4B(iii)). Thus, orexins do not act by causing the release of noradrenaline and activation of α₁ adrenoceptors.

Inhibitors of signal transduction do not block the effect of phenylephrine or orexin A

Activation of receptors coupled to G_q-proteins leads to activation of PLC. Phospholipase C cleaves phosphatidyl-4,5–bisphosphate to generate two second messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP₃). DAG potentiates the activity of protein kinase C, whereas IP₃causes the release of calcium from intracellular calcium stores. We investigated the role of these second messenger pathways in the effect of phenylephrine and orexin A using pharmacological tools.

In our experiments the inward current elicited by phenylephrine or orexin A was not blocked by the protein kinase C inhibitor chelerythrine chloride (10 μm; three of three experiments each) or the inhibitor of the intracellular calcium-ATPase, thapsigargin (n = 3 of 3 for thapsigargin;n = 2 of 2 for orexin A). It was also not blocked by the protein kinase A inhibitor KT5720 (1 μm;n = 2 for phenylephrine;n = 3 for orexin A), which we have found to block the excitatory action of orexin A in the substantia nigra pars reticulata (Korotkova et al., 2002), or by the MAP kinase inhibitor PD98059 (5 μm;n = 2 of 2 for each agonist). In contrast to the findings ofPan and colleagues (1994), we found that the inward current induced by phenylephrine (n = 2 of 2) or orexin A (n = 2 of 2) was not blocked by the phorbol ester, phorbol 12-myristate 13-acetate (100 nm), although an increase of miniature EPSCs was observed after phorbol ester application.

Histamine excites serotonin neurons similarly to orexins and noradrenaline

A third arousal-related system that innervates the dorsal raphe nucleus is the histaminergic system originating in the tuberomammillary nucleus of the hypothalamus (Panula et al., 1989). Histamine H₁ receptors are the most important histamine receptors in mediating the arousing effects of histamine (Monti, 1993;Lin, 2000;Brown et al., 2001b). Like orexin receptors and α₁ adrenoceptors, histamine H₁ receptors are coupled to phospholipase C (Hill et al., 1997). Histamine (10 or 50 μm) depolarized serotonin neurons in current clamp (data not shown) and caused an inward current under voltage clamp (seven of seven cells) (Fig.5A) associated with an increase in current noise. On average, 50 μmhistamine caused an inward current of 37.4 ± 9.0 pA (n = 7). In six cells in which voltage ramps were applied, one type A, three type B, and two type C responses were observed.

Occlusion experiments with phenylephrine were performed using single-unit recordings. Application of histamine (50 μm) in the presence of 3 μm phenylephrine did not lead to an increase in the firing rate of serotonin neurons (Fig.5B(i)) (six of six), whereas in experiments in which phenylephrine was washed out, histamine (50 μm) increased the firing rate to a similar level as seen in the presence of phenylephrine in four of six cases (Fig.5B(ii)). The effect of histamine was not dependent on the release of noradrenaline and subsequent activation of α₁ adrenoceptors, because histamine (50 μm) increased the spontaneous firing rate in the presence of the α₁ adrenoceptor antagonist prasozin (1 μm) in four of seven cases (Fig.5B(iii)). In the continued presence of histamine in the bath, the firing rate remained steady. Subsequent application of the histamine H₁ receptor antagonist, mepyramine (1 μm), strongly depressed the firing rate (n = 4) (Fig.5B(iii)), indicating that histamine H₁ receptors are responsible for the histamine effect.

The excitation of serotonin neurons by orexin A and phenylephrine is dependent on external sodium

The fact that the current–voltage curves for orexins, phenylephrine, and histamine did not cross at the potassium equilibrium potential indicated that the blockade of leak potassium channels could not (on its own) account for the inward current/depolarization. We tested for the involvement of sodium ions in the orexin A-induced current by replacing 124 mm of the external sodium ions with NMDG. Under these conditions, the orexin A-induced inward current and increase in current noise were strongly attenuated (−10.7 ± 3.9 pA;n = 7) (Fig.6A). In experiments in which only 15 mm of the external NaCl was replaced by NMDG.Cl, an inward current could still be observed in four of five cases, the magnitude of which (−31.1 ± 7.8 pA;n = 4) was not significantly different from control. Thus, it is unlikely that the effect of 124 mmNMDG was attributable to a direct blocking effect on the channels but rather was caused by substitution of the external sodium. Similar to orexin A, in 124 mm NMDG, the inward currents induced by 3 μm phenylephrine (−16.5 ± 5.6 pA;n = 7) and 50 μmhistamine (−12.7 ± 3.7 pA;n = 6) were significantly (p < 0.05) reduced in comparison with the currents recorded in control solution.

The increase in current noise and the dependence on external sodium suggested that a sodium-permeable ion channel mediates the inward current, either alone or with concurrent blockade of leak potassium channels (Pan et al., 1994). We attempted to determine the reversal potential of this putative sodium-permeable ion channel by applying voltage ramps from −100 to +20 mV in the presence of potassium channel blockers (either 2 mm external barium or using an intracellular CsCl-based patch solution). Barium (2 mm) did not block the effect of orexin A (100 nm; −37.2 ± 7.6;n = 5) or phenylephrine (3 μm; −37.0 ± 3.6 pA;n = 5). However, these ramps were disturbed by calcium spikes, which appeared around −30 mV. Addition of 2 mmNiCl₂ or 100 μmCdCl₂ to the bathing solution blocked the calcium spikes but also reduced the effect of orexin A or phenylephrine (n = 5). In contrast, application of NiCl₂ or CdCl₂ after the inward current had already been induced did not reverse the effect (n = 3).

Because it was not possible to use Cd or Ni, we conducted experiments in calcium-free solution. The concentration of magnesium was increased to 3.3 mm to preserve the concentration of divalent cations. Under these conditions the orexin A-induced current was not blocked (−61.4 ± 12.8 pA;n = 9) so we were able to apply long voltage ramps (−100 to +20 mV) without contamination with calcium spikes. Despite a slow rundown of outward currents in the range −30 to +20 mV, in five of nine experiments, the orexin-induced current reversed at −23 ± 7 mV (Fig.6B). In the remaining four experiments the curves converged and came very close to each other in the range −30 to 0 mV but then diverged again at positive potentials. In the range −100 to −30 mV, the orexin A-induced current decreased linearly in four cases (type C response). In four cases the current was voltage independent between −100 and −70 mV and then decreased linearly (type B response). In the final case (type A response; illustrated in Fig.6B), there was a region of negative slope conductance between −100 and −60 mV, a maximum at −60 mV, and a decrease of the current between −60 and −10 mV (where it reversed).

The value that we obtained for the reversal potential of these channels (−23 mV) is far from the reversal potential for sodium, so a pure sodium conductance is unlikely to account for our results. We calculated the relative permeability of sodium to potassium (pNa/pK) by inserting the experimentally obtained reversal potentials and the concentrations of sodium and potassium into the Goldmann–Hodgkin–Katz equation. This gave a pNa/pK of 0.43 ± 0.1 (n = 5). In calcium-free extracellular solution containing reduced sodium (124 mm NMDG⁺/25.6 mm Na⁺/0 Ca²⁺/3.3 Mg²⁺), the reversal potential for sodium is +25.9 mV, and the predicted reversal potential for a cationic current with the relative permeabilities calculated above is −64 ± 5 mV (n = 5). Reversal of the current was seen in six of nine experiments in this solution. The reversal potential (−49 ± 6 mV;n = 6) (Fig.6C) was not significantly different from the predicted value but was significantly (p < 0.05) more negative than the value obtained in solution containing the normal extracellular sodium concentration. The current did not reverse in the remaining three experiments.

It should be noted that the calculated reversal potentials are based on a voltage-insensitive nonselective cation current; however, at least some of the conductances of the responses that we obtained were clearly voltage sensitive (type A responses). In addition, the reversal potential measurements are likely to suffer from voltage-clamp and space-clamp errors caused by the use of the single-electrode voltage-clamp technique and recordings from neurons with elongated dendrites, which are unlikely to have been adequately clamped. Thus, these values can only be considered a first approximation.

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