Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline)

Abstract

Dorsal raphe serotonin neurons fire tonically at a low rate during waking. In vitro, however, they are not spontaneously active, indicating that afferent inputs are necessary for tonic firing. Agonists of three arousal-related systems impinging on the dorsal raphe (orexin/hypocretin, histamine and the noradrenaline systems) caused an inward current and increase in current noise in whole-cell patch-clamp recordings from these neurons in brain slices. The inward current induced by all three agonists was significantly reduced in extracellular solution containing reduced sodium (25.6 mm). In extracellular recordings, all three agonists increased the firing rate of serotonin neurons; the excitatory effects of histamine and orexin A were occluded by previous application of phenylephrine, suggesting that all three systems act via common effector mechanisms. The dose-response curve for orexin B suggested an effect mediated by type II (OX2) receptors. Single-cell PCR demonstrated the presence of both OX1 and OX2 receptors in tryptophan hydroxylase-positive neurons. The effects of histamine and the adrenoceptor agonist, phenylephrine, were blocked by antagonists of histamine H1 and alpha1 receptors, respectively. The inward current induced by orexin A and phenylephrine was not blocked by protein kinase inhibitors or by thapsigargin. Three types of current-voltage responses were induced by all three agonists but in no case did the current reverse at the potassium equilibrium potential. Instead, in many cases the orexin A-induced current reversed in calcium-free medium at a value (-23 mV) consistent with the activation of a mixed cation channel (with relative permeabilities for sodium and potassium of 0.43 and 1, respectively).

Fig. 1.

Responses to orexins under current clamp.A, Input resistance changes in serotonin neurons in response to orexin A. Chart recordings of membrane potential are shown. Downward deflections are from hyperpolarizing pulses (−50 pA, 500 msec) used to test the input resistance. Bath application of orexin A (100 nm, 3 min) led to an increase in the input resistance of the neuron in (i) but not that shown in(ii). B, Orexin B excites serotonin neurons. Chart recordings of membrane potential are shown. Downward deflections are from hyperpolarizing pulses (−25 pA, 500 msec) used to test the input resistance. B(i), Melanin concentrating hormone (MCH) does not affect the membrane potential, whereas in the same cell orexin B causes a large depolarization and elicits tetrodotoxin-sensitive action potentials.B(ii), In a different cell, in the presence of tetrodotoxin (0.5 μm), orexin B causes a large depolarization and the appearance of high-amplitude calcium spikes (asterisks) at the end of hyperpolarizing current pulses.

Fig. 2.

Orexin A causes an inward current under voltage clamp. A(i), Chart recording of holding current. Downward deflections represent the responses to voltage ramps from the holding potential of −75 to −130 mV (illustrated inA(ii)). Orexin A (100 nm) causes an inward current and an increase in current noise. In A(iii) the voltage ramps have been converted to a current–voltage plot. InA(iv) the control current responses have been subtracted from the responses in the presence of orexin A. In this cell the net orexin A-induced current declines in amplitude in the hyperpolarizing direction. B and C show the responses in two other serotonin neurons. In B the orexin A-induced current is voltage independent in the range −75 to −130 mV, whereas in C the current declines in amplitude in the depolarizing direction.

Fig. 3.

Orexin receptors involved in the excitation of dorsal raphe neurons. A, Orexin B induces an inward current and increase in current noise under voltage clamp.A(i), Chart recording of holding current. Downward deflections represent the responses to voltage ramps from the holding potential of −75 to −130 mV. A(ii), The voltage ramps have been converted to current–voltage plots in control and in the presence of orexin B. A(iii), Dose–response relationship for the orexin B-induced inward current indicates that the OX₂ receptor is involved. B, Representative results from the single-cell RT-PCR study. Dissociated neurons were tested for the expression of tryptophan hydroxylase (Tph) and with primers for both orexin receptors (OX₁ andOX₂). Most Tph-positive neurons expressed both orexin receptors. The results of an amplification of mRNA from five single cells (captured video images of 3 dissociated neurons are shown at the right side of the gel photographs), as well as positive control (pc) (whole tissue from the dorsal raphe nucleus region) and negative control (nc) (template for the amplification is an electrode solution from the experiment in which the electrode was submerged in the bath with isolated cells but patching was omitted), were analyzed by electrophoresis and visualized with the help of ethidium bromide staining of 2% agarose gels. M, Weight markers [100 bp step DNA ladder (Promega) with the 500 bp band present at trifold intensity].

Fig. 4.

Activation of α₁ adrenoceptors occludes the effect of orexin A. A(i), Chart recording of holding current. Downward deflections represent the responses to voltage ramps from the holding potential of −75 to −130 mV. Phenylephrine (3 μm) causes an inward current and an increase in current noise. Subsequent application of orexin A (100 nm) does not lead to a further change in holding current. In A(ii) the voltage ramps have been converted to a current–voltage plot. In A(iii) the control current responses have been subtracted from the responses in the presence of phenylephrine. In this cell the net phenylephrine-induced current remains fairly constant over the voltage range tested.B, Extracellular single-unit recordings from serotonin neurons. The mean firing frequency ± SEM is plotted against time.B(i), Orexin A does not affect the firing frequency in the presence of phenylephrine (3 μm;n = 8). B(ii), If phenylephrine is washed out, orexin A (100 nm) can increase the firing frequency (n = 7). B(iii), Orexin A (100 nm) can also increase the firing frequency in the presence of the α₁ adrenoceptor antagonist prasozin (1 μm). Insets, Examples of extracellularly recorded action potentials from single experiments. Each trace is an average of 100 individual responses. Calibration: vertical, 0.5 mV; horizontal, 1 msec.

Fig. 5.

Histamine excites serotonin neurons.A(i), Chart recording of holding current. Downward deflections represent the responses to voltage ramps from the holding potential of −75 to −130 mV. Histamine (50 μm) causes an inward current and an increase in current noise. InA(ii) the voltage ramps have been converted to a current–voltage plot. In A(iii) the control current responses have been subtracted from the responses in the presence of histamine. In this cell the net histamine-induced current declines in amplitude in the hyperpolarizing direction.B, Extracellular single-unit recordings from serotonin neurons. The mean firing frequency ± SEM is plotted against time.B(i), Histamine (50 μm) does not affect the firing frequency in the presence of phenylephrine (3 μm; n = 6). B(ii), If phenylephrine is washed out, histamine (50 μm) can increase the firing frequency (in 4 of 6 cases). B(iii), Histamine (50 μm) can also increase the firing frequency in the presence of the α₁ adrenoceptor antagonist, prasozin (1 μm; 4 of 7 cases). Furthermore, this effect is blocked by the histamine H₁ receptor antagonist, mepyramine (1 μm). Insets, Examples of extracellularly recorded action potentials from single experiments. Each trace is an average of 100 individual responses. Calibration: vertical, 0.5 mV; horizontal, 1 msec.

Fig. 6.

Involvement of a nonselective cation channel in the effect of orexin A. A, Chart recording of membrane potential. Downward deflections represent voltage ramps from −75 to −130 mV. When orexin A is applied in an extracellular solution in which 124 mm of NaCl has been replaced byN-methyl-d-glucamine chloride (NMDG.Cl), only a small inward current and increase in current noise are observed. When NMDG-containing solution is replaced by normal extracellular solution during the period when the orexin A effect is normally still active, a larger inward current and increase in noise slowly appear. B(i), In one experiment performed in extracellular solution containing 0 Ca²⁺/3.3 Mg²⁺, slow (20 sec) voltage ramps from −100 to +20 mV in control and in the presence of orexin A cross at −10 mV. B(ii), The control curve has been subtracted from the response obtained in the presence of orexin A to give the orexin A-induced current. C(i), In one experiment performed in extracellular solution containing 124 mm NMDG⁺/25.6 mmNa⁺/0 Ca²⁺/3.3 Mg²⁺, slow (20 sec) voltage ramps from −100 to +20 mV in control and in the presence of orexin A cross at −55 mV.C(ii), The control curve has been subtracted from the response obtained in the presence of orexin A to give the orexin A-induced current.