Effects of Serotonin on Caudal Raphe Neurons: Activation of an Inwardly Rectifying Potassium Conductance

Douglas A. Bayliss, Yu-Wen Li, and Edmund M. Talley

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Bayliss, Douglas A., Yu-Wen Li, and Edmund M. Talley. Effects of serotonin on caudal raphe neurons: activation of an inwardly rectifying potassium conductance. J. Neurophysiol. 77: 1349-1361, 1997. We used whole cell current- and voltage-clamp recording in neonatal rat brain stem slices to characterize firing properties and effects of serotonin (5-HT) on neurons(n = 225) in raphe pallidus (RPa) and raphe obscurus (ROb). Of a sample of 51 Lucifer yellow-filled neurons recovered after immunohistochemical processing to detect tryptophan hydroxylase (TPH), 34 were found to be TPH immunoreactive (i.e., serotonergic). Serotonergic neurons had long-duration action potentials and fired spontaneously at low frequency (~1 Hz) in a pattern that was often irregular; at higher firing frequencies the discharge became more regular. These neurons displayed spike frequency adaptation, with maximal steady-state firing rates of <4 Hz. The overwhelming majority of identified serotonergic neurons was hyperpolarized by bath-applied 5-HT (94%; n = 32 of 34); conversely, most cells in this sample that were hyperpolarized by 5-HT were serotonergic (78%; n = 32 of 41). TPH-immunonegative neurons were separated into two populations. One group had properties that were indistinguishable from those of serotonergic caudal raphe neurons. The other group was truly distinct; those neurons had more hyperpolarized resting membrane potentials, were not spontaneously active, had shorter-duration action potentials, and were depolarized by 5-HT. Caudal raphe neurons responded to 5-HT (1-5 µM) with membrane hyperpolarization in current clamp (-13.4 ± 1.1 mV, mean ± SE) or with outward current in voltage clamp (16.0 ± 1.4 pA). The current induced by 5-HT was inwardly rectifying and associated with an increase in peak conductance and was highly selective for K+. It was completely blocked by 0.2 mM Ba2+ but not by glibenclamide, an inhibitor of ATP-sensitive K+ channels. Effects of 5-HT were dose dependent, with an EC50 of 0.1-0.3 µM. The 5-HT1A agonist 8-OH-DPAT mimicked, and the 5-HT1A antagonists (+)WAY 100135 and NAN 190 blocked, effects of 5-HT. The 5-HT2A/C antagonist ketanserin did not inhibit the effects of 5-HT. Fewer 5-HT-responsive neurons were encountered in slices exposed acutely to pertussis toxin (~13%) than in adjacent control slices not exposed to pertussis toxin (~85%). In addition, in neurons recorded with pipettes containing GTPgamma S (0.1 mM), 5-HT induced an inwardly rectifying current that did not reverse on washing. In many cells recorded with GTPgamma S, a current developed in the absence of agonist that had properties identical to those of the 5-HT-sensitive current; when followed for extended periods, the agonist-independent GTPgamma S-induced conductance desensitized, returning toward control levels with a time constant of ~18 min. Together these results indicate that serotonergic neurons of ROb and RPa are spontaneously active in a neonatal rat brain stem slice preparation and that hyperpolarization of those neurons by 5-HT1A receptor stimulation is due to pertussis toxin-sensitive G protein-mediated activation of an inwardly rectifying K+ conductance. In addition, we identified a group of nonserotonergic medullary raphe neurons that had distinct electrophysiological properties and that was depolarized by 5-HT.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The neurotransmitter serotonin (5-HT) has a widespread distribution and multiple effects in the mammalian CNS. The numerous central effects of 5-HT result from the panoply of 5-HT receptors (Hoyer et al. 1994) and the diversity of ion channels that those receptors modulate (Andrade and Chaput 1991; Bobker and Williams 1990), whereas the extensive fiber system emanates from discrete groups of serotonergic neurons located in the midline raphe nuclei of the brain stem (Jacobs and Azmitia 1992).

The midline raphe cell groups of the brain stem appear to subserve different functions, which is reflected in the organization of the projections that arise from each of the distinct raphe nuclei. The bulk of the ascending projections to rostral brain regions, which are believed to modulate higher-order functions (e.g., cognitive function, mood, etc.), derive from the most prominent raphe cell group, nucleus raphe dorsalis (RDo) (Jacobs and Azmitia 1992). Descending projections to more caudal brain regions and the spinal cord emanate from raphe cell groups located in the medulla oblongata, specifically in nucleus raphe magnus (RMg), nucleus raphe obscurus (ROb), and nucleus raphe pallidus (RPa) (Jacobs and Azmitia 1992; Skagerberg and Bjorklund 1985).

Of the medullary raphe nuclei, RMg preferentially innervates spinal dorsal horn regions and may be involved in modulation of sensory input (Basbaum and Fields 1984; Skagerberg and Bjorklund 1985). In contrast, the caudal medullary raphe nuclei, ROb and RPa, provide the major serotonergic projection to the ventral and intermediolateral horns of the spinal cord, where they innervate motoneurons and sympathetic preganglionic neurons (Jacobs and Azmitia 1992; Skagerberg and Bjorklund 1985). The direct effects of 5-HT on motoneurons and sympathetic preganglionic neurons are predominantly excitatory (Aghajanian and Rasmussen 1989; Lewis et al. 1993; Pickering et al. 1994), and the activity of ROb and RPa neurons in vivo is highly correlated with motor output, being enhanced during rhythmic motor behaviors (e.g., treadmill exercise) (Veasey et al. 1995) and depressed during the atonia of rapid eye movement-like sleep (Jacobs and Azmitia 1992; Woch et al. 1996). Thus neurons of ROb and RPa have projections distinct from the other raphe cell groups and their activity may influence the degree of serotonergic excitatory bias imposed on motor and sympathetic outflow pathways (reviewed in Chalmers and Pilowsky 1991; Jacobs and Azmitia 1992).

At the present time, although there is a substantial amount of information regarding electrophysiological properties of neurons in RDo and RMg, there is very little information on the caudal raphe neurons in ROb and RPa. Extracellular recording in the caudal raphe indicated that ROb and RPa neurons were spontaneously active and inhibited by 5-HT or the 5-HT1A agonist R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), a so-called "autoinhibition" (Clement and McCall 1991; McCall and Clement 1989; Woch et al. 1996). Here we used whole cell recording of visualized caudal raphe neurons in a thin slice preparation, in combination with post hoc immunohistochemistry of recorded neurons, to characterize firing properties of identified serotonergic caudal raphe neurons and to demonstrate that 5-HT1A-mediated inhibition of caudal raphe neurons is mediated by activation of an inwardly rectifying K+ conductance through a pertussis toxin (PTX)-sensitive G protein-mediated mechanism. Some of these results have been presented in preliminary form (Bayliss 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

General Preparation

Whole cell recordings were obtained from caudal raphe neurons using thin brain stem slices obtained from neonatal rats (postnatal day 8 or younger) as described previously (Bayliss et al. 1995; Edwards et al. 1989). In short, neonatal Sprague-Dawley rats were anesthetized by chilling on ice and decapitated, and the brain stem was removed and placed in an ice-cold Ringer solution (see below for composition of solutions) that was bubbled with 95% O2-5% CO2. Deep hypothermia has been described as effective for general anesthesia (Phifer and Terry 1986) and this protocol was approved by the University of Virginia Animal Research Committee. Transverse slices (150 µm) were prepared with the use of a microslicer (DSK-1500E, Dosaka). Slices were incubated for 45 min to 1 h at 37°C and then maintained at room temperature (23-25°C) in Ringer solution (see below) equilibrated with O2 and CO2.

Recording

Slices were submerged in a chamber mounted on a fixed-stage microscope (Axioskop FS, Zeiss) equipped with Nomarski optics and a ×40 water immersion objective. Caudal raphe neurons were identified visually by their location along the midline in the medulla oblongata and by their size and shape [medium to large (i.e., ~15-20 µm × 20-30 µm), bipolar or multipolar cells (Steinbusch and Nieuwenhuys 1983)]. In some cases we filled cells with Lucifer yellow for post hoc identification and immunostaining with antibodies to 5-HT or its synthesizing enzyme, tryptophan hydroxylase (TPH) (see below). Once the cell was identified as a raphe cell, a patch pipette under positive pressure was placed on the surface of the neuron; release of the pressure followed by gentle suction allowed formation of a seal with resistance >1 GOmega , and additional suction ruptured the membrane, permitting whole cell access. Patch electrodes were fabricated from borosilicate glass capillaries (Clark Electromedical) on a horizontal puller (Sutter P-97) to a DC resistance of 2-7 MOmega and connected to the headstage of an Axopatch 200A patch-clamp amplifier (Axon Instruments). Voltage commands were applied, and whole cell currents were recorded and analyzed with the use of the pCLAMP suite of programs (Axon). Currents were filtered at 2-5 kHz and digitized at 5-10 kHz. Series resistance was typically <20 MOmega and was compensated by ~70%. Electrical recordings were performed at room temperature.

In current clamp, spontaneously occurring action potentials were recorded at the resting membrane potential. In some cases, firing was induced by depolarizing intracellular current injection. Action potential duration was measured at half-amplitude. Under whole cell voltage clamp, currents were elicited by applying voltage ramp commands to -120 mV from a holding potential of -40 mV (0.1 V/s) at varying intervals. In preliminary experiments, we found that this voltage ramp paradigm provided current-voltage (I-V) curves that were identical to steady-state I-V curves derived from voltage step commands. The current induced by 5-HT was determined at the holding potential (usually -40 mV). Peak conductance was determined from the slope of a linear fit to the ramp current measured between -90 and -110 mV; the 5-HT-induced conductance was the difference between slope conductance in control and 5-HT.

Data are presented as means ± SE. Data were analyzed statistically with the use of paired t-tests or one-factor analysis of variance (ANOVA) as indicated in the text; to evaluate prior hypothesized differences among group means, ANOVAs were followed by a Bonferroni modification of the t-test (Wallenstein et al. 1980). In all cases significance was accepted if P < 0.05.

Solutions

The solution used for preparation of slices contained (in mM) 130 NaCl, 3 KCl, 5 MgCl2, 1 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Slices were maintained in a similar solution, but with CaCl2 and MgCl2 both at 2 mM and with lactic acid added to a final concentration of 4 mM (Takahashi 1992). For current-clamp recordings we used an external N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-based Ringer solution that contained (in mM) 140 NaCl, 3 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, and 10 glucose, pH adjusted to 7.3 with NaOH; the internal solution contained (in mM) 17.5 KCl, 122.5 potassium gluconate, 10 HEPES, 0.2 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 9 NaCl, 1 MgCl2, 3 MgATP, and 0.3 guanosine 5'-triphosphate (GTP)-tris(hydroxymethyl)aminomethane (Tris), pH adjusted to 7.2 with KOH. For voltage-clamp recordings of inwardly rectifying potassium currents, the external solution was as above but with the addition of 1.0 µM tetrodotoxin. In some experiments, external K+ was raised by equimolar substitution of KCl for NaCl. The internal solution contained (in mM) 120 KCH3O3S, 4 NaCl, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 10 EGTA, 3 MgATP, and 0.3 GTP-Tris, pH 7.2. In some experiments we substituted 0.1 mM GTPgamma S for GTP in the pipette.

Drugs

5-HT was obtained from Sigma, prepared as a 10 mM stock solution in water, and stored at -20°C; it was added to the perfusate at concentrations between 0.1 and 100 µM. 8-OH-DPAT (1 mM in water), 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) (10 mM in dimethyl sulfoxide), and 3-[2-]4-(4-fluorobenzoyl)-1-piperdinyl]ethyl]-2,4(1H,3H)-quinazolinedione tartrate (ketanserin) (5 mM in water) were obtained from RBI and prepared as frozen stock solutions as listed. A specific 5-HT1A antagonist, N-tert-butyl 3-4-(2-methoxyphenyl)piperazin-1-yl-2-phenylpropanamide dihydrochloride [(+)WAY 100135] was a gift from Wyeth-Ayerst Research (Princeton, NJ) and was prepared as a 10 mM stock (in water) (Fletcher et al. 1993). PTX was a generous gift from Dr. E. Hewlett (Univ. Virginia). Drugs were added to the perfusate at final concentrations provided in the text and vehicle concentrations never exceeded 0.01%.

Immunohistochemical identification of recorded neurons

We labeled cells intracellularly by including Lucifer yellow (0.01%) in our pipette solution. Slices containing recorded neurons were immersed in fixative (4% paraformaldehyde) until they could be processed for immunohistochemical detection of 5-HT or TPH, usually within 1 wk. We initially attempted to stain recorded cells with the use of a rabbit antisera to 5-HT (Eugene Tech), following the protocol described by Kangrga and Loewy (1994). Although many immunoreactive neurons were evident in the slice, usually in close proximity to the recorded neuron, we found no examples of double-labeled cells (i.e., cells that contained both Lucifer yellow and 5-HT immunoreactivity). These uniformly negative results suggested that the antigen (i.e., 5-HT) was being lost from neurons under our whole cell recording conditions, and prompted us to attempt identification of serotonergic neurons with the use of antibodies to a potentially less labile antigen. To this end, we obtained a mouse monoclonal antibody to TPH, the 5-HT synthesizing enzyme, which recently became available (Sigma). Initial control experiments in which a double-labeling immunohistochemical protocol was used on perfusion-fixed tissue with this TPH antibody showed that it recognized a population of caudal raphe neurons that was identical to that stained by the 5-HT antibody (data not shown). To stain recorded neurons, immersion-fixed slices were rinsed three times (5 min) in 0.1 M phosphate buffer and incubated in 1% sodium borohydride in phosphate buffer (10 min), followed by two rinses in phosphate buffer (5 min). Slices were then equilibrated in 50 mM Tris and 150 mM NaCl, pH 7.4 (TS) (3 × 5 min), and blocked for 1 h in in TS containing 5.0% normal goat serum (NGS) and 0.5% Triton X-100. They were incubated for one to three nights at 4°C with the anti-TPH antibody (1:500) in TS containing 1% NGS and 0.5% Triton X-100 (TS/NGS/TX). Following rinses in TS/NGS/TX (4 × 5 min), slices were incubated for 1 h at 22°C with biotinylated rabbit anti-mouse IgG3 antisera (1:100; Zymed) for 1 h in TS/NGS/TX, rinsed with TS (4 × 5 min), and then incubated for 1 h at 22°C with avidin-Texas Red conjugate (1:200; Molecular Probes) in TS. After rinses in TS and then Tris, slices were coverslipped in Krystalon (EM Science) before being examined and photographed with a Zeiss fluorescence microscope.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

We studied the basic electrophysiological properties (i.e., membrane potential, input resistance, capacitance, and spontaneous and evoked firing behavior) as well as the response to 5-HT of neurons in the medullary raphe nuclei in neonatal rat brain stem slices. The present report is based on whole cell current- and voltage-clamp recordings from 225 caudal raphe neurons located in either ROb or RPa. Of those raphe neurons tested with 5-HT agonists (n = 187), the majority (75%; n = 141) responded with hyperpolarization or outward current; in the remaining cells, 5-HT caused a depolarization or inward current (11%; n = 20) or had no clear effect (14%; n = 26).

Serotonergic caudal raphe neurons

We performed immunohistochemical experiments to determine whether recorded raphe neurons were serotonergic. Initial attempts in which an antibody to 5-HT was used were met with uniformly negative results (see METHODS). However, with the use of a new monoclonal TPH antibody we were able to stain a subpopulation of recorded neurons (Fig. 1; Table 1). The TPH-immunoreactive neurons were considered serotonergic because in control experiments the TPH antibody labeled an identical population of neurons as those stained with a 5-HT antibody (see METHODS).


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FIG. 1. Serotonergic caudal raphe neuron: action potential characteristics. A caudal raphe neuron was filled with Lucifer yellow via the recording pipette and subsequently processed for immunohistochemical detection of tryptophan hydroxylase (TPH), the serotonin (5-HT) synthesizing enzyme. A: low-power photomicrograph showing the recorded neuron (arrowhead) among other TPH-immunoreactive neurons in the slice (arrows: selected TPH-positive cells). B and C: higher-power photomicrographs illustrating that the recorded neuron (arrowhead) that contained Lucifer yellow (C) was TPH immunoreactive (B). Scale bar: 35 µm (A), 25 µm (B and C). D: camera lucida drawing of the distribution of TPH-immunoreactive neurons in this medullary slice (down-triangle). Arrow: location of the recorded neuron (bullet ). IO, inferior olive; NA, nucleus ambiguus. E: average of 10 spontaneously occurring action potentials in this neuron. Inset: action potential on a faster time base. F: spontaneous irregular discharge pattern of the neuron (~1.4 Hz) characteristic of serotonergic caudal raphe neurons. Dashed line: point taken for membrane potential measurements (-47 mV).

 
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TABLE 1. General properties of caudal raphe neurons

We recovered 51 Lucifer yellow-labeled neurons after immunohistochemical processing for TPH immunoreactivity. This sample was representative of the total population of cells studied inasmuch as 41 of the Lucifer yellow-filled neurons (80%) were hyperpolarized by superfusion with 5-HT (1-5 µM), whereas 10 either did not respond or were depolarized by 5-HT (19%). Of those 41 neurons hyperpolarized by 5-HT, 32 were TPH immunoreactive (78%). Conversely, of the 34 cells identified as serotonergic, 32 were hyperpolarized by 5-HT (94%). Other properties of the remaining 2 serotonergic cells that did not respond to 5-HT were not obviously different from those of the 32 serotonergic caudal raphe neurons that were hyperpolarized by 5-HT (Table 1).

A serotonergic raphe neuron that was hyperpolarized by 5-HT is shown in Fig. 1. This multipolar cell (arrowhead) was located along the midline of the caudal brain stem, among a population of other TPH-immunoreactive neurons (Fig. 1, A and D, arrows). As was characteristic of other serotonergic caudal raphe neurons, this cell fired spontaneously at a low frequency (~1.4 Hz; Fig. 1F), had a long-duration, overshooting action potential with a slight shoulder on the repolarizing phase, and was followed by a pronounced afterhyperpolarization (Fig. 1E). The midpoint between the afterhyperpolarization and the spike threshold was at about -47 mV. The pattern of firing in serotonergic caudal raphe neurons was often irregular, especially at low firing rates. However, when the cell was induced to fire at faster frequencies, for example by DC injection (Fig. 2, A and B), the pattern became highly regular. Caudal raphe neurons also displayed spike frequency adaptation (Fig. 2, C-E). Rectangular depolarizing current pulses of increasing intensity caused increases in firing frequency early in the pulse that decreased with time (Fig. 2C). This adaptation is clearly evident in Fig. 2D, which provides a plot of instantaneous firing frequency as a function of time in the pulse for three intensities of current injection. The averaged relationship between injected current and both initial and steady-state firing frequency is given in Fig. 2E. The amount of adaptation, reflected by the difference between initial and steady-state curves, is clearly a function of the magnitude of injected current. Note also that the steady-state firing frequency appears to saturate at a very low firing frequency (<4 Hz), even with large current injections.


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FIG. 2. Firing behavior in serotonergic caudal raphe neuron. A and B: serotonergic caudal raphe neuron recorded under current-clamp conditions. Injection of depolarizing current transformed the irregular, low-frequency spontaneous firing in a serotonergic caudal raphe neuron (A) to a more regular discharge pattern (B). C: long rectangular depolarizing current pulses (1.8 s) of increasing intensity were delivered to a different serotonergic neuron. Increases in current injection caused increases in firing frequency, particularly early in the pulse. D: plot of the instantaneous firing frequency (for the traces in C) as a function of time in the pulse shows the time-dependent decrease in spike frequency (i.e., adaptation). E: averaged relationship between injected current and firing frequency for the 1st (black-diamond ) and last (black-square) interspike intervals in serotonergic caudal raphe neurons (n = 8). Depolarizing current pulses were injected from the resting potential of the neuron (-48.5 ± 1.1 mV; range -44 to -52 mV).

TPH-immunonegative caudal raphe neurons

We recovered 17 caudal raphe neurons after histological processing that were not TPH immunoreactive; aside from the 1 cell that showed no response to 5-HT, these neurons appeared to represent two populations (Table 1). One group was hyperpolarized by 5-HT and had basic electrophysiological properties that were indistinguishable from those of TPH-immunoreactive cells (n = 9; Table 1). It is unclear whether this group represents a population of 5-HT-sensitive raphe neurons that are truly nonserotonergic or a "false negative" immunohistochemical result due to washout of the antigen during whole cell recording.

The second population of caudal raphe neurons that were not TPH immunoreactive appeared to have truly distinct electrical properties (n = 7; Table 1). These infrequently encountered neurons were easily recognized by their more hyperpolarized resting potential, lack of spontaneous activity, and shorter-duration action potentials. Moreover, cells of this type depolarized rather than hyperpolarized in response to 5-HT. Because this response was only rarely observed, the mechanism of this depolarization was not studied in detail.

Effect of 5-HT on caudal raphe neurons

Effects of 5-HT on membrane potential and current that were typical of those observed in the majority of caudalraphe cells are illustrated in Fig. 3. Under current-clamp recording conditions, 1 µM 5-HT caused a large hyperpolarization (~24 mV) and a cessation of action potential firing in this serotonergic neuron. On wash, the membrane potential repolarized and the cell resumed firing. A second application of 5-HT caused a similar response. In cells that responded to 5-HT (1-5 µM) with a hyperpolarization, the averaged change in potential was -13.4 ± 1.1 mV (n = 48). When the same cell was recorded under voltage clamp at a holding potential of -40 mV, 5-HT caused the development of an outward current (~20 pA) that followed a time course similar to that of the hyperpolarization. The remainder of this report characterizes the receptor and ionic mechanisms underlying the 5-HT-induced outward current in caudal raphe neurons. Although 5-HT was effective in external solutions containing 3 mM K+, as is evident in Fig. 3, the rectification induced by5-HT was more pronounced with higher external K+ concentrations. Therefore, except where noted, all voltage-clamp recordings were carried out in raised external K+ (6 mM).


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FIG. 3. Effect of 5-HT on serotonergic caudal raphe neuron. A: under current-clamp recording conditions, 1 µM 5-HT caused a reversible and repeatable hyperpolarization (~24 mV) in this spontaneously active serotonergic caudal raphe neuron. B: under voltage clamp in the same cell, 2 applications of 5-HT caused the development of an outward current (~20 pA) that each followed a similar time course.

The current induced by 5-HT displayed all the characteristics of inwardly rectifying K+ currents activated by neurotransmitters in other preparations (Fig. 4) (Pan et al. 1990, 1993; Penington et al. 1993a; Williams et al. 1988). Hyperpolarizing ramp voltage commands from a holding potential of -40 to -120 mV were used to characterize the I-V relationships of caudal raphe neurons before, during, and after exposures to 5-HT. As depicted in Fig. 4A, 5-HT caused an outward current at the holding potential. Associated with the change in holding current, the I-V relationship was transformed from an essentially linear profile to one that was markedly alinear, passing inward current more readily than outward (i.e., inwardly rectifying). The component of current activated by 5-HT was derived by digital subtraction of the control current from that recorded in 5-HT (Fig. 4B). This 5-HT-sensitive current averaged 16.0 ± 1.4 pA (n = 72) at the holding potential (-40 mV) and also showed inward rectification. To quantitate the effects of 5-HT on conductance, the I-V relationship between -90 and -110 mV (indicated by vertical bars in Fig. 4A) was fitted by linear regression and the slope taken as the peak conductance. A plot of peak conductance as a function of time for the cell shown in Fig. 4, A and B, is given in Fig. 4C. From a control level of ~0.5 nS, conductance increased to nearly 13 nS in the presence of 5-HT before recovering fully after wash. The initial peak conductance averaged 0.92 ± 0.07 nS (corresponding to an input resistance ~1 GOmega ) and increased to 5.27 ± 0.38 nS in the presence of 1 µM 5-HT (n = 72).


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FIG. 4. 5-HT activates a barium-sensitive inwardly rectifying K+ conductance. A: currents were evoked by ramp hyperpolarizations (from -40 to -120 mV) in control, during application of 1 µM 5-HT, and after wash. Ramp currents were plotted as a function of membrane potential. B: 5-HT-sensitive current was obtained by digital subtraction of the control current from that obtained in 5-HT and plotted as a function of membrane potential; the 5-HT-sensitive current is inwardly rectifying and reverses near -72 mV (in 6 mM K+; arrowhead). C: peak conductance was taken as the slope of the current-voltage (I-V) relationship between -90 and -110 mV (bold vertical lines in A) and plotted as a function of time. 5-HT caused a large reversible increase in peak conductance. D: reversal potential of the 5-HT-sensitive current was measured with extracellular K+ concentration ([K+]o) of 3, 6, and 12 mM (n = 5, 11, and 6, respectively) and plotted on a semilog scale. The data points are well represented by the Nernst equation (------), indicating that the 5-HT-sensitive current is K+ selective. E and F: effect of barium on the 5-HT-induced increase in peak conductance (E) and on the 5-HT-sensitive current (F). The effect of Ba2+ was partially reversible on washing (not shown).

The 5-HT-sensitive current measured in 6 mM external K+ reversed at -72 mV in this cell (Fig. 4B, arrowhead), very close to the predicted potassium reversal potential (-71 mV), suggesting that the 5-HT-induced current was carried predominantly by K+. To test this possibility further, the reversal potential (Erev) of the 5-HT-sensitive current was measured with different concentrations of K+ in the bath; these data are plotted in Fig. 4D along with the expected relationship for a pure K+ current given by the Nernst equation. In 3, 6, and 12 mM K+ the measured Erevs were-86.6 ± 1.9 mV, -69.2 ± 1.0 mV, and -51.3 ± 1.3 mV, respectively. The slope of a regression line relating measured values of Erev to the log extracellular K+ concentration was 58.7, close to the value predicted by the Nernst equation (58.8 at 23°C), indicating that the current is carried exclusively by K+.

A characteristic feature of inwardly rectifying K+ currents is their sensitivity to low concentrations of barium (Pan et al. 1990, 1993; Penington et al. 1993a; Williams et al. 1988). Accordingly, we found that the effects of 5-HT in caudal raphe neurons were also blocked by barium. The 5-HT-induced increase in peak conductance was completely blocked by 0.2 mM Ba2+ (Fig. 4E), as was the inwardly rectifying current activated by 5-HT (Fig. 4F). In a group of five cells tested before and during barium application, the 5-HT-induced current was reduced from 20.8 ± 9.8 pA under control conditions to 2.8 ± 1.6 pA in 0.2 mM Ba2+(P < 0.05). Likewise, the peak conductance increase activated by 5-HT in the same cells was reduced by barium from 6.2 ± 2.9 nS to 0.1 ± 0.05 nS (P < 0.05).

A spontaneously activating outward current was observed in a small population of caudal raphe neurons at the holding potential. This current appeared to be an ATP-sensitive K+ current because it was blocked by glibenclamide (10 µM; n = 5). It is unlikely that this current represents the 5-HT-sensitive current, because 5-HT was still effective in the presence of 10 µM glibenclamide (n = 3) and the I-V relationship of the glibenclamide-sensitive current showed no evidence of inward rectification over the same voltage range at which the 5-HT-sensitive current was strongly rectifying.

Receptor pharmacology of 5-HT effects on caudal raphe neurons

The effects of 5-HT on the inwardly rectifying K+ conductance and the holding current were dependent on the concentration of 5-HT. As shown in Fig. 5, the relationship was similar whether determined in terms of effects on peak conductance or current, consistent with the idea that effects on the inwardly rectifying conductance account for the 5-HT-sensitive current. The dose response curve was fairly steep; there was essentially no effect of 5-HT at a concentration of 0.05 µM, whereas at 1 µM its effects were apparently saturated. These data were fitted to a logistic equation of the form y = (a - c)/ [1 + ([5-HT]/EC50)b] + c, where a and c are the theoretical maximum and minimum, respectively, and b is a slope function. The EC50s determined from these curves were 0.1 and 0.3 µM, respectively.


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FIG. 5. Effects of 5-HT on inwardly rectifying K+ conductance are concentration dependent. A: effects of increasing concentrations of 5-HT on holding current (at -40 mV in 6 mM K+) were determined in caudal raphe neurons; averaged concentration-response data for 5-HT-induced current is plotted. B: peak conductance is plotted as a function of 5-HT concentration. Solid lines: fit of the data to a logistic equation with an EC50 for effects on current and conductance of 0.1 and 0.3 µM, respectively. Each data point represents the mean ± SE from >= 4 cells.

Pharmacological experiments were performed to determine the 5-HT receptor subtype that mediates activation of the inwardly rectifying K+ conductance in caudal raphe neurons. As shown in Fig. 6A, the 5-HT1A agonist 8-OH-DPAT (0.1 µM) activated a conductance with an I-V relationship similar to that induced by 5-HT. In another raphe neuron (Fig. 6B), the 5-HT1A antagonist (+)WAY 100135 (1 µM) was able to completely block the effect of 5-HT on peak conductance (Fig. 6B). By contrast, the 5-HT2A/C antagonist ketanserin (1 µM) did not significantly attenuate the current activated by 5-HT (Fig. 6C). These data are summarized in Fig. 6D. The average peak conductances in the presence of 5-HT (5.3 ± 0.4 nS; n = 72) and 8-OH-DPAT (4.2 ± 1.0; n = 7) were not different from each other, and each was significantly greater than control(0.9 ± 0.1 nS; P < 0.001). The peak conductance increase induced by 5-HT was blocked by the 5-HT1A antagonists (+)WAY 100135 (1.0 ± 0.3 nS; n = 6) and NAN 190(1.1 ± 0.7 nS; n = 3). In the presence of ketanserin, 5-HT caused a significant increase in peak conductance (5.0 ± 1.5; n = 6; P < 0.001) that was not different from that induced by 5-HT alone. The initial peak conductances among the groups were not different. Together these data indicate that a 5-HT1A receptor mediates activation of the inwardly rectifying conductance by 5-HT.


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FIG. 6. Effects of 5-HT on inwardly rectifying K+ conductance are mediated by the 5-HT1A receptor. A: effect of 1 µM 5-HT and the 5-HT1A agonist 8-OH-DPAT (0.1 µM) on a caudal raphe neuron. The I-V characteristics of the current induced by 8-OH-DPAT were similar to those of the 5-HT-sensitive current. B and C: peak conductance is plotted as a function of time. The increase in peak conductance induced by 1 µM 5-HT was blocked by 1.0 µM (+)WAY 100135, a 5-HT1A receptor antagonist (B), but not by ketanserin, a 5-HT2A/C receptor antagonist (C). D: averaged data showing effects of 5-HT (1.0 µM; n = 72) and 8-OH-DPAT (0.1 µM; n = 7) on peak conductance and the effects of 5-HT in the presence of 5-HT receptor antagonists (+)WAY 100135 (1.0 µM; n = 6), NAN 190 (1.0 µM; n = 3), and ketanserin (1 µM; n = 6). Differences were highly significant [F(5,142) = 26.6; P < 0.0001]. Asterisks: groups that were significantly different from control, but not from each other. There was no difference among groups in initial conductance.

PTX-sensitive G proteins mediate effects of 5-HT on caudal raphe neurons

To test whether 5-HT1A activation of inwardly rectifying K+ currents was mediated by PTX-sensitive G proteins in caudal raphe neurons, two types of experiments were performed. In the first, alternate slices were incubated in the presence or absence of PTX (1-2 µg/ml) at 37°C for >= 2 h before recording. Only 13% of cells treated with PTX(n = 23) responded to 5-HT, whereas 85% of cells in alternate slices that had not been treated with PTX responded to 5-HT (n = 13). This was similar to the percentage of cells from the total population that responded to 5-HT (75%).

In the second set of experiments, cells were recorded with electrodes that contained the nonhydrolyzable GTP analogue GTPgamma S (Fig. 7). In the presence of this analogue, mechanisms mediated by G proteins are expected to be irreversible. Accordingly, 5-HT caused rapid and irreversible activation of an inwardly rectifying current (see Fig. 7A, inset) in caudal raphe neurons that were recorded with GTPgamma S-containing pipettes (n = 2; Fig. 7A). When tested in these cells, a subsequent application of 5-HT had no additional effect (n = 1; not shown). A slower, time-dependent increase in the inwardly rectifying conductance was also seen even in the absence of added transmitter (Fig. 7B). Maximal activation of the conductance by GTPgamma S in the absence of transmitter occurred within 2-5 min of whole cell access and averaged 5.9 ± 1.5 nS with an associated outward current of 20.6 ± 5.9 pA (n = 8), similar to that following 5-HT application. Once the conductance was fully activated by GTPgamma S, 5-HT agonists had little additional effect, indicating that the response was occluded (Fig. 7B). Interestingly, as shown in Fig. 7C, activation of the inwardly rectifying conductance by intracellular GTPgamma S (in the absence of transmitter) was followed by a slower time-dependent decrease in peak conductance. The conductance decays were fitted with a single-exponential function; the average of the decay time constants was 1,082.2 ± 272.2 s (n = 9). The decay was not associated with a return of transmitter sensitivity, because 5-HT was ineffective even after the peak conductance had completely recovered (n = 4; not shown). These data indicate that PTX-sensitive G proteins mediate effects of 5-HT on caudal raphe neurons and further suggest that this mechanism undergoes a desensitization process that is independent, and downstream, of receptor activation.


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FIG. 7. G proteins mediate effects of 5-HT on inwardly rectifying K+ conductance. Cells were recorded with a pipette solution that included the nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue GTPgamma S (100 µm). Peak conductance is plotted as a function of time. Insets: sample ramp currents evoked by hyperpolarizing ramp voltage commands at the times indicated in the conductance plots. A: 5-HT (1.0 µM) caused an increase in inwardly rectifying K+ conductance that did not reverse on wash of 5-HT. B: in this cell, peak conductance increased even in the absence of transmitter; after nearly maximal activation by GTPgamma S, 8-OH-DPAT had little further effect. C: agonist-independent activation of the inwardly rectifying K+ conductance by GTPgamma S was followed by a slow, time-dependent decrease in conductance.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We applied whole cell patch-clamp recording techniques in combination with immunohistochemical analysis of recorded neurons to provide the first characterization of the firing properties and response to 5-HT of identified serotonergic neurons in ROb and RPa, the caudal medullary raphe cell groups. We showed that most of these caudal raphe neurons have firing properties that are similar to those of neurons in other raphe cell groups (Jacobs and Azmitia 1992; Pan et al. 1990, 1993; VanderMaelen and Aghajanian 1983), and that 5-HT inhibits serotonergic caudal raphe neurons via 5-HT1A receptors through a G protein-mediated mechanism involving activation of an inwardly rectifying K+ conductance.

Serotonergic caudal raphe neurons

Identified serotonergic neurons in ROb and RPa had high resting input resistance (~1 GOmega ) under whole cell recording conditions, and were spontaneously active, generating broad action potentials (~5-6 ms in duration at room temperature) at a low, irregularly paced frequency (~1 Hz). In addition, >90% of serotonergic caudal raphe neurons responded to 5-HT with a marked hyperpolarization. The properties that we have described for serotonergic neurons of the caudal raphe are generally similar to those of identified serotonergic neurons in other raphe nuclei (e.g., RMg and RDo) (Pan et al. 1990, 1993; VanderMaelen and Aghajanian 1983). Those other raphe neurons also generate broad action potentials, and in RDo, neurons show a marked spike frequency adaptation associated with a pronounced afterhyperpolarization similar to that we have described in RPa and ROb (cf. VanderMaelen and Aghajanian 1983). Neurons in RDo and RMg are spontaneously active in vivo, but unlike the neurons we recorded, serotonergic neurons in RMg and RDo are often silent in slice preparations in vitro (Pan et al. 1990, 1993; VanderMaelen and Aghajanian 1983). The lack of spontaneous activity in those neurons in vitro has been attributed to the deafferentation that is inevitable in slice preparations (VanderMaelen and Aghajanian 1983). The fact that most serotonergic neurons in ROb and RPa were spontaneously active in our thin slice preparation may reflect a reduced reliance on afferent input.

The properties we observed in serotonergic caudal raphe neurons were also consistent with those previously described for presumed serotonergic neurons recorded extracellularly in the caudal raphe of anesthetized or intact, sleeping cats (Clement and McCall 1991; Jacobs and Azmitia 1992; Veasey et al. 1995; Woch et al. 1996). In those studies, medullary neurons located on the ventral midline that were hyperpolarized by 5-HT1A agonists had broad action potentials and a relatively slow rate of discharge (~1-2 Hz) (Clement and McCall 1991; Jacobs and Azmitia 1992; Woch et al. 1996). Accordingly, we found that the vast majority of slow-firing caudal raphe neurons hyperpolarized by 5-HT were indeed serotonergic (~78%), supporting the assumption that most of the neurons recorded in those earlier in vivo studies were probably serotonergic.

TPH-immunonegative caudal raphe neurons

A number of recorded caudal raphe neurons were not immunoreactive for TPH and therefore could not be identified as serotonergic. These negative immunohistochemical findings must be viewed with some caution. Neurons were studied with the whole cell recording technique and therefore some amount of intracellular dialysis would be expected; this dialysis could, in some cases, result in dilution or loss of the antigen from the cell and lead to a false negative result (as apparently was the case when we attempted to stain cells with the use of an antibody to 5-HT). Thus it is difficult to say with certainty that all of the nonimmunoreactive cells are truly nonserotonergic. In this regard, the basic electrophysiological properties and response to 5-HT ofthese nonimmunoreactive cells suggested that they were of two populations; one that was indistinguishable from the positively identified serotonergic raphe neurons and the other with distinctly different properties. The former group may represent a false negative result. Cells in that group do not appear to be analogous to the so-called secondary cells in RMg. Nonserotonergic secondary cells in RMg, although also spontaneously active and hyperpolarized by 5-HT, were substantially smaller and had much shorter action potentials (~1 ms) than the neighboring serotonergic RMg neurons (Pan et al. 1990, 1993), whereas the group of spontaneously active TPH-immunonegative cells that were hyperpolarized by 5-HT in the caudal raphe had electrophysiological properties that were not different from those of the serotonergic caudal raphe cells.

In contrast to the first group of nonimmunoreactive neurons described above, we believe that the second group represents a population of truly nonserotonergic caudal raphe neurons, on the basis of their distinct electrophysiological properties. These cells had a more hyperpolarized resting membrane potential than serotonergic cells, they were not spontaneously active, and they depolarized in response to 5HT. A similar group of neurons has not previously been described in either RMg or RDo. Although the cellular mechanism of the 5-HT-induced depolarization was not studied in detail in these infrequently encountered raphe cells, the I-V relationship of the underlying current suggested that it may result from closure of an outwardly rectifying K+ conductance (Bayliss and Li, unpublished observation). A current with similar properties is modulated by 5-HT in rat sympathetic preganglionic neurons (Pickering et al. 1994).

Mechanism of 5-HT-induced hyperpolarization

The majority of caudal raphe neurons responded to 5-HT with a hyperpolarization (or outward current) that resulted from activation of an inwardly rectifying K+ conductance. The conductance was barium sensitive and highly selective for K+, and was activated by 5-HT1A receptors through a PTX-sensitive G protein-mediated mechanism. A similar mechanism underlies the 5-HT-induced hyperpolarization of neurons in RMg and RDo (Pan et al. 1993; Penington et al. 1993a; Williams et al. 1988).

It was reported that neurons of the caudal medullary raphe were less sensitive to 5-HT than those in other raphe cell groups (Trulson and Frederickson 1987). However, we found that the average hyperpolarizing response to a saturating concentration of 5-HT (~13 mV) was not different from that of RMg and RDo neurons (~10-15 mV) (Pan et al. 1993; Williams et al. 1988). This is true despite the fact that we recorded from neonatal rat caudal raphe neurons in thin slices (150 µm) at room temperature under whole cell conditions whereas recordings from the other raphe cell groups were made from adult rats in thicker slices (300 µm) with the single-electrode voltage-clamp technique at 37°C. Although the maximal hyperpolarizing response was similar in these studies, the sensitivity to 5-HT was different. The EC50 for effects of 5-HT under our recording conditions was 0.1-0.3 µM, whereas in adult RMg and RDo neurons in thick slices the EC50 was >= 1 order of magnitude higher (Pan et al. 1993; Williams et al. 1988). This higher EC50 could reflect differences in the 5-HT1A receptor-mediated mechanism related to age or to the specific raphe nuclei. However, we favor the possibility that it is simply a result of differences in the preparations. For example, increased metabolism of 5-HT in thicker slices would lower the effective 5-HT concentration and shift the apparent EC50 to higher concentrations. Consistent with this interpretation, the EC50 for activating the same mechanism in dissociated RDo neurons from adult rats, where metabolism would be minimal, was an order of magnitude lower than what we found in the thin slice preparation (Penington et al. 1993a).

Inwardly rectifying K+ channels were activated by 5-HT in outside-out patches of RDo neurons, indicating that soluble second messengers were not required for this membrane-delimited, G protein-mediated effect in those cells (Penington et al. 1993b). Recently molecular cloning has identified a number of G protein-coupled inwardly rectifying K+ channels (GIRKs) that form heteromultimers that are activated by beta gamma -, rather than alpha -, subunits of G proteins in heterologous expression systems (Doupnik et al. 1995). Likewise, in hippocampal neurons where GIRKs are expressed at high levels (Karschin et al. 1996), activation by 5-HT of native inwardly rectifying K+ channels in inside-out patches appears to be mediated by Gbeta gamma (Oh et al. 1995). It remains to be determined whether a similar mechanism mediates activation by 5-HT of inwardly rectifying K+ channels in caudal raphe neurons where, of the cloned GIRKs, only expression of GIRK3 (Kir3.3) has been detected at significant levels (Karschin et al. 1996).

Agonist-independent desensitization

Maximal activation of the inwardly rectifying K+ conductance was observed in the absence of transmitter in cells recorded with pipettes containing the nonhydrolyzable GTP analogue GTPgamma S. Under these conditions, addition of agonists caused little or no further increase in conductance (i.e., the response was occluded). After maximal activation by GTPgamma S, a slow, steady decrease in the inwardly rectifying K+ conductance ensued, which was not associated with a renewed responsiveness to transmitter application. A similar, agonist-independent desensitization phenomenon induced by GTPgamma S has also recently been described in neurons of the substantia nigra (Kim et al. 1995) and in Xenopus oocytes expressing GIRK1 (Kovoor et al. 1996). The mechanism for this agonist-independent desensitization has not been determined. It may reflect inactivation of the G protein or the K+ channel itself, as proposed by Kovoor et al. (1996). Interestingly, it is possible that the mechanism mediating this desensitization might also be activated physiologically by other neurotransmitter receptors. For example, substance P inhibits inwardly rectifying K+ channels in locus coeruleus neurons through a PTX-insensitive G protein-mediated mechanism (Velimirovic et al. 1995); inhibition of the channels by substance P is associated with a dramatic decrease in the ability of somatostatin or enkephalin to activate the same channels (Velimirovic et al. 1995). Thus it is possible that the pattern of effects on inwardly rectifying K+ channels that we observed in caudal raphe cells dialyzed with GTPgamma S---first activation, then inhibition---reflects the sequential activation of G protein mechanisms that converge on the channels with different kinetics and opposing effects.

Functional considerations of raphe neuronal firing properties and effects of 5-HT

The firing rate of raphe neurons in intact or decerebrate cats is sensitive to sleep-wake state, whether sleep is natural or experimentally induced (Jacobs and Azmitia 1992; Veasey et al. 1995; Woch et al. 1996). The average firing rate in presumed serotonergic caudal raphe neurons of the intact cat was maximal at ~4 Hz during active waking and decreased to ~1 Hz during sleep (Jacobs and Azmitia 1992; Veasey et al. 1995); a similar low rate of discharge was reported in anesthetized cat (Clement and McCall 1991; McCall and Clement 1989). Interestingly, we found that spontaneous discharge of caudal raphe neurons in the neonatal rat brain stem slice was also ~1 Hz and that, even with injections of large amounts of depolarizing current, the steady-state firing frequency of these neurons was maximal at or about 4 Hz. This suggests that intrinsic mechanisms of the neurons may dictate the maximal steady discharge rates of these cells. The nature of the intrinsic currents that cause the spike frequency adaptation was not investigated in detail. However, the presence of an enhanced afterhyperpolarization following repetitive high-frequency discharge and the fact that higher firing frequencies can be sustained under conditions in which calcium currents are inhibited (see accompanying paper; Bayliss et al. 1997), suggests that calcium-dependent K+ conductances activated during repetitive firing may be involved.

The inhibitory effects of 5-HT1A receptor activation on caudal raphe neurons are similar to those reported in other groups of raphe neurons (Pan et al. 1993; Penington et al. 1993a; Williams et al. 1988). In RDo, serotonergic inhibitory postsynaptic potentials can be evoked by extracellular stimulation in brain slices, suggesting that those neurons collateralize to provide a feedback autoinhibition (Pan et al. 1989; Wang and Aghajanian 1978). Furthermore, a tonic autoinhibition by 5-HT has been demonstrated in recordings of dorsal raphe neurons in vivo, where exogenous application of 5-HT1A antagonists increased neuronal activity, especially when the activity of the neurons was high (Fornal et al. 1996). It is thought that such an autoinhibitory mechanism might serve to limit the output of those raphe neurons. Although caudal raphe neurons clearly express functional 5-HT1A receptors and hyperpolarize in response to 5-HT1A stimulation, it remains to be determined whether they send collaterals that feed back to release 5-HT and inhibit other caudal raphe cells.

    ACKNOWLEDGEMENTS

  The authors gratefully acknowledge the contributions of N. Sadr and R. Ogle in immunostaining tissue sections, and thank Drs. P. G. Guyenet and R. L. Stornetta for the use of the imaging system for camera lucida and photomicrography. The authors are also grateful to Dr. Albert Berger and J. Singer for comments on the manuscript, to Dr. John Haycock for information on use of the TPH antibody, to Dr. Erik Hewlett for the gift of pertussis toxin, and to Wyeth-Ayerst for (+)WAY 100135.

  This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS-33583) and The Council for Tobacco Research.

    FOOTNOTES

  Address reprint requests to D. A. Bayliss.

  Received 5 August 1996; accepted in final form 12 November 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society