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The Journal of Neuroscience, October 15, 2002, 22(20):8850-8859
Convergent Excitation of Dorsal Raphe Serotonin Neurons by
Multiple Arousal Systems (Orexin/Hypocretin, Histamine and
Noradrenaline)
Ritchie E.
Brown,
Olga A.
Sergeeva,
Krister S.
Eriksson, and
Helmut L.
Haas
Institut für Neurophysiologie,
Heinrich-Heine-Universität, D-40001 Düsseldorf, Germany
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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 
1
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).
Key words:
arousal; narcolepsy; tuberomammillary; voltage clamp; orexin; dorsal raphe
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INTRODUCTION |
The activity of the brain during
waking is maintained by the concerted action of a number of
neuromodulators. These substances are released from diffuse activating
systems located in the brain stem (serotonin and noradrenaline),
hypothalamus (histamine and orexins), and basal forebrain
(acetylcholine) (McCormick, 1992
; Kilduff and Peyron, 2000; Lin, 2000).
One of the important neuromodulators involved in the control of waking
physiology is the monoamine, serotonin (5-HT). Serotonin cooperates
with acetylcholine and other amines during waking to cause
desynchronization of the cortex (Dringenberg and Vanderwolf, 1998).
Behavioral experiments and clinical findings have shown that an
adequate level of serotonergic tone is essential for normal brain
function. Hypofunction of this system leads to a loss of "behavioral
control" and psychiatric problems such as excessive aggressiveness,
eating disorders, and suicide (Lucki, 1998).
The serotonergic innervation of the forebrain originates primarily in
the dorsal raphe nucleus of the brainstem (Jacobs and Azmitia, 1992).
Like noradrenaline neurons in the locus coeruleus (Aston-Jones and
Bloom, 1981) and histamine neurons in the tuberomammillary nucleus
(Sakai et al., 1990), most of the serotonin neurons in the dorsal raphe
fire tonically at a slow rate during waking, fire considerably less
during slow-wave sleep, and cease firing during rapid eye movement
(REM) sleep (Trulson and Jacobs, 1979; Jacobs and Fornal, 1991; Sakai
and Crochet, 2001). In contrast to the in vivo situation,
most serotonin neurons do not fire spontaneously in vitro
(Vandermaelen and Aghajanian, 1983). Much evidence supports a role for
the noradrenergic system in maintaining tonic firing of serotonin
neurons. Systemic or local administration of
1-adrenoceptor antagonists reduces 5-HT neuron
firing in vivo (Baraban and Aghajanian, 1980), whereas bath
application of the 1-adrenoceptor agonist, phenylephrine, restores tonic firing in vitro (Vandermaelen
and Aghajanian, 1983).
In comparison with noradrenaline, little is known about how other
arousal systems affect the firing of serotonin neurons. Recently we
have shown that orexin A strongly excites dorsal raphe serotonin
neurons (Brown et al., 2001a). Orexin neurons are located in the
lateral hypothalamus and project particularly strongly to
arousal-related systems such as the dorsal raphe (Chemelli et al.,
1999; Date et al., 1999). C-fos expression in orexin neurons and
preproorexin mRNA levels show circadian variations, with the strongest
expression being observed during waking (Taheri et al., 2000;
Estabrooke et al., 2001), whereas intracerebroventricular application
of orexin A potently enhances arousal (Piper et al., 2000). On the
other hand, destruction of orexin neurons or disruption of the orexin
type II receptor causes the sleep disorder narcolepsy, the symptoms of
which include excessive daytime sleepiness and disrupted nighttime
sleep (Lin et al., 1999; Thannickal et al., 2000).
While investigating the effects of orexins on dorsal raphe neurons, it
became apparent that there were considerable similarities between the
effects of orexins and 1 receptor agonists on
5-HT neurons. Here we show that the orexin system, the noradrenaline system, and a third arousal-related system, the histamine system (Lin,
2000; Brown et al., 2001b), converge on common ionic mechanisms to
excite dorsal raphe serotonin neurons.
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MATERIALS AND METHODS |
Preparation and maintenance of slices. Brain slices
were prepared from 2- to 4-week-old male Wistar rats. All experiments were conducted in compliance with German law and with the approval of
the Bezirksregierung Düsseldorf. The animals were quickly decapitated, and the brains were transferred to a modified artificial CSF (ACSF) containing (in mM): sucrose 209, KCl
1.8, KH2PO4 1.2, MgSO4 1.3, CaCl2 2, NaHCO3 25.6, D-glucose 10 (pH 7.4 when bubbled with 95% O2/5%
CO2, 300 mOsm) (Aghajanian and Rasmussen, 1989). Three 400-µm-thick coronal brain slices were cut from
anterior-posterior level 8.1 to 9.3 with respect to bregma,
according to the atlas of Kruger et al. (1995), using a Vibratome (TPI,
St. Louis, MO). Subsequently, the ventral portions of the slices were
removed, and they were transferred to a holding chamber filled with
standard ACSF containing (in mM): NaCl 124, KCl
1.8, KH2PO4 1.2, MgSO4 1.3, CaCl2 2, NaHCO3 25.6, D-glucose 10 (pH 7.4 when bubbled with 95% O2/5%
CO2, 295 mOsm). Slices remained in the holding
chamber at room temperature until use, when they were transferred
individually to a recording chamber (submerged-type) and perfused with
ACSF at a flow rate of 3 ml/min. Recordings were made at 31°C.
Electrophysiological recordings. Extracellular single-unit
recordings were made from serotonergic neurons using glass pipettes filled with ACSF (12-20 M
). Signals were recorded using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), filtered at 0.1-10
kHz, sampled at 20 kHz, and analyzed with pClamp8 software (Axon
Instruments). The frequency of extracellular action potentials was
determined on-line in bins of 15 sec duration. The
1 adrenoceptor agonist, phenylephrine (3 µM), was added to the perfusion solution to
enhance the number of spontaneously active serotonin neurons (Vandermaelen and Aghajanian, 1983). Under these conditions cells were
considered to be serotonergic if (1) their firing rate was below 3.5 Hz, (2) the waveform of extracellularly recorded action potentials
exhibited a large initial positivity followed by a smaller multiphasic
tail with a total waveform duration >3 msec, (3) their firing was
reduced by >50% by application of the 5-HT1A agonist 8-OH-DPAT (100 nM), removal of
phenylephrine from the bath, or application of the
1 adrenoceptor antagonist, prasozin (1 µM). Non-serotonergic neurons were encountered
only infrequently with phenylephrine in the bath. They fired at faster
rates (>5 Hz), had biphasic action potentials of shorter duration,
were unaffected by application of 8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT), and were only weakly affected by removal of
phenylephrine from the bath.
Intracellular recordings were made from neurons in the nucleus dorsalis
raphe using the "blind" whole-cell patch-clamp technique (Staley et
al., 1992). Patch pipettes (3-6 M) were pulled from borosilicate
glass (GB150F-8P, Science Products, Hofheim, Germany) and filled with
an intracellular solution containing (in mM): potassium
gluconate 135, NaCl 5, MgCl2 2, HEPES 10, EGTA
0.1, Na2ATP 2, NaGTP 0.5 (pH 7.25 with KOH, 275 mOsm). Intracellular signals were recorded using an Axoclamp 2B
amplifier (Axon Instruments) in bridge mode or in the continuous,
single-electrode, voltage-clamp mode. Serotonergic neurons were
identified by their characteristic electrophysiological properties as
described previously (Brown et al., 2001a). Membrane potential
measurements were adjusted for a 15 mV liquid junction potential
between pipette solution and bath solution (calculated using pClamp8
software; Axon Instruments). Series resistance varied from 6 to 40 M; the bridge balance was maintained continuously and monitored
during current-clamp experiments. Long, hyperpolarizing steps (25 or
50 pA, 500 msec) were applied every 30 sec to monitor the input
resistance. Recordings were low-pass filtered at 10 kHz.
Voltage-clamp recordings were made only from neurons having a stable
series resistance of <20 M. Series resistance compensation was not
used. Tetrodotoxin (TTX; 0.5 µM),
6-cyano-7-nitroquinoxaline-2,3 dione (CNQX; 5 µM),
D-2-aminophosphonopentanoic acid (AP5; 50 µM), and bicuculline methiodide (10 µM)
were added to the bathing solution to block voltage-gated sodium
channels and spontaneous synaptic potentials mediated by glutamate AMPA
and NMDA receptors and GABAA receptors,
respectively. Long (4 sec) voltage ramps from 75 to 130 mV were
applied every 30 sec. Current-responses were sampled at 2 kHz and
low-pass filtered at 1 kHz. In 0 Ca2+/3.3
Mg2+-containing solution, longer (20 sec)
ramps from 100 to +20 mV were applied every 40 sec and sampled at 200 Hz. Continuous recordings of membrane voltage or current were made
using a Gould TA550 chart recorder (Gould Electronics, Cleveland, OH).
Cellular RNA harvest and reverse transcriptase-PCR. For
preparation of isolated cells, the nucleus dorsalis raphe was dissected from the slice and incubated with papain (Sigma, Deisenhofen, Germany)
in crude form (0.3-0.5 mg/ml) for 40 min at 37°C. Subsequently the
tissue was placed in a bathing solution of the following composition (in mM): NaCl 150, KCl 3.7, CaCl2 2.0, MgCl2 2.0, HEPES
10, pH 7.4. Cells were separated by gentle pipetting. Neurons visually selected on an inverted microscope were digitally photographed and
approached with a patch electrode, and a gigaohm seal was obtained. The
thick-walled borosilicate glass electrodes had resistances of 2-5 M
after filling with the following solution containing (in
mM): CsCl 140, MgCl2 2, CaCl2 0.5, EGTA 5, HEPES/CsOH 10 (sterilized by
autoclaving and adjusted to pH 7.2). The cells were voltage clamped by an EPC-9 amplifier (HEKA Electronics, Lambrecht, Germany) at
70 mV. After the whole-cell configuration was established, the
electrode with the patched neuron was lifted into the application system where the cytoplasm was sucked into the electrode. Cell identification was verified by reverse transcriptase (RT)-PCR analysis
of tryptophan hydroxylase (Tph) expression. The protocols of the RT
reaction and PCR amplification were similar to those described
previously (Vorobjev et al., 2000). The primers designed to recognize
Tph and orexin (hypocretin) receptor cDNAs are listed in Table
1. The thin-walled PCR tubes contained a
mixture of first-strand cDNA template (2-5 µl), 10× PCR buffer (5 µl), 10 pM each of sense and antisense primer,
and 200 µM each of deoxyNTP (dNTP) and
2.5 U Taq polymerase. The final reaction volume was adjusted
to 50 µl with nuclease-free water (Promega). The
Mg2+ concentration was 1.5 mM for all reactions. The Taq enzyme,
PCR buffer, Mg2+ solution, and four dNTPs
were all purchased from Qiagen (Erkrath, Germany). All oligonucleotides
were synthesized by MWG-Biotech (Ebersberg, Germany), and amplification
was performed on a thermal cycler (GenAmp 9600, Perkin-Elmer,
Weiterstadt, Germany). A two-round amplification strategy was used in
each protocol. In each round, 35 cycles of the following thermal
programs were used: denaturation at 94°C for 48 sec, annealing at
53°C for 1 min, and extension at 72°C for 90 sec. For the second
amplification round, 1 µl of the product from the first amplification
was used as a template.
The results of amplification were analyzed by agarose gel (2%)
electrophoresis and staining with ethidium bromide. All products of the
second round of amplification were purified (PCR purification kit from
Qiagen) in water and subjected to sequencing in both directions (which
was done on an automatic sequencer; model 377, ABI, Weiterstadt,
Germany). Sequencing of the amplification products revealed their
identity with the known rat cDNA sequences (see GenBank access numbers
in Table 1).
Drugs and statistics. Bicuculline methiodide, chelerythrine
chloride, CNQX, ethylene glycol-bis (
-aminoethyl ether) EGTA, HEPES,
histamine dihydrochloride, Na2ATP, NaGTP,
NiCl2,
N-methyl-D-glucamine (NMDG),
phenylephrine hydrochloride, potassium gluconate, and pyrilamine
(mepyramine) maleate were obtained from Sigma; 8-OH-DPAT, AP5,
(9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo- 9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720), phorbol 12-myristate 13-acetate,
2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059),
prasozin hydrochloride, and thapsigargin were from Tocris Cookson
(Bristol, UK) and tetrodotoxin citrate was from Alomone Laboratories
(Jerusalem, Israel). Orexin A and B peptides and melanin
concentrating hormone (Bachem) were dissolved in 0.9% saline to a
concentration of 10 µM, and the resulting
solution was stored as frozen aliquots at 20°C. CNQX, KT5720,
PD98059, and thapsigargin were dissolved in dimethylsulfoxide (final
bath concentration <0.1%). Other drugs were dissolved in distilled water and stored as stock solutions (100 or 1000× final
concentrations) at 4°C. Drugs were bath applied.
All values are given as the mean ± SEM. Statistical comparisons
were made using the unpaired or paired Student's t test, as appropriate.
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RESULTS |
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 nM
orexin 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).
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Figure 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.
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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 the I-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-HT1A 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).
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Figure 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 in
A(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. In
A(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.
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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.
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Figure 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 and
OX2). 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].
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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 1 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
1 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 1
adrenoreceptors are coupled to Gq
G-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
1 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 µM
phenylephrine 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 Figure 4 where phenylephrine had a relatively weak
effect.
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Figure 4.
Activation of 1 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 1 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.
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In addition to causing an inward current, noradrenaline modulates the
firing of serotonin neurons by inhibition of the voltage-gated potassium current, IA, 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 1 adrenoceptor agonist,
phenylephrine. A concentration of phenylephrine (3 µM) was chosen that elicits a rate of tonic firing similar to that seen in 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 1 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 1 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
1 adrenoceptors.
Inhibitors of signal transduction do not block the effect of
phenylephrine or orexin A
Activation of receptors coupled to
Gq-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 IP3
causes 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 of Pan 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
1 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 µM
histamine 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.
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Figure 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. 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 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 1 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.
|
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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 1 adrenoceptors, because histamine (50 µM) increased the
spontaneous firing rate in the presence of the
1 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 mM
NMDG 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 µM
histamine (12.7 ± 3.7 pA; n = 6) were
significantly (p < 0.05) reduced in comparison
with the currents recorded in control solution.
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Figure 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 by
N-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 mM
Na+/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.
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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 mM
NiCl2 or 100 µM
CdCl2 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.
| |
DISCUSSION |
We have shown here that three different arousal systems, the
orexin, histamine, and noradrenaline systems, excite dorsal raphe serotonergic neurons. These systems act through postsynaptically located OX1, OX2,
H1, and 1 receptors, respectively.
Previous studies have demonstrated the presence of orexin fibers in the
dorsal raphe nucleus (Peyron et al., 1998; Chemelli et al., 1999). We
found that both orexin A and orexin B excited 5-HT neurons, whereas
MCH, which is located in neurons intermingled with orexin neurons in
the lateral hypothalamic area (Bittencourt et al., 1992), was without
effect. Orexin A has high (nanomolar) affinity for both orexin
receptors, whereas orexin B has high affinity for the type II orexin
receptor (OX2) and only low (micromolar) affinity
for the type I receptor (Sakurai et al., 1998). In our study, the
potency of orexin A and orexin B tested at 100 nM was very
similar in current-clamp and voltage-clamp experiments, and the
EC50 for orexin B in voltage-clamp experiments
(22 nM) was in the range expected for an effect mediated by
OX2 receptors (Sakurai et al., 1998). Occasional
cells did not respond to orexin B but did subsequently respond to
orexin A or the 1 receptor agonist,
phenylephrine. Furthermore, in single-cell PCR experiments we found
that all tryptophan hydroxylase-positive cells had a strong signal for
OX1, whereas the signal for
OX2 was more variable, and in some cells no
signal could be detected. Taken together, these results suggest that
most of the 5-HT cells in the dorsal raphe contain both types of orexin
receptors, whereas some cells contain only OX1
receptors. This conclusion is in accordance with the findings using
in situ hybridization, which demonstrated the presence of
mRNA for both receptor types in the dorsal raphe (Trivedi et al., 1998;
Marcus et al., 2001), and with the presence of the orexin 1 receptor
protein, as shown by immunohistochemistry (Hervieu et al., 2001).
The similarities between the effects of orexins and phenylephrine on
dorsal raphe neurons (see below), together with the fact that orexin
receptors, like 1 adrenoceptors, are coupled
to phospholipase C (Sakurai et al., 1998), led us to perform occlusion
experiments and also to test a further arousal-related system, the
histamine system, the H1 receptors of which are
also coupled to this signal transduction cascade (Hill et al., 1997).
The presence of H1 receptors in the dorsal raphe
has been demonstrated previously in the guinea pig (Bouthenet et al.,
1988), and here we found in extracellular recordings that the effect of
histamine on serotonin neurons was blocked by the
H1 receptor antagonist, mepyramine.
Several lines of evidence suggest that the three systems act on common
effector mechanisms. First, orexin A, phenylephrine, and histamine all
induced an inward current in voltage-clamp recordings that was
associated with a prominent increase in current noise. Second, the
current-voltage relationships for all three agonists in the range
130 to 75 mV were similar. Third, the inward current for all three
agonists was significantly reduced in extracellular solution in which
most of the sodium had been replaced by the impermeant ion NMDG.
Finally, the inward current caused by orexin A and the excitatory
effects of orexin A and histamine in extracellular recordings were
occluded by previous activation of 1 receptors.
Both orexin receptors, 1 adrenoceptors, and
histamine H1 receptors all normally couple to
Gq G-proteins and to phospholipase C (Hill
et al., 1997; Sakurai et al., 1998). However, in our experiments, the
inward current induced by orexin A or phenylephrine was not blocked by
inhibitors of the two major phospholipase C signaling pathways: release
of calcium from IP3-sensitive stores and
activation of protein kinase C. Further experiments will be required to
determine whether the coupling between receptors and effectors is
mediated directly, through G-proteins alone, or through so far
unidentified second messengers.
In current-clamp experiments, orexins on average increased the input
resistance measured by application of hyperpolarizing pulses. This
suggested that a blockade of leak potassium conductance was responsible
for the excitation (Brown et al., 2001a). However, closer examination
of the data revealed that in individual experiments, this increase was
extremely variable and not correlated with the amplitude of the
depolarization. In voltage-clamp experiments, voltage ramps did not
cross at the potassium equilibrium potential. Furthermore, the observed
inward current was accompanied by a large increase in current noise,
suggesting that channels were in fact being opened by orexins. These
findings were reminiscent of those found by Pan and colleagues (1994)
in their investigation of 1 adrenoceptor
effects on serotonin neurons. These authors found that the inward
current caused by phenylephrine did not reverse in sharp-electrode
recordings or in whole-cell recordings when the drug was tested shortly
after the whole-cell configuration was obtained. However, in many cells
they found that if they waited 15-20 min after obtaining the
whole-cell configuration, then phenylephrine caused an inward current
that did cross at the potassium equilibrium potential, indicating that
modulation of a potassium channel is a component of the response. In
the remainder of the cells, the I-V curves did
not cross, similar to the results that we present here. In contrast to
Pan and colleagues (1994), in our experiments we discarded neurons that
did not have a stable holding current, and the effects of
phenylephrine, orexin, or histamine were usually tested within 20 min
of obtaining whole-cell access.
Our experiments suggest that activation of a mixed cation channel is an
important component of the action of orexins, histamine, and
noradrenaline. The effects of orexin A, phenylephrine, and histamine
were strongly reduced by replacement of 124 mM of the external sodium by the impermeant ion NMDG. This is similar to results
in the tuberomammillary neurons, where we concluded that a
sodium-calcium exchanger mediates the effect of orexins (Eriksson et
al., 2001); however, here the inward currents were always associated with a prominent increase in current noise that was generated by the
stochastic opening of ion channels. In just over half the recordings in
calcium-free solution, current-voltage plots were found to cross at
23 mV, which is consistent with relative permeabilities for sodium
and potassium of 0.43 and 1, respectively. Furthermore, the reversal
potential was significantly shifted in the hyperpolarizing direction in
calcium-free extracellular solution containing low sodium.
Recently a family of nonselective cation channels, which are activated
by receptors that can couple to phospholipase C and generate
"noisy" currents, have been identified in invertebrates and
mammalian tissues (the transient receptor potential channels) (Harteneck et al., 2000). In preliminary single-cell PCR experiments, we have found that tryptophan hydroxylase-positive neurons from the
dorsal raphe region express several members of this family of channels
(O. Sergeeva, unpublished observations).
In some experiments we were unable to obtain a clear reversal of the
current. This could be explained by a contribution caused by the
blockade of leak potassium channels, as described by Pan and colleagues
(1994). As the holding potential becomes more positive, this
contribution would become larger, whereas the contribution of the
nonselective cation current becomes smaller (or reverses). Alternatively, the failure to observe reversal may have been caused by
the rundown of outward currents.
The convergence of multiple arousal systems on common effector systems
seen here is reminiscent of the convergence of these systems on
thalamic relay cells and cortical pyramidal neurons (McCormick, 1992)
and emphasizes once again the importance of the serotonin system during
waking. A further implication of these findings is that if the effector
mechanisms are fully activated by one of the systems, then the other
systems will be without effect on the firing of serotonin neurons. This
does not appear to be the case, because intracerebroventricular
administration of orexin A leads to a grooming response that can be
blocked by antagonists of 5-HT2C receptors (Duxon
et al., 2001).
The orexin system has aroused considerable interest in the last few
years because of its involvement in the sleep disorder, narcolepsy,
which involves a dysregulation in the timing of REM sleep episodes
(Nishino and Mignot, 1997; Chemelli et al., 1999; Lin et al., 1999;
Thannickal et al., 2000; Hara et al., 2001). In particular, the orexin
type II receptor, which we have shown here to be involved in the
effects of orexins on raphe neurons, is of importance. Canines or mice
lacking the type II receptor are narcoleptic, whereas mice lacking only
the type I receptors are not (Lin et al., 1999; Willie et al., 2001).
Serotonin systems play an important role in suppression of REM sleep,
in particular the rapid eye movements and pontine-geniculate-occipital
spikes that accompany dreaming (Hobson et al., 1975; Lydic et
al., 1987). Lack of orexin modulation of dorsal raphe neurons in
narcolepsy would not be critical if noradrenergic and histaminergic
inputs could compensate, but unfortunately these two systems are also normally excited by orexins (Hagan et al., 1999; Eriksson et al., 2002), and thus their input to serotonin neurons is also likely to be
weakened. The weakened input from these systems to dorsal raphe
serotonin neurons in narcolepsy is likely to be important in several
aspects of the disease, including sleep-onset REM periods and
sleep-associated hallucinations.
| |
FOOTNOTES |
Received Nov. 7, 2001; revised July 15, 2002; accepted July 31, 2002.
Correspondence should be addressed to Dr. Ritchie Brown,
Department of Psychiatry, Harvard Medical School, Brockton Veterans Administration Hospital, Research 151 C, 940 Belmont Street,
Brockton, MA, 02301. E-mail:
Ritchie_Brown{at}hms.harvard.edu.
| |
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ABSTRACT
INTRODUCTION
