2002 Nature Publishing Group http://www.nature.

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The sedative component of

anesthesia is mediated by GABAA
receptors in an endogenous sleep
pathway
L. E. Nelson1,2, T. Z. Guo1, J. Lu3, C. B. Saper3, N. P. Franks1,2 and M. Maze1,2
1Department of Anaesthetics & Intensive Care, Chelsea & Westminster Hospital, Imperial College School of Medicine, London SW10 9NH, UK
2Biophysics Section, Blackett Laboratory, Department of Biological Sciences, Imperial College of Science, Technology & Medicine,

London SW7 2BW, UK

3Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA

Correspondence should be addressed to N.P.F. (n.franks@ic.ac.uk)

Published online: 26 August 2002, doi:10.1038/nn913

We investigated the role of regionally discrete GABA (-aminobutyric acid) receptors in the sedative
response to pharmacological agents that act on GABAA receptors (muscimol, propofol and pentobarbital; GABAergic agents) and to ketamine, a general anesthetic that does not affect GABAA
receptors. Behavioral studies in rats showed that the sedative response to centrally administered
GABAergic agents was attenuated by the GABAA receptor antagonist gabazine (systemically administered). The sedative response to ketamine, by contrast, was unaffected by gabazine. Using c-Fos as a
marker of neuronal activation, we identified a possible role for the tuberomammillary nucleus (TMN):
when gabazine was microinjected directly into the TMN, it attenuated the sedative response to
GABAergic agents. Furthermore, the GABAA receptor agonist muscimol produced a dose-dependent
sedation when it was administered into the TMN. We conclude that the TMN is a discrete neural locus
that has a key role in the sedative response to GABAergic anesthetics.

General anesthesia is comprised of behavioral elements, including

sedation or hypnosis, amnesia, analgesia and muscle relaxation.
As the sedative and the analgesic properties of the same agent may
be modulated at independent sites1, it is imperative that each component be explored separately. We investigated the neurological
mechanisms underlying the sedative, or sleep-inducing, actions of
three general anesthetics that are thought to act mainly on GABAA
receptors (GABAergic agents).
To date, most of the research on the actions of GABAergic
general anesthetics has been directed at the molecular level2,3.
Clinically relevant concentrations of anesthetics (such as barbiturates, etomidate, propofol, neuroactive steroids and volatile
anesthetics) markedly enhance the chloride current that is mediated by GABA A receptors in neurons and in recombinant
expression systems46. The intravenous anesthetic propofol
(PRO) and the barbiturate anesthetic pentobarbital (PTB) both
enhance GABAA receptor function or GABAergic neurotransmission in a variety of systems711.
The connection between the activation of GABAA receptors
and the sedative response is still, however, obscure. Agonists with
reputed selectivity for the GABAA receptor, such as THIP (4,5,6,7tetrahydroisoxazolo[5,4,-c]-pyridin-3-ol), produce the loss-ofrighting reflex (LORR; see Methods) in rats12, but direct evidence
linking this action to GABAA receptors is lacking. Moreover, it is
not known at which anatomical sites the GABAA receptors that
nature neuroscience volume 5 no 10 october 2002

mediate the sedative response are located. According to some brain

imaging studies, PRO depresses metabolism throughout the brain
and, most prominently, in the cortex13. Other studies, however,
emphasize the importance of certain sub-cortical14,15 as well as
cortical14 structures, which supports the contention that specific
neuronal networks are particularly affected.
Here we address the question of whether the sedative actions of
general anesthetics involve some of the neuronal pathways thought
to be involved in sleep. Distinct neuronal pathways in at least three
discrete regions of the brain are involved in non-rapid eye movement (NREM) sleep. It has been suggested that sleep-promoting16,
GABA-containing neurons in the ventrolateral preoptic nucleus
(VLPO) are under tonic inhibition from noradrenergic neurons in
the locus coeruleus (LC) of the pons. According to this proposed
pathway, inhibition of LC neurons would result in activation of
VLPO neurons and ultimately induce sleep. These GABAergic
VLPO neurons, in turn, innervate the ipsilateral TMN17, a posterior hypothalamic cell group thought to be important in promoting arousal1821. These arousal-promoting, histaminergic TMN
neurons are wake-active17,19,22 and are inhibited by the release of
GABA and galanin by VLPO neurons. It is thought that this TMN
inhibition is pivotal in causing sleep2325 (Fig. 1).
Here we examined the role of discrete GABAergic pathways in
the sedation induced by agents putatively acting on GABAA receptors. The results of these experiments lead us to propose that the
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Fig. 1. Simplified NREM sleep-promoting

pathway. An inhibition of noradrenergic neurons in the LC, which accompanies endogenous NREM sleep22,33, releases a tonic
noradrenergic inhibition of the VLPO35. The
activated VLPO16 is believed to release GABA
into the TMN17,2325, which inhibits its release
of arousal-promoting histamine into the cortex, and thus induces loss of consciousness18,45. A number of pathways are involved
in NREM sleep; the sleep-active VLPO16 projects to all the ascending monoaminergic,
cholinergic and orexinergic arousal nuclei
(TMN, LC, DR, PPTg, LDTg, PeF)17,25, which
project to the cortex where they release
arousal-promoting neurotransmitters to promote wakefulness16. We focused on the TMN
as representative of the arousal centers inhibited by the VLPO during sleep. The LC widely
innervates the brain, but only projections
associated with NREM sleep are shown here.
A simplified version of this circuitrythe portion of the pathway highlighted in redwas the focus of our research. ACh, acetylcholine; GABA, -aminobutyric acid; DR, dorsal raphe nuclei; His, histamine; 5-HT, serotonin; LC, locus coeruleus; LDTg, laterodorsal tegmental nuclei; NE, norepinephrine; NREM, non-rapid eye movement; OX, orexin
(hypocretin); PeF, perifornical area; PPTg, pedunculopontine tegmental nuclei; TMN, tuberomammillary nucleus; VLPO, ventrolateral preoptic nucleus.

actions of GABAergic anesthetics on the GABAA receptors in the

TMN are centrally involved in inducing the sedative or hypnotic
component of anesthesia.

RESULTS
Gabazine attenuates GABAergic-induced sedation
Using behavioral measures, we first tested whether systemic
administration of the GABAA receptor antagonist gabazine (GBZ)
could attenuate the sedative action of various centrally administered anesthetics. We used the LORR as our primary measure for
sedation because it reflects one important facet of the sedative
state in humansimmobility. Moreover, the concentrations of
anesthetics that are necessary to produce loss of consciousness
in humans are similar to those needed to induce LORR in animals4. As expected, subcutaneous (SC) administration of GBZ
(5 mg/kg) decreased the percentage of animals exhibiting LORR
in response to muscimol (MUS; 0.52.5 g/10 l, ICV; Fig. 2a).
MUS alone had a median effective dose (ED50) of 1.23 0.12 g
(mean s.e.m.); MUS in the presence of GBZ showed a significantly larger ED50 of 2.04 0.06 g (P < 0.05).
GBZ pretreatment similarly resulted in a large rightward shift
of the LORR dose-response curve to the intravenous anesthetic
PRO (0.559.9 mg/20 l, ICV). The ED50 of PRO alone was
2.1 0.3 mg, (mean s.e.m.) and the ED50 of PRO in the presence of GBZ was 7.2 0.42 mg (P < 0.05, Fig. 2b). Sedation
induced by the barbiturate PTB (0.752.20 mg/10 l, ICV) was
significantly, but less markedly, attenuated by similar GBZ pretreatment (Fig. 2c). The ED50 of PTB alone was 1.31 0.08 mg
(mean s.e.m.) and the ED50 of PTB in the presence of GBZ was
1.57 0.08 mg (P < 0.05).
Another class of anesthetics, exemplified by ketamine
(KET) 26 , nitrous oxide 27 and xenon 28 , has little effect on
GABAA receptors, but reduces excitatory neurotransmission
by inhibiting the NMDA subtype of glutamate receptors. Systemic GBZ (5 mg/kg, SC) did not affect the sedative response to
KET (0.751.75 mg/10 l, ICV; Fig. 2d); the ED50 (1.15 0.07 mg)
was identical with or without GBZ. This finding shows that
systemic GBZ does not act non-specifically to attenuate
980

responses to all anesthetic agents (for example, by causing a

generalized increase in neuronal excitability).
These data indicate that MUS, PRO and PTB are less effective as sedatives in the presence of a GABAA receptor antagonist
and are probably exerting their sedative effects, at least in part,
through the GABAA receptor. Notably, GBZ was even more effective at attenuating the LORR induced by PRO than it was at attenuating the LORR induced by the selective GABAA receptor agonist
MUS. The effect of GBZ on the LORR induced by PTB, however,
was considerably smaller than that for both MUS and PRO. These
differences can most simply be accounted for by the fact that the
potencies of these agents for inhibiting or activating GABAA
receptors depend on the receptor subunit composition29,30. Each
agent acts on a somewhat different, but overlapping, population
of GABAA receptors, so the degree of the antagonism by GBZ is
likely to vary between anesthetics. In addition, it is reasonable to
suppose that PTB may have other targets in addition to the
GABAA receptor that mediate its sedative effects (for example,
PTB depresses glutamate-induced excitation31).
Changes in c-Fos expression during sedation
Next, we used immunohistochemistry to assess whether GABAergic agents act on a pathway that is known to mediate NREM sleep
(Fig. 1). Activation of the cellular immediate-early gene c-fos, and
subsequent accumulation of expressed c-Fos protein, is often
used as evidence of neuronal activity32. Although the link between
c-Fos expression and neuronal activation is indirect, c-Fos levels have been shown to change in different parts of the brain during spontaneous sleepwake episodes22,33,34, and these changes
are taken as surrogate markers for neuronal activity. During the
sedation induced by the systemic administration of the GABAergic agents tested (MUS, PRO and PTB), similar patterns of
changes in c-Fos expression were evident in the NREM sleeppromoting pathway. Intraperitoneal (IP) administration of MUS
(10 mg/kg), PRO (140 mg/kg) and PTB (100 mg/kg) increased
c-Fos expression in the VLPO, and decreased c-Fos expression
in the TMN, as compared to saline control (Fig. 3ac). Interestingly, none of the GABAergic agents (MUS, PRO or PTB) directnature neuroscience volume 5 no 10 october 2002

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ly affected neuronal activity in the LC, unlike during NREM sleep,

when LC activity is significantly decreased22,33,34.
We interpret these data to indicate that GABAergic agents
activate VLPO neurons, which then release GABA into the TMN,
thus inhibiting the activity of this arousal-producing nucleus.
GABAergic agents thus affect the NREM sleep pathway in two
distinct ways. The first is by acting on some unidentified target
upstream from the VLPO, where anesthetic action results in excitation of the VLPO. This might, for example, involve GABAA
receptors in a pathway that normally inhibits VLPO firing, and
whose enhancement would result in disinhibition of VLPO.
VLPO is densely innervated by monoaminergic arousalpromoting centers in the brain: histaminergic neurons in the
TMN, noradrenergic neurons in the LC, ventrolateral medulla
and nucleus of the solitary tract, and serotoninergic neurons in
the dorsal, median and central linear raphe35. TMN neurons also
contain GABA and galanin, which could influence VLPO activity36 (histamine does not influence VLPO firing rates37). Although
acetylcholine strongly inhibits VLPO neurons in vitro37, VLPO
does not receive significant cholinergic afferent projections35.
The second mechanism is likely to be by enhancing the inhibitory effect of endogenous GABA that is released into the VLPO.
Either way, it follows that if GABAA receptors in the TMN
are directly involved in the sedative response to GABAergic anesthetics, then microinjecting a GABAA receptor agonist directly
into the TMN should cause sedation.
Administration of MUS into the TMN induces sedation
Discrete bilateral injection of MUS into the TMN (24 g/
0.2 l/side, for 48 g total bilateral injection) produced a dosedependent sedative response (Fig. 4a). In contrast, bilateral

Fig. 2. Systemic gabazine attenuates sedation induced by

GABAergic agents but not by NMDA receptormediated
agents. Pretreatment with the systemically administered
GABAA receptor antagonist gabazine (GBZ; 5 mg/kg, SC)
induced a rightward shift in the percentage of animals
exhibiting LORR to centrally administered (a) muscimol
(MUS; 0.52.5 g/10 l, ICV), (b) propofol (PRO;
0.559.9 mg/20 l, ICV) and (c) pentobarbital (PTB;
0.752.2 mg/10 l), indicating that the sedative effects
induced by these compounds are mediated by GABAA
receptors. In contrast, systemically administered GBZ
(5 mg/kg, SC) did not attenuate the loss-of-righting
response (LORR) to (d) centrally administered NMDA
receptor antagonist ketamine (KET; 0.751.75 mg/10 l,
ICV). Note that in (d), KET and KET + GBZ curves are
superimposed. Minimum cohort size is six.

injections of the GABA A receptor agonist MUS

(dose range from 50 ng40 g/0.2 l/side) into the
LC (n = 6 animals per dose), 3 mm lateral to the
TMN (in the amygdalohippocampal area; n = 4),
or 2 mm dorsal to the TMN (in the dorsal region
of the posterior hypothalamus; n = 4) did not
induce loss of consciousness. These findings suggest that, while GABAA receptors are distributed throughout
the brain, GABAA agonists produce a sedative response in a
regionally specific manner. The lack of effect of MUS in the LC
is consistent with our observation (see above) of the lack of any
change in c-Fos expression in the LC during sedation. This
would appear to rule out the LC as a critical element in the sedative response induced by GABAergic agents (in marked contrast

Fig. 3. c-Fos expression induced by GABAergic agents. Effects of systemically administered (a) muscimol (MUS; 10 mg/kg, IP), (b) propofol
(PRO; 140 mg/kg, IP) and (c) pentobarbital (PTB; 100 mg/kg, IP) on c-Fos
expression (as a marker of neuronal activation) in the locus coeruleus
(LC), ventrolateral preoptic nucleus (VLPO) and tuberomammillary
nucleus (TMN). Minimum cohort size is six; results shown as mean
s.e.m., *P < 0.05 compared to normal saline (NS) control.
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direct effects at the TMN, there may need to be concomitant

release of GABA from the VLPO into the TMN.
Administration of GBZ into the TMN attenuates sedation
As a corollary to the experiment in which MUS microinjected
into the TMN induced LORR, one would predict that a GABAA
receptor antagonist similarly discretely injected into the TMN
would attenuate the sedation induced by systemically administered GABAergic agents. The GABAA receptor antagonist GBZ
was discretely injected bilaterally into the TMN (0.2 g/0.2 l/
side for 0.4 g total bilateral injection), followed immediately
by systemically administered GABAergic anesthetic. The duration of LORR induced in response to systemically administered
PRO (100 mg/kg; Fig. 4b) and PTB (50 mg/kg; Fig. 4c) was
decreased by GBZ microinjections into the TMN, although, as
with its effects on LORR (Fig. 3), GBZ was more effective
against PRO than PTB. This further supports the hypothesis
that during the sedation caused by GABAergic anesthetic
agents, GABAA receptors on histaminergic neurons in the TMN
are causally involved.

DISCUSSION

Fig. 4. Effects of discrete injections of GABAA receptor agonist and

antagonist into the TMN. (a) The sedative effects of discrete bilateral
injection of GABAA receptor agonist muscimol (MUS; 4 g, 8 g) into
the TMN. Discrete bilateral injection of the GABAA receptor antagonist
gabazine (GBZ, 0.2 g/0.2 l/side) into the TMN decreased the duration
of loss of righting response (LORR) induced by systemically administered
(b) propofol (PRO; 100 mg/kg, IP) and (c) pentobarbital (PTB; 50 mg/kg,
SC). These results indicate that PRO and PTB are less effective as sedatives when a GABAA receptor antagonist is injected discretely into the
posterior hypothalamus. Minimum cohort size is six, results shown as
mean s.e.m., *P < 0.05 compared to normal saline (NS) control.

to the situation for 2 adrenoceptor agonists38, where inhibition of the LC is pivotal).

Whereas direct injection of MUS into the TMN induced
LORR, other GABAergic anesthetic agents (PTB and PRO) only
caused marked sedation. We determined this quantitatively by
recording activity with a video camera (Methods). We found
that the bilateral injection of PTB (160 g/0.2 l/side) or PRO
(60 g/0.2 l/side) into the TMN reduced activity compared to
control (saline or intralipid injection, respectively) by
67.5 13.6% in the case of PTB (mean s.e.m., n = 8 animals,
P < 0.05) and by 43.2 17.2% for PRO (n = 6, P < 0.05). The
inability of PTB or PRO discretely administered into the TMN
to induce LORR is perhaps not surprising because general anesthetics are far more effective at enhancing the actions of GABA
at GABAA receptors than they are at directly activating the receptors in the absence of GABA. For example, PRO is approximately
ten times more potent in enhancing GABAA receptors than it is
as a direct activator5,39. Therefore, for PRO and PTB to exert
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The aims of this study were threefold: (i) to determine the role
of regionally discrete GABAA receptor-mediated pathways in
the sedative response to general anesthetics, (ii) to identify the
discrete sites at which GABAA receptors mediate this response
and (iii) to determine whether GABAA receptors in the TMN
are causally involved in the sedative component of anesthetic
action. Four test compounds were chosen to represent a wide
spectrum of sedatives. They were MUS (a pharmacologically
selective GABAA receptor agonist), PRO and PTB (two intravenous anesthetics that putatively modulate GABA A receptors911), and finally KET, which has little or no direct effect
on GABA receptors and is thought to act mainly through the
NMDA receptor26.
We first conducted systemic behavioral studies to demonstrate the involvement of the GABAA receptor in the transduction of the sedative response. Second, through immunohistochemical studies of c-Fos expression (as a marker for neuronal activation), we established the TMN as a possible site at
which GABAA receptors could transduce the sedative response.
Next, we showed that the GABAA receptor agonist MUS, administered directly into the TMN, produced a dose-dependent sedation. Notably, injection of MUS into other sites (such as the LC
and hypothalamic sites near the TMN) had no effect, indicating the anatomically discrete nature of the response. Finally, we
microinjected the GABAA receptor antagonist GBZ into the
TMN, which attenuated the sedation induced by systemically
administered GABAergic anesthetic agents.
We interpret these findings to suggest several key mechanistic
features of sedation induced by GABAergic anesthetic agents.
First, the sedative response to PRO and PTB involves the activation of GABAA receptors. Second, sedation is not due to a generalized inhibition throughout the CNS, but rather involves
actions on specific neuronal pathways, particularly those in the
hypothalamus that underlie NREM sleep. Third, GABAA receptors in the TMN are of key importance in one pathway mediating
sedation induced by GABAergic anesthetic agents.
The implications of these findings may be far-reaching.
Most research into the involvement of the GABAA receptor in
general anesthesia has thus far been focused at the membrane
level. The elucidation of the mechanisms of anesthesia at the
level of neuronal networks could prove invaluable in placing
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this molecular description in a broader context. While it seems

likely that specific GABAergic pathways in several regions of
the brain, in addition to the TMN, may converge and contribute to the induction of sedation and general anesthesia, the
results described here identify the TMN and VLPO as key elements in one of these critical pathways.

METHODS
Animals. Adult male Fischer rats (250300 g; n = 166) purchased from
B&K Universal (Grimston Aldbrough Hull, UK) were given access to
food and water ad libitum and housed under controlled conditions (12
hours of light starting at 7 p.m.; 2022C) in an isolated ventilated chamber. All animal procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. All efforts were
made to minimize animal suffering and the number of animals used.
Drug preparation. Muscimol hydrobromide (MUS; Sigma-Aldrich,
Poole, Dorset, UK), ketamine hydrochloride (KET; Ketalar, Parke-Davis,
Hampshire, UK), pentobarbital sodium salt (PTB; Sigma-Aldrich) and
gabazine hydrobromide (GBZ; SR-95531, Tocris, Bristol, UK) were prepared in normal saline solution (NS). Propofol (PRO; Tocris, Bristol, UK
and Sigma-Aldrich) was dissolved in 20% intralipid purified soybean oil
solution (Intralipid, Fresenius Kabi, Dublin, Ireland). PTB (J.M.
Loveridge, Southampton, UK) was also used as 20% solution (w/v) in
NS. Halothane (Fluothane, Zeneca, Cheshire, UK) was administered via
an anesthesia machine.
Cannulae implantation. Animals were anesthetized using halothane
(35%) in a plexiglass chamber, prepared for aseptic surgery and secured
into a stereotaxis frame. Cannulae (22G, 15 mm length for intracerebroventricular (ICV) cannulation, 20 mm length for TMN cannulation;
Tomlinson Tubes, Bidford-on-Avon, UK) were positioned for injection
into either the ICV space or TMN (ICV coordinates, 1.0 mm mediolateral, 5.2 anteroposterior, 9.1 dorsoventral from Bregma; TMN coordinates, 1.0 mm mediolateral, 1.0 mm anteroposterior, 4.0 mm
dorsoventral40), and affixed with dental resin (Orthoresin, Dentsply, Surrey, UK) and animals were allowed to recover for at least three days.
Discrete drug administration. Drugs were discretely administered into
either the ICV space or TMN at least four days after implantation of the
cannulae, during the day. The GABAA receptor agonist MUS (24 g/0.2
l/side) or the GABAA receptor antagonist GBZ (0.2 g/0.2 l/side) were
microinjected directly into the TMN using a CMA microinjection pump
(CMA/100, CMA/Microdialysis) at a rate of 0.4 l/min over 30 s. The
needle was removed 30 s after completion of the injection. PTB (0.752.0
mg/10 l) or PRO (0.559.9 mg/20 l) was administered unilaterally into
the ICV space at a rate of 5 l/min.
Assessment of sedation/hypnosis. Our primary endpoint for sedation
was the loss-of-righting reflex (LORR). LORR was defined as the inability of animals to right themselves when positioned in a supine position.
In experiments measuring sleep, righting reflex was considered restored
when animals first regained an upright position, standing on their feet.
Doseresponse data were fitted as previously described41 to a logistic
equation of the form
p=

100Dn
Dn + (ED50)n

where p is the percent of the population anesthetized, D is the drug dose, n

is the slope parameter and ED50 is the drug dose for a half-maximal effect.
It was necessary in some experiments to quantify the activity of animals
that were sedated significantly, but not deeply enough to exhibit LORR.
Activity was measured at night (under red light) using a video camera
with night vision (Sony Digital Handycam, DCR-PC100E, Japan) filming
from below through a plexiglass sheet (30 cm 50 cm area) marked with
a 10 10 cm grid. Activity was quantified by counting grid crossings over
a 5-min epoch.
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Perfusion and tissue sectioning. Animals were administered MUS (10

mg/kg, IP), PRO (140 mg/kg, IP) or PEN (100 mg/kg, IP), or saline at
approximately 8 p.m. (the early part of rodent waking cycle, to ensure
that control animals were awake). Two hours later, they were transcardially perfused with phosphate saline buffer (PBS; 100 ml; 0.1 M
phosphate buffer, 0.9% NaCl; pH 7.4) followed by 4% paraformaldehyde (500 ml; BDH) in 0.1 M phosphate buffer. Whole brains were
removed, post-fixed in 10% formalin or 4% paraformaldehyde
overnight (until tissue sank), incubated in 20% sucrose overnight, and
coronally cryosectioned (1:4 series, 30 m).
Immunohistochemistry. Sections were double-immunostained using
previously described methods16,17,42. All sections were stained for c-Fos
(goat polyclonal antibody; 1:20,000; Santa Cruz, Insight Biotechnology,
Wembley, UK or 1:150,000; rabbit; Oncogene, CN Biosciences UK, Nottingham, UK) using secondary donkey anti-goat IgG (1:200; Chemicon,
Harrow, UK), VectaStain Elite ABC solution (Vector, Peterborough, UK)
visualized using 3,3-diaminobenzidine (DAB) with nickel (Vector). Next
they were separated into those containing the LC, VLPO and TMN, and
counter-stained for dopamine -hydroxylase (DBH; rabbit polyclonal
antibody; 1:20,000; Affiniti Research Products, Exeter, UK), galanin (rabbit polyclonal antibody; 1:50,000; Bachem UK, St. Helens, UK) and
adenosine deaminase (ADA; rabbit polyclonal antibody; 1:20,000, Chemicon), respectively, using donkey anti-rabbit IgG (1:200; Chemicon), and
visualized using DAB without nickel. Galanin staining was enhanced
using tyramide amplification (TSA Biotin System; Perkin-Elmer Life Sciences, Hounslow, UK) according to manufacturers protocol. In some
instances, VLPO sections were first stained for c-Fos protein using
immunohistochemistry (DAB brown stain) and subsequently for galanin
mRNA using in situ hybridization methodology previously described43,44
to further verify the anatomical location of the VLPO.
Cell counting. Using light microscopy, c-Fos positive neurons were identified by dense black nuclear staining and the DBH-, galanin-, and ADApositive neurons in the LC, VLPO, and TMN by brown cytosolic staining,
confirmed by reference to a rat brain atlas40. Four sections per animal
were counted (blind to treatment) and averaged. Data were analyzed
using one-way ANOVA and the Bonferonni test, and are presented as
means standard errors of the means (s.e.m.). Differences were considered significant at P < 0.05.
Acknowledgments
The Medical Research Council (UK; G9817980 and G9100635), the
Westminster Medical Trust (UK) and the National Institutes of Health (USA;
HL60292 and AG09975) supported this work.

Competing interests statement

The authors declare that they have no competing financial interests.

RECEIVED 2 JULY; ACCEPTED 2 AUGUST 2002

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nature neuroscience volume 5 no 10 october 2002