Download Leptin Signaling in the Nucleus Tractus Solitarii

Leptin Signaling in the Nucleus Tractus Solitarii Increases
Sympathetic Nerve Activity to the Kidney
Allyn L. Mark, Khristofor Agassandian, Donald A. Morgan, Xuebo Liu,
Martin D. Cassell, Kamal Rahmouni
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Abstract—The hypothalamic arcuate nucleus was initially regarded as the principal site of leptin action, but there is
increasing evidence for functional leptin receptors in extrahypothalamic sites, including the nucleus tractus solitarii
(NTS). We demonstrated previously that arcuate injection of leptin increases sympathetic nerve activity (SNA) to brown
adipose tissue and kidney. In this study, we tested the hypothesis that leptin signaling in the NTS affects sympathetic
neural outflow. Using a stereotaxic device in anesthetized rats, we microinjected leptin (0.25 to 1.00 ␮g) or saline into
the NTS while recording SNA to kidney and brown adipose tissue. Microinjection of leptin into the commissural and
medial subnuclei of the caudal NTS at the level of the area postrema in Sprague-Dawley rats produced a dose-related
increase in renal SNA (⫹112⫾15% with leptin 1 ␮g; n⫽7; P⬍0.001) but did not increase SNA to brown adipose tissue
(⫺15⫾12%; P value not significant). This effect depended on intact functional leptin receptors, because it was not
observed in Zucker obese rats that have a missense mutation in the leptin receptor. Rostral NTS injection of leptin failed
to increase SNA, indicating that leptin signaling in the NTS is probably confined to the caudal NTS at the level of the
area postrema. In summary, this study demonstrates that leptin signaling in the caudal NTS increases SNA to the kidney
but not to the brown adipose tissue. The study strengthens the concept of a distributed brain network of leptin action
and demonstrates that these distributed brain sites can mediate contrasting sympathetic responses to leptin.
(Hypertension. 2009;53[part 2]:375-380.)
Key Words: leptin 䡲 nucleus tractus solitarii 䡲 sympathetic nerve activity 䡲 kidney 䡲 brown adipose tissue
L
eptin is an adipocyte-derived hormone that plays a
critical role in the regulation of energy homeostasis. The
hypothalamic arcuate nucleus was initially regarded as the
principal site of leptin action, but there is increasing evidence
for a distributed brain network of leptin action.1
Among these distributed brain sites, there is increasing
evidence for functional leptin receptors in the nucleus tractus
solitarii (NTS), albeit in lower levels than in the arcuate
nucleus. Functional leptin receptor mRNA is expressed in the
NTS of mice and rats.2– 4 Peripheral administration of leptin
increases phosphorylated signal transducers and activators of
transcription 34,5 and c-fos6,7 immunoreactivities in the NTS.
Proopiomelanocortin neurons, which are prominent in mediating leptin action in the arcuate nucleus, also occur in the
NTS.8 –10 There is controversy centering on whether these
NTS proopiomelanocortin neurons do10 or do not1,11 mediate
some of the NTS leptin action, but there is agreement that
there are leptin-responsive neurons in the NTS.1–7,9 –12 For
example, Grill et al3 demonstrated that a low dose of leptin
injected into the dorsal vagal complex including the NTS
decreased food intake and body weight in rats. In addition, a
number of NTS neurons that are activated by gastric disten-
sion are also activated by leptin,12,13 and leptin acts in the
hindbrain to potentiate the satiety response to gastric
distension.12
In addition to effects on appetite and body weight, leptin
increases sympathetic nerve activity (SNA).14 –17 With systemic,15 cerebroventricular,7,16 or intra-arcuate administration
of leptin,17 there are increases in SNA to brown adipose tissue
(BAT), which is involved in thermogenic metabolism, and to
the kidney, which is involved in cardiovascular regulation.
Because the NTS is critically involved in sympathetic regulation and given the emerging evidence for leptin signaling in
the NTS, we tested the hypothesis that leptin receptor
activation in the NTS regulates sympathetic neural activity.
Methods
Animals
Male Sprague-Dawley rats and Zucker obese and lean rats (Harlan
Sprague-Dawley), age 14 to 16 weeks, were studied. Rats were
housed in a temperature-controlled room with a 12:12 hour lightdark cycle. Standard laboratory rat chow and tap water were
provided ad libitum. The studies and procedures were approved by
the University of Iowa Animal Research Committee. All of the
Received October 3, 2008; first decision October 24, 2008; revision accepted November 25, 2008.
From the Departments of Internal Medicine (A.L.M., D.A.M., X.L., K.R.) and Anatomy and Cell Biology (K.A., M.D.C.) and Center on Functional
Genomics of Hypertension (A.L.M., K.A., D.A.M., X.L., M.D.C., K.R.), Carver College of Medicine, University of Iowa, Iowa City.
Correspondence to Allyn L. Mark, 3187 MERF, University of Iowa Carver College of Medicine, Iowa City, IA 52242-1101. E-mail
[email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.124255
375
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February 2009, Part II
studies were conducted in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Experimental Preparation
Rats were anesthetized with IP ketamine (91.0 mg/kg) and xylazine
(9.1 mg/kg). After ketamine and xylazine anesthesia and insertion of
a venous catheter, ␣-chloralose 50 mg/kg was administered intravenously in 0.5 mL over 45 minutes followed by 25 mg/kg per hour in
0.5 mL/h. Arterial pressure and heart rate were monitored with a
catheter inserted into a caudal artery and a pressure transducer
connected to a computer. The trachea was cannulated, and each rat
was allowed to breathe oxygen-enriched air spontaneously. Rectal
temperature was maintained at 37.5°C using a temperaturecontrolled surgical table and lamp. The animal was instrumented for
sympathetic nerve recordings as described below and was then
placed in a stereotaxic frame (David Kopf Instruments) and prepared
for stereotaxic microinjections into the NTS as described below.
Sympathetic Nerve Recordings
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After the induction of anesthesia, we obtained simultaneous multifiber recordings of SNA from a nerve to the kidney and to the BAT.
The left kidney was exposed through a retroperitoneal flank incision.
Using a dissecting microscope, a renal nerve was carefully dissected
free and placed on a bipolar 36-gauge platinum-iridium electrode.
When an optimal recording of renal SNA was obtained, the electrode
was covered with silicone gel (Kwik-Sil, World Precision Instruments, Inc). Interscapular BAT was then exposed through a nape
incision. A nerve fiber innervating BAT was identified, placed on the
bipolar electrode, and secured with silicone gel for recordings of
SNA to BAT.
Each electrode was attached to a high-impedance probe (HIP-511,
Grass Instruments), and the nerve signal was amplified 105 times
with a Grass P5 AC preamplifier. After amplification, the nerve
signal was filtered at a 100- and 1000-Hz cutoff with a nerve traffic
analysis system (Model B600c, University of Iowa Bioengineering).
The nerve signal was then routed to an oscilloscope (Model 54501A,
Hewlett-Packard) for monitoring the quality of the sympathetic nerve
recording and to a resetting voltage integrator (Model B600c,
University of Iowa Bioengineering).
Brain Microinjections
Once the surgery for SNA recordings was complete, the head of the
rat was placed in a stereotaxic frame. The head of the rat was flexed
⬇30° in the stereotaxic device to level the caudal brain stem. For
microinjections into the caudal NTS, after surgical exposure, the
pipette was placed in the brain stem 0.4 to 0.5 mm rostral to the
calamus scriptorius, 0.4 to 0.6 mm lateral to the midline, and 0.5 to
0.7 mm beneath the surface of the brain stem. After injections, the
pipette was left in place for 5 minutes and then removed. For
microinjection into the rostral NTS, the pipette was placed in the
brain stem 0.8 to 1.0 mm rostral to the calamus scriptorius, 0.8 to
1.0 mm lateral to the midline, and 0.5 to 0.7 mm beneath the surface
of the brain stem.
We mixed fluorescent beads (0.2% FluoSphere, 1 ␮m, blue
fluorescent, Invitrogen) with leptin and vehicle for each injection to
assess the accuracy of the injection site. At the end of each
experiment, the animal was perfused with 4.0% paraformaldehyde
and 0.5% glutaraldehyde. The brain was sectioned, and brain
sections were reviewed by an investigator (M.D.C.) who was blinded
to the experimental protocol. Only experiments in which the injection site was verified were included.
Experimental Protocols
After surgical preparation, rats were allowed to stabilize for 15 to 20
minutes. Baseline recordings of sympathetic nerve activities, arterial
pressure, and heart rate were collected twice during a 10-minute
control period and averaged for a single value for the control period.
In the parent protocol, Sprague-Dawley rats received either
vehicle (saline, 50 nL) or recombinant mouse leptin (0.25, 0.50, or
1.00 ␮g in 50 nL saline; R&D Systems, Inc) into the left caudal NTS.
Baseline
5 hrs after leptin (0.5 μg) into NTS
Renal SNA
BAT SNA
15 sec
Figure 1. Segments of record showing neurograms of renal and
BAT SNA at baseline and 5 hours after injection of leptin (0.5
␮g) into the caudal NTS. NTS leptin increased renal but not BAT
SNA.
In 6 experiments, we injected leptin 0.5 ␮g in 50 nL of saline into
both the left and right caudal NTS. In the above and subsequent
protocols, recordings of SNA, arterial pressure, and heart rate were
obtained every 15 minutes for 5 hours after the injections.
To determine whether the responses were leptin receptor mediated, we performed experiments in Zucker obese rats that have a
missense mutation in the leptin receptor18 to determine whether the
effects of leptin are receptor mediated. We injected leptin (0.5 ␮g in
50 nL of saline) into the left caudal NTS of Zucker obese and lean
rats.
To determine whether other regions of the NTS beside the caudal
NTS mediate leptin signaling of SNA, we injected leptin (0.5 ␮g in
50 nL of saline) into the left rostral NTS in Sprague-Dawley rats.
Data Analysis
To ensure that electronic noise was excluded from the measurement
of SNA, each SNA recording was corrected for postmortem background activity. Sympathetic nerve responses are expressed as the
percentage of change from baseline values. Results are presented as
means⫾SEMs. Data were analyzed using unpaired t test or 1- or
2-way ANOVA. Time-course responses were analyzed using
repeated-measures 2-way ANOVA. Fisher least significant difference posthoc test was used for pairwise comparisons, as appropriate.
A value of P⬍0.05 was considered significant.
Results
Responses to Caudal NTS Injections of Leptin
Injection of leptin (0.25 to 1.00 ␮g in 50 nL of saline) into the
caudal NTS of Sprague-Dawley rats resulted in a dose-related
increase in renal SNA (Figures 1 and 2). Bilateral injection of
0.50 ␮g of leptin into the caudal NTS produced an increase in
renal SNA (⫹85⫾13%; n⫽5) that did not differ (P value not
significant) from that seen with injection of 1.00 ␮g of leptin
unilaterally (⫹112⫾15%).
In contrast to the effects on renal SNA, unilateral caudal
NTS injection of leptin did not increase SNA to BAT
(Figures 1 and 2). Bilateral caudal NTS injection of 0.5 ␮g of
leptin also failed to increase SNA to BAT (⫹2⫾13%; n⫽5).
Caudal NTS injection of vehicle (50 nL of saline) did not alter
either renal or BAT SNA (Figure 2A through 2D).
To ascertain that the lack of increase in SNA to BAT was
not caused by a lack of responsiveness of the preparation, we
studied responses to cooling at the end of 12 experiments in
which NTS leptin injection failed to increase SNA to BAT.
Cooling the rats from 37.5° to 30.0°C increased SNA to BAT
by ⫹122⫾41% (P⬍0.05). Cooling increased SNA to BAT in
each of these 12 experiments. These results indicate that the
failure of leptin to increase SNA to BAT cannot be explained
by a lack of responsiveness of the preparation.
Mark et al
Vehicle
Leptin (1 μg)
100
* ** *
C
* *
Δ Renal SNA (%)
Δ Renal SNA (%)
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A
Leptin Signaling in the Nucleus Tractus Solitarii
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377
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*
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Leptin (μg)
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Δ BAT SNA (%)
Vehicle
Leptin (1 μg)
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Figure 2. Effects of leptin injection into the caudal NTS on renal and BAT SNA in rats. The time course of renal and BAT SNA
responses to NTS injection of vehicle (50 nL of saline) or leptin (1.0 ␮g in 50 nL of saline) is shown in the left panels (A and B). Caudal
NTS injection of leptin, 1.0 ␮g, produced a significant increase in renal (P⬍0.001) but not BAT (P value not significant) SNA. Summary
data for percentage of change in renal and BAT SNA 5 hours after NTS injection of vehicle (50 nL of saline) or leptin (0.25, 0.50, or 1.00
␮g in 50 nL of saline) are shown in the right panels (C and D). Vehicle (labeled leptin 0) had no effect. Caudal NTS injection of leptin
produced a dose-related increase in renal SNA (P⬍0.001) but failed to increase BAT SNA (P value not significant). Data are
means⫾SEMs of n⫽4 to 7 per group. *P⬍0.05 vs vehicle.
The location of the injection sites confirmed to be within
the caudal NTS is shown in Figure 4. The injections were
distributed in the lateral part of the commissural subnucleus
and the dorsomedial part of the medial subnucleus. There was
no notable difference in the responses to leptin injection into
the lateral part of the commissural subnucleus versus the
dorsomedial part of the medial subnucleus.
As shown in Figure 3, mean arterial pressure increased
significantly (P⬍0.05 versus vehicle) with the high dose of
leptin but not with the low or middle dose when compared
with responses to vehicle. Baseline mean arterial pressure
was 87⫾11, 95⫾4, 86⫾4, and 94⫾2 mm Hg in the vehicle
and 0.25-, 0.50-, and 1.00-␮g leptin groups, respectively.
Heart rate increased in both the vehicle and leptin groups, but
the increases did not differ between groups (P value not
significant). For example, heart rate increased from 229⫾16
to 299⫾12 bpm in the vehicle group and from 242⫾13 to
302⫾8 bpm in the leptin 0.50-␮g group.
Δ MAP (mmHg)
25
Vehicle
Leptin (1 μg)
15
To determine whether the SNA responses to caudal NTS
leptin are receptor mediated, we compared effects of caudal
* **
*
*
*
** * * *
5
-5
-15
B
*
25
Δ MAP (mmHg)
A
Responses to NTS Injection of Leptin in Zucker
Lean and Obese Rats
*
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Δ BAT SNA (%)
B
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-25
0
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Time (min)
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-25
Leptin (μg)
0
0.25
0.5
1.0
Figure 3. Effects of leptin injected into the caudal NTS on arterial pressure. A, Time course of mean arterial pressure (MAP) responses
to NTS injection of vehicle or leptin (1.0 ␮g). B, Summary data for changes in MAP 5 hours after NTS injection of vehicle (50 nL of
saline shown as leptin 0) or leptin (0.25, 0.50, and 1.0 ␮g). Data are means⫾SEMs of n⫽4 to 7 per group. *P⬍0.05 vs vehicle.
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February 2009, Part II
Sprague-Dawley rats failed to increase SNA to either BAT
(⫺21⫾17%) or kidney (⫹5⫾25%). A representative photomicrograph of the florescent beads in the rostral NTS is
shown in Figure S1 (available in the online data supplement
at http://hyper.ahajournals.org). The lack of SNA response to
rostral NTS leptin is consistent with previous reports that
leptin receptor signaling in the NTS is confined to the caudal
NTS.1,10,11
AP
cNTS
sol
iNTS
cc
mNTS
DMX
Discussion
1 mm
NTS injection of leptin (0.5 ␮g in 50 nL of saline) between
Zucker obese rats that have a missense mutation in the leptin
receptor18 and lean controls. As shown in Figure 5A, injection
of leptin into the caudal NTS increased renal SNA in the
Zucker lean but not in the Zucker obese rats. NTS injection of
leptin failed to increase BAT SNA in either Zucker lean or
obese rats (Figure 5B). These findings support the conclusion
that the renal SNA responses to caudal NTS in the SpragueDawley and Zucker lean control rats are dependent on an
intact leptin receptor.
Responses to Rostral NTS Injections of Leptin
We also studied responses to leptin injection into the rostral
NTS to determine whether this region of the NTS might
participate in leptin signaling of sympathetic outflow, because viral tracing has indicated that the rostral and intermediate NTS, but not the caudal NTS, are polysynaptically
connected with BAT.19 In the current study, injection of
leptin (0.5 ␮g in 50 nL of saline) into the rostral NTS in
B
150
120
100
*
50
0
ZL
ZO
Δ BAT SNA (%)
A
Δ Renal SNA (%)
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Figure 4. Schematic diagram of the NTS showing the sites of
leptin injection assessed from the location of fluorescent microspheres coinjected with leptin. The injections were localized in the lateral part of the commissural subnucleus and the
dorsomedial part of the medial subnucleus of the NTS. The
size of each marker reflects the borders of the deposit of microspheres. cNTS indicates commissural subnucleus; iNTS,
intermediate subnucleus; mNTS, medial subnucleus; DMX,
dorsal vagal motor nucleus; AP, area postrema; sol, solitary
tract.
70
20
-30
ZL
ZO
Figure 5. Effects of 0.5 ␮g of leptin injected into the caudal
NTS on renal and BAT SNA in leptin receptor mutant Zucker
obese (ZO) and control Zucker lean (ZL) rats. A, Comparison
of renal SNA response to leptin injection into the NTS
between the Zucker obese and lean control rats. B, Comparison of BAT SNA response to leptin injection into the NTS
between the Zucker obese and lean control rats. Data are
means⫾SEMs of n⫽4 to 7 per group. *P⬍0.05 vs lean
controls.
The major new findings from this study are as follows: (1) leptin
receptor signaling in the NTS increases SNA to kidney but not
to BAT; (2) sympathoactivation to microinjection of leptin into
the NTS was associated with an increase in arterial pressure; and
(3) the renal sympathetic nerve responses to NTS leptin appear
to emanate from the caudal region of the NTS at the level of the
area postrema. Leptin signaling in the NTS produces a strikingly
different pattern of sympathetic responses than that emanating
from leptin signaling in the hypothalamic arcuate nucleus in our
previous study.17 The current study adds to evidence for functional leptin receptors in the NTS and thereby strengthens the
concept of a distributed brain network of leptin action. The
results further demonstrate that these distributed brain sites can
mediate contrasting responses to leptin.
As discussed in the introduction, our study was prompted
by emerging evidence for functionally significant leptin
receptors in the NTS.1–13 NTS injection of leptin failed to
increase SNA in Zucker obese rats that have a missense
mutation in the leptin receptor that renders the leptin receptor
dysfunctional, indicating that the renal SNA responses to
NTS leptin in the Sprague-Dawley and Zucker lean control
rats were leptin receptor mediated. Injection of leptin into the
caudal NTS increased renal SNA in both Sprague-Dawley
and Zucker lean or wild-type rats but failed to increase SNA
to BAT.
We considered 2 possible explanations for the failure of
caudal NTS to increase SNA to BAT. The first was a
nonspecific lack of responsiveness of BAT SNA in our
preparation. This was excluded by demonstrating that cooling
the rats, which is a stimulus to thermogenic metabolism,
increased SNA to BAT. In addition, in our previous report,
using the same approach as in the current study, microinjection of leptin into the hypothalamic arcuate nucleus increased
both BAT and renal SNA.17 Second, we considered the
possibility that the rostral, as opposed to the caudal, NTS
region might regulate SNA to BAT, but injection of leptin
into the rostral NTS failed to increase SNA to either the
kidney or BAT. Injections of pseudorabies virus, a transneuronal tracer, into interscapular BAT result in early infection
of the rostral and intermediate NTS, and many neurons so
infected are responsive to cold.19 However, injection of leptin
into the rostral NTS failed to increase SNA to either the
kidney or BAT.
Our data, therefore, suggest that leptin receptor activation
in the NTS does not initiate sympathetic activation to thermogenic BAT. Our studies do not exclude a role for the NTS
in the neural pathways mediating leptin-induced increases in
SNA to BAT, but the findings suggest that leptin signaling in
the NTS does not trigger sympathetic activation to BAT. This
Mark et al
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is supported by the findings that increased leptin transgenic
expression in the dorsal vagal complex, including the NTS,
failed to affect uncoupling protein-1 mRNA expression in the
BAT.20
The finding that caudal NTS leptin increased renal SNA,
whereas rostral NTS injection of leptin failed to increase
SNA, supports previous suggestions1,10,11 that leptin signaling
in the NTS occurs primarily in the caudal NTS at the level of
the area postrema. Our injections into the caudal NTS were
clustered in the lateral part of the commissural subnucleus
and the dorsomedial part of the medial subnucleus. There
were no notable differences in the renal SNA responses to
leptin in these 2 areas. Anatomic studies suggest that the
caudal NTS may be indirectly connected with postganglionic
sympathetic neurons innervating the kidney,21 and there is
evidence that visceral afferents from the kidney ultimately
end in the commissural NTS.22
We cannot completely exclude the possibility that the
area postrema and/or dorsal motor nucleus of the vagus
contributed to the renal SNA responses to the injection of
leptin in the caudal NTS. The vast majority of the injection
sites into the caudal NTS were centered lateral to the area
postrema and dorsal to the dorsal motor nucleus of the
vagus (Figure 4), but some leptin may have diffused, albeit
at decreasing concentration, into the area postrema or the
nucleus of the vagus. Nevertheless, given the distribution
of the center of the injection sites in the caudal NTS, the
most likely interpretation of our data is that the responses
emanated mainly from the commissural and medial subnuclei of the caudal NTS.
From these studies, we cannot identify the NTS neurons
activated by leptin to increase SNA. NTS leptin increased
renal SNA (which is under baroreceptor control) but not SNA
to BAT (which is not under baroreceptor control). Our NTS
injection sites include neurons in receipt of baroreceptor
afferents.23 These observations suggest that leptin could be
acting in the NTS to increase renal SNA by acting on
baroreceptor neurons, but our NTS injection sites also include
neurons subserving visceral afferents,24 and NTS neurons that
are activated by gastric distension are also activated by
leptin.12,13
Caudal NTS injection of leptin increased arterial pressure. However, this effect was significant only with the
highest dose of leptin. This is consistent with the modest
pressor action of leptin, particularly in acute studies.16,17
The pressor action of caudal NTS injection of leptin may
be attributable to sympathetically mediated vasoconstriction or a consequence of the renal sympathetic antinatriuretic actions of leptin. However, additional studies are
needed to test this hypothesis.
Perspectives
A striking and surprising finding was the contrast between
the pattern of sympathetic activation with NTS leptin in
this study and the pattern of sympathetic responses to
arcuate administration of leptin in our previous study.17
NTS injection of leptin increased renal SNA but failed to
increase SNA to BAT. In contrast, in our previous study,
arcuate injection of leptin (in the same dose injected into
Leptin Signaling in the Nucleus Tractus Solitarii
379
the NTS) increased SNA to both BAT and kidney with
greater increases in BAT SNA.17 The contrasting pattern of
responses to NTS versus arcuate leptin suggests that the
responses to NTS leptin are not emanating from the
hypothalamic arcuate nucleus. The finding that both NTS
and arcuate leptin trigger SNA strengthens the concept of
a distributed brain network of leptin action. The contrasting responses to NTS versus arcuate leptin suggest that
these distributed brain sites can mediate qualitatively
distinct leptin actions. Our study extends the understanding of the brain regions involved in the sympathetic neural
actions of leptin and the concept of a distributed brain
network of leptin action.
Acknowledgments
We acknowledge the assistance of David Dellsperger in data
management and the secretarial assistance of Jan Rogers.
Sources of Funding
This work was supported by the National Institutes of Health grant
HL084207 to A.L.M. and K.R. and by American Heart Association
grant 0530274N to K.R.
Disclosures
None.
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Leptin Signaling in the Nucleus Tractus Solitarii Increases Sympathetic Nerve Activity to
the Kidney
Allyn L. Mark, Khristofor Agassandian, Donald A. Morgan, Xuebo Liu, Martin D. Cassell and
Kamal Rahmouni
Downloaded from http://hyper.ahajournals.org/ by guest on April 29, 2017
Hypertension. 2009;53:375-380; originally published online December 22, 2008;
doi: 10.1161/HYPERTENSIONAHA.108.124255
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Copyright © 2008 American Heart Association, Inc. All rights reserved.
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Online supplement
LEPTIN SIGNALING IN THE NUCLEUS TRACTUS SOLITARII INCREASES
SYMPATHETIC NERVE ACTIVITY TO THE KIDNEY
Allyn L. Mark(1,3), Khristofor Agassandian(2,3), Donald A. Morgan(1,3), Xuebo Liu (1,3),
Martin D Cassell(2.3), Kamal Rahmouni(1,3)
Departments of Internal Medicine(1) and Anatomy and Cell Biology(2) and Center on
Functional Genomics of Hypertension(3), Carver College of Medicine, University of
Iowa, Iowa City, Iowa
Short title: Leptin signaling in the nucleus tractus solitarii.
Correspondence to:
Allyn L. Mark, M.D.
3187 MERF
University of Iowa Carver College of Medicine
Iowa City, IA, 52242-1101, USA
Email: [email protected]
Tel: 319 353 5674
Fax: 319 353 5350
Supplemental data:
A representative photomicrograph of the florescent beads in the rostral NTS
sol
NTS
4V
Figure S1. Photomicrograph of a representative coronal section showing the site of
fluorescent microspheres during a unilateral microinjection into the rostral nucleus tractus
solitarii. Abbreviations: 4V, fourth ventricle; NTS, nucleus tractus solitarii; sol, solitary
tract.