Ventral Lamina Terminalis Mediates Enhanced
Cardiovascular Responses of Rostral Ventrolateral Medulla
Neurons During Increased Dietary Salt
Julye M. Adams, Megan E. Bardgett, Sean D. Stocker
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—Increased dietary salt enhances sympathoexcitatory and sympathoinhibitory responses evoked from the rostral
ventrolateral medulla (RVLM). The purpose of the present study was to determine whether neurons of the forebrain
lamina terminalis (LT) mediated these changes in the RVLM. Male Sprague-Dawley rats with and without LT lesions
were fed normal chow and given access to water or 0.9% NaCl for 14 to 15 days. Unilateral injection of L-glutamate
into the RVLM produced significantly larger increases in renal sympathetic nerve activity and arterial blood pressure
of sham rats ingesting 0.9% NaCl versus water. However, these differences were not observed between ventral
LT-lesioned rats drinking 0.9% NaCl versus water. Similar findings were observed when angiotensin II or
␥-aminobutyric acid was injected into the RVLM. Interestingly, a subset of animals drinking 0.9% but with damage
restricted to the organum vasculosum of the lamina terminalis did not show enhanced responses to L-glutamate or
␥-aminobutyric acid. In marked contrast, RVLM injection of L-glutamate or ␥-aminobutyric acid produced exaggerated
sympathetic nerve activity and arterial blood pressure responses in animals drinking 0.9% NaCl versus water after an
acute ventral LT lesion or chronic lesion of the subfornical organ. Additional experiments demonstrated that plasma
sodium concentration and osmolality were increased at night in rats ingesting 0.9% NaCl. These findings suggest that
neurons of the ventral LT mediate the ability of increased dietary salt to enhance the responsiveness of RVLM
sympathetic neurons. (Hypertension. 2009;54:00-00.)
Key Words: brain 䡲 sodium 䡲 hypertension 䡲 blood pressure 䡲 sympathetic nerve activity
E
levated dietary salt intake does not invariably increase
arterial blood pressure (ABP) but does contribute to the
development of hypertension or the severity of hypertension
in salt-sensitive individuals and experimental models. Compelling data in several models indicate that dietary salt acts
centrally with other factors to increase sympathetic nerve
activity (SNA) and peripheral resistance.1–3 Moreover, dietary salt potentiates the sympathetic and/or pressor responses to stress,4,5 hyperinsulinemia,6 and activation of
somatic afferents.7,8 Collectively, these observations suggest
that dietary salt may alter the gain of central sympatheticregulatory networks. This hypothesis is supported by data
from several laboratories that SNA and ABP responses to
microinjection of various excitatory and inhibitory neurotransmitters into the rostral ventrolateral medulla (RVLM)
are enhanced in animals chronically maintained on a high-salt
diet.7,9 –11
Elevated dietary salt intake causes widespread changes in
neurohumoral profiles, including suppression of the peripheral renin-angiotensin (Ang) system12 and increases in plasma
sodium concentration or osmolality.13–15 One of the major
sites where the central nervous system detects such changes
in neurohumoral stimuli is the forebrain lamina terminalis
(LT).16,17 The LT consists of several interconnected structures
located along the rostral wall of the third ventricle, including
the median preoptic nucleus, subfornical organ (SFO), and
organum vasculosum of the LT (OVLT). The latter 2 structures lack a complete blood-brain barrier and are thereby
responsive to a number of circulating factors. LT lesions
severely disrupt physiological responses to a number of
neurohumoral stimuli, including osmolality and circulating
Ang II.16,18 –21 Interestingly, lesions of the anteroventral third
ventricle region (AV3V), which encompasses the LT, prevent
the development or reverse hypertension in Dahl saltsensitive,22 DOCA-salt,23 Grollman,24,25 and Goldblatt25,26
hypertensive rats. Although limited data exist, these models
show exaggerated cardiovascular responses to the injection of
L-glutamate in the RVLM.27,28 Collectively, these observations suggest that the responsiveness of RVLM sympatheticregulatory neurons can be modulated by the forebrain LT.
The purpose of the present study was to determine whether
the forebrain LT mediated the ability of dietary salt to
enhance sympathetic and cardiovascular responses from the
RVLM.
Received December 10, 2008; first decision January 7, 2009; revision accepted May 14, 2009.
From the Department of Physiology, University of Kentucky, Lexington.
Correspondence to Sean D. Stocker, Department of Physiology, University of Kentucky, 800 Rose St MS-508, Lexington, KY 40536-0298. E-mail
[email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.127803
1
2
Hypertension
August 2009
A
+0.6 mm
LV
DBB
+0.3 mm
DBB
0.0 mm
MnPO
B
LV
DBB
DBB
OVLT
AC
AC
OVLT
-0.3 mm
f
3V
f
AC
3V
OVLT
0.0 mm
-0.3 mm
i
ii
iii
iv
OC
OVLT
MnPO
AC
MnPO
C
MnPO
OC
Figure 1. Schematic drawings of ventral LT lesions for rats drinking (A) water or (B) 0.9% NaCl. The lesion boundary is outlined in
black; control animals had no lesion or received a directed misplaced lesion (gray). C, Digital photomicrographs of 2 rostral-caudal levels of the LT for control (i and ii) and ventral LT-lesioned (iii and iv) rats. Scale bar: 500 ␮m. Coordinates are in reference to bregma. LV
indicates lateral ventricle; DBB, diagonal band; AC, anterior commissure; MnPO, median preoptic nucleus; f, fornix; 3V, third ventricle;
OC, optic chiasm.
Materials and Methods
Animals
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
All of the experimental procedures conform to the National Institutes
of Health Guide for the Care and Use of Laboratory Animals and
were approved by the University of Kentucky Institutional Animal
Care and Use Committee. Male Sprague-Dawley rats (200 to 250 g,
Charles River Laboratories) were housed in a temperature-controlled
room (22⫾1°C) with a 12:12-hour light-dark cycle (lights on 7 AM to
7 PM). Rats were fed standard rat chow containing 0.23% NaCl
(Harlan Teklad Global Diet 2018) and given access to deionized
water for ⱖ7 days before experiments began.
Lesion of the LT
Rats were anesthetized with 3% isoflurane and placed into a
stereotaxic frame with the skull level between lambda and bregma.
After a small craniotomy, a Teflon-coated tungsten electrode (50- or
250-␮m tip, 0.008 OD, AM Systems) angled 8° from the midsagittal
plane was lowered into the ventral LT using coordinates in reference
to bregma: 0.0- to 0.5-mm rostral, 1.0-mm lateral, and 8.0-mm
ventral to dura. DC current (100 or 500 ␮A) was applied for 30
seconds. Electrode tip size and current intensity were varied to
produce small (OVLT) versus large (ventral LT) lesions, respectively. Sham control rats consisted of 2 groups: identical procedures
except no current was applied or lesions were placed lateral to the
ventral LT by applying DC current (500 ␮A, 30 seconds) using
identical coordinates, except the electrode was parallel to the
midsagittal plane. SFO lesions were produced by applying DC
current (500 ␮A, 30 seconds) to a tungsten electrode (250-␮m tip)
angled 8° from the midsagittal plane at 2 different sites in reference
to bregma: 0.8 versus 1.1 mm caudal, 0.7 versus 0.7 mm lateral, and
5.2 versus 4.9 mm ventral to dura. The craniotomy was filled with
bone wax and the incision closed with suture. Rats were given
ampicillin (100 mg/kg, IM), returned to home cages, and given
access to 10% sucrose solution until water and food intakes returned
to prelesion levels (⬇2 to 5 days).
Experimental Design
Rats were fed normal chow and water for ⱖ7 days and then
randomly assigned to drink water or 0.9% NaCl solution for 14 to 15
days. Food and fluid intakes were monitored daily. Then, animals
were anesthetized with a mixture of urethane/chloralose and prepared for renal SNA recordings and RVLM microinjections, as
described elsewhere9,10 (see the data supplement online at http://
hyper.ahajournal.org). In experiment 1, rats with and without a
chronic ventral LT lesion received a unilateral injection of
L-glutamate (0.1, 1.0, and 3.0 nmol) into the RVLM in a randomized
manner with ⬎5 minutes between injections. ␥-Aminobutyric acid
(GABA; 0.03, 0.10, and 10.00 nmol) was injected on the contralateral side. In experiment 2, rats with and without a chronic ventral LT
lesion received a unilateral injection of Ang II (0.6 and 6.0 pmol)
into the RVLM. One dose was tested per side, and 6.0 pmol were
injected ipsilateral to the SNA recording. Control groups in experi-
ments 1 and 2 were either sham lesioned or received lesions placed
lateral to the midline. In experiment 3, rats drinking water or 0.9%
NaCl for 14 to 15 days received an acute ventral LT lesion ⬇60
minutes before microinjection of L-glutamate and GABA. In
experiment 4, rats with and without chronic SFO lesions received
injections of L-glutamate and GABA. For experiments 1, 2, and 4,
RVLM injections were performed at 24 to 26 days after the initial
lesion. Plasma electrolytes, hematocrit, plasma protein, and blood
volume were measured in a subset of animals as described
previously.9
Circadian Analysis of Plasma Electrolytes,
Osmolality, and Food and Fluid Intakes
Control and ventral LT-lesioned rats were fed normal chow and
given access to water or 0.9% NaCl for 14 days. At 1 PM or 1 AM, rats
were anesthetized with 3% isoflurane, and blood (0.5 mL) was
collected by aortic puncture into heparinized tubes and analyzed for
plasma electrolytes by an I-STAT1 analyzer and 6⫹ cartridges
(Abbott). Plasma osmolality was determined in duplicate by
freezing-point depression (Advanced Instruments). Food and fluid
intake measurements were monitored daily except in a subset of animals
where daytime and nighttime measurements were performed.
Statistical Analysis
All of the data are expressed as mean⫾SE. Changes in integrated
SNA were calculated by subtracting background noise after hexamethonium (30 mg/kg, IV). The 1-second peak SNA and ABP
responses were compared with a 30-second baseline segment immediately before the injection. Renal SNA was only analyzed when
injections were performed ipsilateral to the nerve recording. All of
the data were analyzed by a 1- or 2-way ANOVA with repeated
measures when appropriate (dose factor). All of the posthoc tests
were performed with independent or paired t tests with a layered
Bonferroni correction. A P⬍0.05 was considered statistically
significant.
Results
Ventral LT Lesion Prevents Salt-Induced
Enhancement of RVLM Responses
A major goal of the present study was to determine whether
LT neurons mediated the enhanced cardiovascular responses
of RVLM neurons during increased dietary salt. Figure 1
illustrates histology for control and ventral LT-lesioned
animals. Lesions of the ventral LT produced extensive
damage to the OVLT and midline preoptic nuclei at the level
of the anterior commissure. In the majority of cases, the
ventral median preoptic nucleus at the commissural level was
intact. Damage was not observed caudal to the median
preoptic nucleus. As reported previously,9 –11 RVLM injec-
Adams et al
∆ Renal SNA ∆ Mean ABP
(%)
(mmHg)
A
Water (n=8)
Salt (n=7)
Lesion+Water (n=10)
Lesion+Salt (n=6)
60
*†
40
*†
0
200
*†
*†
*†
200
Water
Salt
180
100
20 s
Δ Mean ABP
m mHg)
(m
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Water (n=8)
Salt (n=7)
Lesion+Water (n=10)
Lesion+Salt (n=6)
B
0
-20
-40
*†
0.03
ABP
(mmHg)
*†
0.1
*†
10
GABA (nmol)
Figure 2. A, Peak change in mean ABP
and renal SNA during RVLM injection of
L-glutamate in rats with chronic lesion of
the ventral LT. B, Individual examples of
ABP, mean ABP, 兰renal SNA, and raw
renal SNA during injection of 1.0 nmol of
L-glutamate. *P⬍0.05 control⫹water vs
control⫹salt; †P⬍0.05 control⫹salt versus lesion⫹salt.
Renal SNA
tion of L-glutamate produced significantly greater renal SNA
and ABP responses in control rats drinking 0.9% NaCl versus
those drinking water (Figure 2). In marked contrast, these
differences were completely absent in rats with ventral LT
lesions. In fact, the sympathoexcitatory responses at every
dose of L-glutamate were not different between ventral
LT-lesioned rats drinking 0.9% NaCl versus control rats
drinking water. As a consequence of the experiments, an
additional set of animals had lesions that missed the ventral
LT (rostral or lateral), and injection of L-glutamate into the
RVLM still produced significantly greater increases in renal
SNA (1 nmol: 150⫾9% versus 105⫾7%; P⬍0.05) and mean
ABP (1 nmol: 47⫾2 versus 27⫾3 mm Hg; P⬍0.05) of rats
ingesting 0.9% NaCl (n⫽4) versus water (n⫽3), respectively
(data not shown for 0.1 and 3.0 nmol). These responses were
not different from control animals drinking 0.9% NaCl or
water, respectively.
To examine whether LT neurons mediated the enhanced
sympathoinhibitory responses evoked from the RVLM during
increased dietary salt, GABA was microinjected into the
contralateral RVLM. As reported previously,9 rats drinking
0.9% NaCl versus water displayed significantly greater depressor responses to every dose of GABA. Again, these
differences were absent in rats with lesions restricted to the
ventral LT (Figure 3) but present in rats with lesions that
missed the ventral LT (data not shown).
In a second group of animals, we examined whether
chronic lesion of the ventral LT prevented the enhanced
sympathoexcitatory responses to Ang II in the RVLM during
increased dietary salt. RVLM injection of Ang II produced
significantly greater increases in renal SNA and mean ABP in
control rats drinking 0.9% NaCl versus those drinking water
(Figure 4). In marked contrast, rats with a lesion of the ventral
A
Salt
300
Renal
SNA (%) 100
0.1
1
3
L-Glutamate (nmol)
3
Lesion
Water
100
Mean ABP
(mmHg)
100
0
Control
ABP
(mmHg)
*†
20
B
Salt Enhances RVLM Responses via Lamina Terminalis
Mean ABP
(mmHg)
Control
175
Water
Salt
LT and drinking 0.9% NaCl showed similar changes in renal
SNA and ABP to RVLM injection of Ang II versus control or
ventral LT-lesioned rats drinking water. Again, injection of
Ang II (6 pmol) into the RVLM of rats drinking 0.9% NaCl
but with lesions that missed the ventral LT (n⫽4) still
produced enhanced renal SNA (66⫾8%) and mean ABP
(19⫾2 mm Hg) responses. Histology for ventral LT lesions is
illustrated in Figure S1.
OVLT Lesion Prevents Salt-Induced Enhancement
of RVLM Responses
A subset of animals had more focal lesions with damage
restricted to the OVLT (Figure 5). Interestingly, chronic
ingestion of 0.9% NaCl did not result in potentiated sympathoexcitatory responses to L-glutamate (Figure 5C) or GABA
(Figure 5D). In fact, the changes in renal SNA or ABP evoked
by injection of L-glutamate or GABA in these animals were
not different from control and ventral LT-lesioned rats
drinking water or ventral LT-lesioned rats drinking 0.9%
NaCl (Figures 3 and 4).
Enhanced RVLM Responses Are not Prevented by
Acute Lesion of the Ventral LT or Chronic
SFO Lesion
Acute lesion of the ventral LT in rats drinking water or 0.9%
NaCl produced a transient decrease in ABP (⫺2⫾4 versus
⫺5⫾2 mm Hg) and renal SNA (⫺31⫾10% versus
⫺31⫾9%); however, both variables returned to baseline
values within 30 minutes. Histology is illustrated in Figure
S2. In marked contrast to chronic lesion of the ventral LT,
RVLM injection of L-glutamate produced significantly
greater renal SNA and ABP responses in rats with acute
lesion of the ventral LT drinking 0.9% NaCl versus water
Lesion
Water
Salt
100
150
100
20 s
Figure 3. Peak change in mean ABP
during RVLM injection of GABA in rats
with chronic lesion of the ventral LT. B,
Individual examples of ABP and mean
ABP during injection of 0.1 nmol of
GABA. *P⬍0.05 control⫹water vs
control⫹salt; †P⬍0.05 control⫹salt vs
lesion⫹salt.
Hypertension
A
Water (n=8)
Salt (n=8)
∆ Renal SNA
(%)
∆ Mean ABP
(mmHg)
4
30
*
20
August 2009
Lesion+Water (n=4)
Lesion+Salt (n=5)
†
*
10
0
6
75
*
50
ABP
(mmHg)
†
180
0.6
†
Water
Lesion
Salt
100
20 s
200
∫ Renal
SNA (%) 100
There were no differences in plasma sodium concentration or
osmolality during the day between control or lesioned rats
drinking water or 0.9% NaCl (Table). However, control and
lesioned rats drinking 0.9% NaCl displayed significant increases in plasma sodium concentration and osmolality at
night. All of the groups ingested significantly more food and
fluid during the dark versus light cycle, and rats drinking
0.9% NaCl ingested significantly more fluid and had higher
daily sodium intakes (Tables S1 and S2). However, there
were no differences in baseline mean ABP, heart rate, renal
SNA, or plasma and/or blood volume (Tables S2 and S3).
Discussion
Increased dietary salt enhances sympathetic and cardiovascular responses evoked and/or mediated by RVLM
sympathetic-regulatory neurons.4 –7,9 –11 However, the mecha-
40
20
0
*†
*†
0.1
Water (n=8)
1
3V
100
3
0
†
*† *
*†
0.1
L-Glutamate (nmol)
Salt (n=8)
i
ii
OC
OVLT
200
MnPO
1
3
Lesion+Water (n=3)
D
∆ Mean ABP
(mmHg)
60
*†
AC
AC
∆ SNA (%)
C
B
f
MnPO
OVLT
∆ Mean ABP
(mmHg)
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nism by which increased dietary salt is detected by the central
nervous system and translates into functional differences in
the regulation of RVLM sympathetic neurons was previously
unknown. The present findings provide several new key
observations: (1) chronic lesions of the ventral LT and OVLT
prevent the enhanced cardiovascular responses to RVLM
stimulation during increased dietary salt intake; (2) acute
lesion of the ventral LT or chronic SFO lesion did not affect
these responses; and (3) increased dietary salt intake elevated
plasma sodium concentration and osmolality at night. Altogether, these findings suggest that ventral LT, and perhaps
OVLT, neurons mediate the ability of increased dietary salt to
enhance the responsiveness of RVLM sympathetic neurons.
The forebrain LT is a specialized group of structures that
permit the central nervous system to detect changes in
neurohumoral factors.16,17 Given the widespread neurohumoral changes associated with increased dietary salt intake,
we hypothesized that forebrain LT neurons indirectly detect
the changes in dietary salt to alter the responsiveness of
RVLM neurons. Indeed, rats with chronic lesion of the
ventral LT (and OVLT) and ingesting 0.9% NaCl had similar
sympathoexcitatory and sympathoinhibitory responses versus
those animals ingesting water. Chronic SFO lesions did not
affect these responses. These findings cannot be explained by
differences in salt intake, because rats with chronic lesions of
the ventral LT or OVLT ingested similar amounts of 0.9%
NaCl as control rats. Furthermore, the ability of chronic
ventral LT and OVLT lesions to prevent the enhanced
Analysis of Plasma Electrolytes and Osmolality
DBB
Figure 4. A, Peak change in mean ABP
and renal SNA during RVLM injection of
Ang II in rats with chronic lesion of the
ventral LT. B, Individual examples of
ABP, mean ABP, 兰renal SNA, and raw
renal SNA during injection of 6 pmol of
Ang II. *P⬍0.05 control⫹water vs
control⫹salt; †P⬍0.05 control⫹salt vs
lesion⫹salt.
Renal SNA
6
AngII (pmol)
LV
DBB
Salt
150
(Figure 6A). Similarly, RVLM injection of L-glutamate produced significantly greater increases in renal SNA and ABP
of SFO-lesioned rats drinking 0.9% NaCl versus water
(Figure 6B). Histology is illustrated in Figure S3. Exaggerated sympathoinhibitory responses to RVLM injection of
GABA were observed in both groups (data not shown). Moreover, the responses observed in acute LT- or chronic SFOlesioned rats drinking 0.9% NaCl were not different from control
rats drinking 0.9% NaCl.
Injection sites for all of the experiments were centered in
the RVLM as defined previously9,10 (Figure S4).
A
Water
100
Mean ABP
(mmHg)
25
0
Control
B
0
†
-20 *
-40
0.03
*†
0.1
*†
10
GABA (nmol)
Lesion+Salt (n=3)
Figure 5. A, Schematic drawings of
OVLT lesions for rats drinking water
(dashed) or 0.9% NaCl (black). Lines
indicate the lesion boundary. B, Digital
photomicrograph of OVLT lesion. Scale
bar: 500 ␮m; arrow indicates lesion. C,
Peak change in mean ABP and renal
SNA of OVLT-lesioned and control rats
during RVLM injection of (C) L-glutamate
or (D) GABA. *P⬍0.05 control ⫹water vs
control⫹salt; †P⬍0.05 control⫹salt versus lesion⫹salt.
40
20
0
*
*
0.1
200
100
*
0
1 3
0.1
L-Glutamate (nmol)
Water (n=8)
* *
1
Salt Enhances RVLM Responses via Lamina Terminalis
B
3
Salt (n=8)
40
20
0
*
*
60
*
0.1
∆ Renal SNA(%)
*
60
∆ ABP (mmHg)
∆ ABP (mmHg)
A
∆ Renal SNA(%)
Adams et al
200
*
*
*
Figure 6. Peak change in mean ABP
and renal SNA during RVLM injection of
L-glutamate in rats drinking water or
0.9% NaCl for 14 days that received (A)
an acute ventral LT lesion or (B) chronic
SFO lesion. *P⬍0.05 water vs salt for
both groups. There were no differences
between the control and lesion groups
within the same diet.
100
0
1 3
0.1
L-Glutamate (nmol)
Lesion+Water (n=3)
1
3
Lesion+Salt (n=3)
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
RVLM responsiveness is likely more complex. First, these
enhanced responses are observed in rats drinking 0.9% NaCl
after 14 or 21 days but not at 1 or 7 days.9 Second, rats
drinking 0.9% NaCl for 14 days still exhibited enhanced
responses when water was returned for 1 day.9 Third, acute
lesion of the ventral LT in the present study did not reverse
the enhanced responses evoked from the RVLM of rats
drinking 0.9% NaCl for 14 days. Collectively, these observations indicate that dietary salt alters the responsiveness
through a chronic change in neuronal function or some form
of neuronal plasticity.
An interesting observation in the current study is that the
ingestion of 0.9% NaCl significantly increased plasma sodium concentration at night but not during the day in both
control and ventral LT-lesioned animals. Other studies have
reported that dietary salt elevates plasma sodium concentration or osmolality in rats13,15 and humans.14 Small increases in
osmolality of 1% to 2% stimulate drinking in mammals,33,34
thereby suggesting that osmosensory cells can detect discrete
changes in osmolality or plasma sodium concentration. These
observations, together with the present study, raise the possibility that increased dietary salt elevates plasma osmolality
to activate osmosensory neurons in the ventral LT and to alter
the responsiveness of RVLM neurons. In fact, an enhanced
responsiveness of RVLM neurons has been reported in
48-hour water-deprived rats.35 Such a model would suggest
that chronic changes in plasma osmolality produced by
dietary salt intake must reach the threshold of osmosensory
neurons. Currently, there are no available data to directly
address this issue; however, the altered responsiveness of
RVLM neurons has been observed over a range of different
salt intakes.7 Clearly, additional evidence is needed to directly link dietary salt intake and the changes in the responsiveness of RVLM neurons with osmotic perturbations or
other circulating factors.
responsiveness of RVLM neurons is likely not attributed to
some chronic adaptation as a result of the lesion per se,
because the sympathoexcitatory and sympathoinhibitory responses were not different between control and lesioned rats
drinking water. Therefore, these findings indicate that ventral
LT or OVLT neurons mediate the ability of increased dietary
salt to enhance the responsiveness of RVLM sympathetic
neurons.
A critical question that arises from these studies is the
nature of the neurohumoral factor(s) that activates LT neurons to alter the responsiveness of RVLM neurons. Indeed,
neurons within these structures express receptors for a variety
of circulating factors.17 Although AV3V lesions in rats
clearly disrupt thirst and vasopressin secretion to a number of
physiological stimuli,16 such lesions produce damage across
the entire forebrain LT. However, discrete lesion of the
OVLT in dogs disrupts thirst and vasopressin secretion
stimulated by elevated plasma sodium concentration and
circulating Ang II,18 whereas lesion of the SFO in rats20 and
dogs21 blunts thirst stimulated by Ang II. Studies in sheep
indicate that combined ablation of several LT structures is
needed to attenuate such responses.19,29 Therefore, the factor(s) by which dietary salt activates ventral LT neurons to
alter the responsiveness of RVLM neurons remains unclear.
The downstream pathways and cellular mechanisms that
mediate the enhanced responsiveness of RVLM neurons
during increased dietary salt are not known. The forebrain LT
densely innervates many hypothalamic nuclei, including the
hypothalamic paraventricular nucleus.16,17 Previous studies
have demonstrated that neurons in the hypothalamic paraventricular nucleus with descending projections are excited by
hyperosmolality.30 Anatomic and functional data indicate that
these neurons use Ang II as a neurotransmitter,31,32 and we
recently reported a greater Ang II type 1 receptor activation in
the RVLM of rats on a high-salt diet.10 Yet, available
evidence suggests that the mechanism of the enhanced
Table. Plasma Sodium Concentration and Osmolality of Control and Ventral LT-Lesioned Rats
Drinking Water or 0.9% NaCl
Plasma Sodium, mEq/L
Group
5
Plasma Osmolality, mosmol/L
Day
Night
Day
Control⫹water
135.2⫾0.4 (9)
136.3⫾0.6 (11)
293⫾2 (9)
294⫾1 (11)
Control⫹salt
134.5⫾0.5 (9)
138.4⫾0.6 (13)*
292⫾1 (9)
297⫾1 (13)*
Lesion⫹water
135.0⫾1.0 (7)
136.0⫾1.0 (8)
292⫾1 (7)
293⫾1 (8)
Lesion⫹salt
136.1⫾0.9 (6)
139.8⫾0.8 (8)*
294⫾1 (6)*
298⫾2 (8)
Values are mean⫾SEM. Parentheses indicate number of animals; mosmol, milliosmol.
*Significant difference between water and 0.9% NaCl within the control or lesion group (P⬍0.05).
Night
6
Hypertension
August 2009
In the present study, lesion of the ventral LT did not
produce profound deficits in fluid ingestion or sodium balance, as reported previously in AV3V-lesioned rats.16 AV3V
lesions damage numerous structures along the rostral wall of
the third ventricle, including the median preoptic nucleus,
fibers of passage from the SFO, and other periventricular
nuclei.16 Ventral LT lesions of the present study did not
damage the median preoptic nucleus or the SFO. Therefore,
the lack of fluid and osmoregulatory deficits in the present
study is likely attributed to the smaller lesions and the
presence of other osmoregulatory nuclei in the central nervous system.
Perspectives
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Increased dietary salt raises plasma (or cerebrospinal fluid)
sodium concentration and contributes to neurogenic forms of
salt-sensitive hypertension in 1 of 2 ways: a direct sodiumdriven increase in SNA and ABP1,2 or a chronic increased
gain of sympathetic-regulatory networks.7,9 –11 The increased
gain of RVLM sympathetic neurons has physiological significance, because increased dietary salt enhances the sympathoexcitatory responses to insulin6 and stimulation of somatic
afferents,7,8 responses that depend on RVLM neurotransmission.36,37 The ability of ventral LT lesions to prevent the
enhanced responsiveness of RVLM neurons during increased
dietary salt intake is reminiscent to the effect of AV3V lesions
on various models of neurogenic hypertension. AV3V lesions
prevent the development of or reverse hypertension in Dahl
salt-sensitive,22 DOCA-salt,23 Grollman,24,25 and Goldblatt25,26
hypertensive rats. The available data suggest that these models
show exaggerated responses to the injection of L-glutamate in
the RVLM.27,28 In marked contrast, AV3V lesions do not affect
hypertension in the spontaneously hypertensive rat,38 and spontaneously hypertensive rats do not display enhanced responses to
L-glutamate injection in the RVLM.39 Altogether, these observations raise the possibility that AV3V lesions attenuate neurogenic hypertension, in part, by preventing an enhanced excitability of RVLM sympathetic neurons.
Sources of Funding
This research was supported by Great Rivers American Heart
Association postdoctoral (J.M.A.) and predoctoral (M.E.B.) fellowships, American Heart Association Scientist Development Grant
(S.D.S), and a National Institutes of Health National Heart, Lung,
and Blood Institute grant HL090826 (S.D.S.).
Disclosures
None.
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enhances sympathoexcitatory and sympathoinhibitory responses from the
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Hypertension. 2008;52:932–937.
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Ventral Lamina Terminalis Mediates Enhanced Cardiovascular Responses of Rostral
Ventrolateral Medulla Neurons During Increased Dietary Salt
Julye M. Adams, Megan E. Bardgett and Sean D. Stocker
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension. published online June 8, 2009;
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ONLINE SUPPLEMENT:
Ventral Lamina Terminalis Mediates Enhanced Cardiovascular Responses
of RVLM Neurons During Increased Dietary Salt
Julye M. Adams. Megan E. Bardgett and Sean D. Stocker
Department of Physiology, University of Kentucky
Address correspondence to:
Sean D. Stocker, Ph.D.
Assistant Professor
Department of Physiology, University of Kentucky
800 Rose St. MS-508
Lexington, KY 40536-0298
Email: [email protected]
Phone: 859-323-4344
Fax: 859-323-1070
Materials and Methods
RVLM microinjections and SNA recordings were performed as described previously in
our laboratory 1, 2. Briefly, rats were anesthetized isoflurane (2-3% in 100% O2) and
then replaced by a mixture of urethane (750 mg/kg, iv) and α-chloralose (75 mg/kg, iv).
Renal SNA recordings were performed using a bipolar stainless steel electrode,
amplified (20,000 x), and filtered (low pass: 100 Hz, high pass: 3 kHz). Signals were
digitized (5 kHz), rectified, and integrated (1s time constant) using a Micro1401 and
Spike 2 software (Cambridge Electronic Design). Animals were artificially ventilated
with oxygen-enriched room air and paralyzed with gallamine triethiodide (25 mg/kg/h, 25
µL/h, iv). End-tidal CO2 and body temperature was maintained at 4-4.5% and 37±1°C,
respectively. An adequate depth of anesthesia was assessed by either the absence of
a withdrawal reflex (before neuromuscular blockade) or a pressor response to foot
pinch. Supplemental doses of anesthetic (10% initial dose) were given as necessary
but rarely needed. Initially, L-glutamate (1 nmol) was injected into the RVLM at 3
different sites separated by 300µm in the rostral-caudal plane to identify the site that
produced the largest increase in ABP; subsequent injections were performed at these
coordinates. All injections (60 nL) were performed over 5 s by an experimenter blind to
the salt and lesion condition. Injection sites were marked at the end of experiments with
0.2% rhodamine beads. At the end of experiments, animals were perfused
transcardially with 4% paraformaldehyde (50 mL). Brains were harvested, post-fixed,
sectioned at 50 µm, and counterstained with cresyl violet. RVLM injection sites and
lesions were analyzed by an experimenter blind to the injection results, salt group, and
lesion group.
References
1.
Adams JM, Madden CJ, Sved AF, Stocker SD. Increased dietary salt enhances
sympathoexcitatory and sympathoinhibitory responses from the rostral
ventrolateral medulla. Hypertension. 2007;50:354-359.
2.
Adams JM, McCarthy JJ, Stocker SD. Excess dietary salt alters angiotensinergic
regulation of neurons in the rostral ventrolateral medulla. Hypertension.
2008;52:932-937.
3.
Fink GD, Johnson RJ, Galligan JJ. Mechanisms of increased venous smooth
muscle tone in desoxycorticosterone acetate-salt hypertension. Hypertension.
2000;35:464-469.
4.
Stocker SD, Meador R, Adams JM. Neurons of the rostral ventrolateral medulla
contribute to obesity-induced hypertension in rats. Hypertension. 2007;49:640646.
Table S1. Food and fluid intakes during the light and dark cycles of control and lesion
rats.
Light Cycle
Food (g) Fluid (mL)
Dark Cycle
Food (g)
Fluid (mL)
Group
n
Control
Water
6
5±1
3±1
22±1
31±2
6
5±1
3±1
23±1
51±4*
Water
8
5±1
5±1
25±2
36±2
0.9% NaCl
8
4±1
6±1
24±1
56±5*
0.9% NaCl
Ventral LT Lesion
Values are mean±SEM. *Significant difference within group between water vs 0.9%
NaCl (P<0.05)
Table S2. Characteristics of rats with various lesions and drinking either water or 0.9% NaCl.
Characteristic
n
Control
Water
0.9%
Chronic LT/OVLT
Water
0.9%
7
Initial BWT (g)
334±8
333±11
356±8
347±16
223±13* 239±14* 273±11* 273±12*
Final BWT (g)
430±14
429±17
411±10
446±7
362±15* 384±8*
28±1
31±1
30±1
29±1
31±4
29±1
29±2
33±3
54±3†
37±2
59±6†
35±3
48±5†
29±4
53±6†
Fluid Intake (mL/day) 33±2
Na+ Intake(mEq/day) 1.1±0.1 9.6±0.5†
14
1.2±0.1 10.2±0.9†
4
3
SFO-Lesion
Water
0.9%
8
Food Intake (g/day)
17
Acute LT
Water
0.9%
4
4
368±19* 380±19*
1.2±0.2 8.4±0.8† 1.2±0.1 9.5±1.0†
Baseline Mean ABP
(mmHg)
121±5
125±3
124±2
119±4
112±6
117±3
124±3
125±1
Baseline HR (bpm) 389±16
366±9
370±11
369±18
392±19
406±19 380±4
391±7
Renal SNA (µv)
131±13 137±19
156±29
134±14
152±13 130±21 160±24
147±35
Value are mean ± SEM. *P<0.05 versus water treatment in same lesion group, †P<0.01 versus water
treatment in same group
Table S3. Characteristics of control or lesioned rats drinking water or 0.9% NaCl
Characteristic
Control Group
Water
Salt
n
8
Hematocrit (%)
45±1
P Protein (g/dl)
6.9±0.1
Chronic SFO
Water
Salt
14
11
4
43±1
43±1
44±1
44±1
45±1
6.9±0.1
6.8±0.2
6.8±0.1
-------
-------
Plasma Na+ (mEq/L) 138±1
139±2
136±1
137±2
138±1
137±2
Plasma K+ (mEq/L)
4.7±0.2
4.6±0.2
4.2±0.3
Plasma Volume (mL) 10.3±0.2 11.0±0.6
11.5±0.4
Blood Volume (mL)
Blood Volume per
100 g body weight
4.3±0.2
7
Chronic Ventral LT
Water
Salt
16.6±0.4 18.3±0.9
4.1±0.2
3.5±0.6
4
4.5±0.4
4.5±0.4
11.8±0.6
10.4±0.3
11.0±0.3
18.2±0.6
19.3±1.0
16.1±0.3
17.2±0.6
4.3±0.1
4.5±0.2
4.4±0.3
4.5±0.1
Values are mean ± SE. Plasma protein was determined by protein refractometry
(Refractometer Veterinary ATC, VWR International), and plasma Na+ and K+ concentration by
flame photometry (Model 2655-10, Cole Palmer Instrument Co.). In a subset of animals,
plasma and blood volume were determined using Evan’s Blue Dye as described previously 1,
3, 4
. Animals with ventral LT and OVLT lesions were combined.
Figure S1. Schematic drawings of VLT lesions for rats drinking (A) water or (B) 0.9% NaCl
and receiving an injection of AngII into the RVLM. The lesion boundary is outlined in black;
control animals receiving a misplaced lesion are in grey. Abbreviations: LV, lateral ventricle;
DBB, diagonal band; AC, anterior commissure; OVLT, organum vasculosum of the lamina
terminalis; MnPO, median preoptic nucleus; f, fornix; 3V, third ventricle; OC, optic chiasm
Figure S2. Schematic drawings of acute ventral LT lesions for rats drinking (A) water or (B)
0.9% NaCl. The lesion boundary is outlined in black.
Figure S3. Schematic drawings of SFO lesions for rats drinking (A) water or (B) 0.9% NaCl.
The lesion boundary is outlined in red. Abbreviations: LV, lateral ventricle; 3V, 3rd ventricle;
SFO, subfornical organ; PVH, hypothalamic paraventricular nucleus; f, fornix; vhc, ventral
hippocampal commissure; PVA, thalamic paraventricular nucleus; PT, paratenial thalamic
nucleus; sm, stria medullaris of the thalamus; Re, reunions thalamic nucleus
Figure S4. Schematic drawings of RVLM injection sites in rats drinking water (open) or 0.9%
NaCl (filled) in one of four groups: A) control, B) chronic ventral LT lesion, C) acute ventral
LT lesion, or D) chronic SFO lesion. Injections sites L-glutamate and AngII are illustrated on
the left side whereas those for GABA are illustrated on the right side. Abbreviations: ST,
spinal trigeminal nucleus; NA, nucleus ambiguus; IO, inferior olive; p, pyramidal tracts