| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2001;37:619.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
-Aminobutyric AcidB Receptor–Mediated Responses in the Nucleus Tractus Solitarius Are Altered in Acute and Chronic Hypertension
From the Department of Pharmacology, The University of Texas Health Science Center at San Antonio.
Correspondence to Steve Mifflin, PhD, Department of Pharmacology, Mail Code 7764, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229-3900. E-mail mifflin{at}uthscsa.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
The increase in mean arterial pressure evoked by injection of the
-aminobutyric acid (GABA)B agonist
baclofen into the nucleus tractus solitarius (NTS) is greater in
spontaneously hypertensive rats and renal wrap chronically hypertensive
(CHT) rats compared with normotensive (NT) controls. We report here
that the baclofen-induced pressor response (BIPR) is enhanced after
acute hypertension (AHT) of only 30 minutes. Sprague-Dawley rats were
anesthetized with Inactin, paralyzed, and artificially
ventilated. As we previously reported, after unilateral electrolytic
ablation of the NTS, microinjection of 40 pmol baclofen into the
contralateral NTS of NT rats resulted in a BIPR of 22±1 mm Hg
(n=12). During the infusion of phenylephrine for 30 minutes
(AHT), the BIPR was 39±5 mm Hg (n=10), significantly greater
than the response in NT rats
(P<0.01) and no different from
the response in CHT rats (39±5 mm Hg, n=7). Baclofen has both
presynaptic and postsynaptic effects. To eliminate the presynaptic
component of the baclofen response, sinoaortic denervation (SAD) was
performed before the microinjections. The magnitude of the BIPR was
12±1 mm Hg in NT-SAD rats (n=8), 12±1 mm Hg in AHT-SAD
rats (n=12), and 20±3 mm Hg in CHT-SAD rats (n=7). The BIPR is
enhanced in both CHT and AHT rats. It appears that the increase in
baroreceptor afferent input to NTS during
phenylephrine-induced AHT provides a greater substrate
for presynaptic inhibition by baclofen because the postsynaptic
component of the baclofen response is the same in NT-SAD and AHT-SAD.
The enhanced BIPR in CHT rats appears to be associated with an
enhancement of both the presynaptic and postsynaptic components of the
response.
Key Words: baroreceptors • baroreflex • electrophysiology • hypertension, renal
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The inhibitory amino acid
-aminobutyric acid (GABA) has been
demonstrated to be a potent modulator of neurons within the nucleus of
the solitary tract (NTS), the initial site of baroreceptor afferent
integration within the central nervous system. In vivo and in vitro
studies have demonstrated that the inhibition of NTS neurons and
afferent evoked discharge in these neurons by the
GABAB receptor agonist baclofen is mediated by
both presynaptic and postsynaptic
mechanisms.1 2 The
microinjection into the NTS of baclofen results in an increase in
arterial pressure, heart rate, and sympathetic nerve
discharge to the
kidney,3 4 5 6
which are predictable effects of inhibition of NTS neurons that
integrate baroreceptor afferent inputs. This baclofen-induced pressor
response (BIPR) is enhanced in chronically hypertensive rats,
specifically the spontaneously hypertensive rats (SHR) and
deoxycorticosterone
(DOCA)-salt5 and 1-kidney,
renal wrap models of
hypertension.6 The duration
that arterial pressure must be elevated before an enhanced
BIPR is observed is currently unknown. Furthermore, the relative
contributions of presynaptic and postsynaptic inhibitory
mechanisms to the BIPR are also unknown. The present study was designed to answer 2 questions. First, is the enhanced BIPR that is observed in chronic hypertension also observed in acute hypertension? Second, what are the relative contributions of presynaptic and postsynaptic inhibition to the BIPR? To answer the first question, we measured the BIPR in acutely hypertensive (30 minutes of phenylephrine infusion) and chronically hypertensive (4 weeks after unilateral nephrectomy, renal wrap surgery) rats. To answer the second question, we performed similar measurements before and after section of baroreceptor buffer nerves. The results demonstrate that after acute hypertension, the BIPR is enhanced by the same amount as previously reported in chronically hypertensive rats. The results suggest that in normotensive rats, the BIPR is an equal mix of presynaptic and postsynaptic inhibition. The results further suggest that during acute hypertension, the enhanced BIPR is mediated primarily by an increase in the presynaptic component of the BIPR, whereas in chronic hypertension, the enhanced BIPR is mediated by an increase in both the presynaptic and postsynaptic components of the BIPR.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Successful experiments were performed on adult male Sprague-Dawley rats (375 to 500 g; Charles River Laboratories or Harlan Sprague-Dawley Inc). Rats were housed 2 per cage in a fully accredited (Association for the Assessment and Accreditation of Laboratory Animal Care International and US Department of Agriculture) laboratory animal room with free access to food and water. All rats were given
1 week to acclimate before being used for any procedures.
The Institutional Animal Care and Use Committee approved all
experimental protocols.
Chronic Hypertensive Model
Hypertension was induced with a 1-kidney renal wrap
procedure. Rats were anesthetized with medetomidine (0.5 mg/kg
IP; Pfizer) and ketamine (75 mg/kg IP; Fort Dodge Laboratory).
A figure-8 Grollman renal wrap and contralateral nephrectomy were
performed on these animals.7
Control animals consisted of sham-operated rats that were similarly
anesthetized and received a unilateral nephrectomy but no wrap
of the contralateral kidney or of rats with no surgical procedures
before the day of the experiment. Because the responses of both groups
of normotensive rats were identical, they were grouped together for
analysis. Anesthesia was terminated by atipamezole
(1 mg/kg IP; Pfizer) at the conclusion of the surgical procedures.
Postoperative analgesic agents (Nubaine IM) were available as
needed.
Acute Surgical Preparation
Four to 6 weeks after the initial surgery,
hypertensive and sham-operated animals were anesthetized with
Inactin (100 mg/kg IP) and placed on a thermostatically controlled
heating pad. Body temperature was monitored with a rectal probe and
maintained at 36° to 38°C throughout the experiment. After
placement of a venous catheter in the femoral vein and cannulation of
the trachea, the rat was artificially ventilated with room air
supplemented with 100% O2. Additional
anesthetic was administered as needed (10 mg IV) to maintain stable
arterial pressure and heart rate at rest and during pinch
of the hind paw. Gallamine triethiodide (20 mg ·
kg-1 · 30
min-1 IV) was administered for paralysis. A
femoral artery was cannulated, and arterial pressure was
measured with a strain-gauge transducer. The rat was then placed in a
stereotaxic head frame, and an occipital
craniotomy was performed to expose the dorsal medulla
in the region of the obex.
Denervation of Reflexogenic Areas
To determine the contribution of
peripheral afferents to the baclofen-induced responses,
after the acute surgical preparation described here, some of the rats
underwent bilateral section of the carotid sinus and aortic nerves. In
some of these rats, a bilateral section of the vagal nerves was also
performed. The microinjection aspects of the protocol did not begin
until mean arterial pressure (MAP) returned to the
predenervation level, typically 30 to 60 minutes. At this time, a bolus
injection of phenylephrine was administered, and the
absence of a reflex bradycardia during a 35- to 50-mm Hg increase in
arterial pressure used to verify the adequacy of the
denervation.
Microinjection Protocol
We repeated the microinjection protocol originally
described by Tsukamoto and
Sved.5 A bipolar stimulating
electrode was placed in the NTS using coordinates provided by these
authors (0.5 mm rostral to calamus, 0.5 mm lateral to the
midline, and 0.5 mm below the surface of the brain). With this
electrode, an electrolytic lesion was placed in the right NTS to
eliminate reflex buffering of responses to drugs injected into the left
NTS. After a recovery period of 30 to 60 minutes, a glass micropipette
(outer tip diameter <50 µm) filled with baclofen (dissolved in
artificial cerebrospinal fluid, pH adjusted to 7.4) was placed into the
contralateral NTS with use of the same stereotaxic
coordinates. Baclofen was injected (40 pmol in a 100-nL volume) slowly
over 1 to 3 minutes with a pressurized source, and
cardiovascular parameters were measured
with a MacLab A/D system.
Acute Hypertension and Hypotension
To examine the effects of acute changes in pressure
on the BIPR, intravenous infusions of
phenylephrine (100 µg/mL) were used to raise the pressure
of normotensive rats to the same level as in chronic hypertension for
30 minutes. Intravenous infusions of nitroprusside (250
µg/mL) were used to lower the pressure of hypertensive rats to
normotensive levels for 30 minutes. To ensure that the enhanced BIPR
was not the result of changes at the vascular level, responses to bolus
injections of norepinephrine (0.1 µg) and
angiotensin II (30 to 40 ng) were examined during the acute
hypertension induced by phenylephrine infusion. A previous
study found that pressor responses to phenylephrine,
angiotensin II, and vasopressin were not altered in chronic
renal wrap
hypertension.8
Data Analysis
Pulsatile pressure and MAP were viewed and saved for
off-line analysis with a MacLab A/D system. Statistical
significance was determined with ANOVA with Tukey’s or Dunn’s tests
used for post hoc comparisons. Student’s
t test was used to
analyze the norepinephrine and
angiotensin II bolus injection data. All values are
expressed as mean±SEM, and significance was accepted at
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
BIPR in Buffer Nerve–Intact Rats
The baseline levels of MAP in the groups of animals studied are presented in Table 1. As previously described in the renal wrap model of hypertension, the microinjection of baclofen into the NTS of chronic hypertensive rats induces a pressor response that is greater than the pressor response induced by identical baclofen injections in normotensive rats (Figure, Table 2).
|
|
|
In the course of these studies, we made the chance observation that acute, spontaneous changes in arterial pressure altered the BIPR. We further examined this phenomenon using phenylephrine to acutely increase MAP in normotensive rats and nitroprusside to acutely lower pressure in chronically hypertensive rats. During phenylephrine infusions that increased the MAP of a normotensive rat to the same MAP as a chronically hypertensive rat for 30 minutes, the BIPR was enhanced (Figure, Table 2). The magnitude of the BIPR measured in acute hypertensive rats was the same as the BIPR observed in chronic hypertensive rats (Figure, Table 2). Furthermore, during nitroprusside infusions that lowered the MAP of chronic hypertensive rats to the same MAP as in normotensive rats for 30 minutes, the BIPR was reduced (Figure). The BIPR measured in these acutely normotensive rats was the same as the BIPR observed in sham, normotensive rats.
To determine whether the changes in the magnitude of the BIPR observed after acute increases in MAP were due to alterations in the contractile properties of blood vessels, the pressor response induced by a bolus intravenous injection of norepinephrine (n=6) or angiotensin II (n=7) was measured before and during a 30-minute phenylephrine infusion. Norepinephrine increased MAP by 23±2 mm Hg at the resting level of MAP and by 28±3 mm Hg during phenylephrine infusion (P=0.11). Angiotensin II increased MAP by 38±4 mm Hg at the resting level of MAP and by 41±3 mm Hg during phenylephrine infusion (P=0.67).
BIPR in Baroreceptor-Denervated Rats
To determine the contribution of presynaptic inhibition
of carotid sinus and aortic afferents to the BIPR, microinjection
experiments were performed in rats after bilateral section of the
carotid sinus and aortic nerves. Sinoaortic denervation (SAD) did not
alter baseline MAP in the 3 groups
(Table 1). The effects of SAD on the magnitude of the BIPR
are presented in
Table 2, and in all 3 groups, the difference in the
magnitude of the BIPR between the buffer nerve–intact and SAD animals
was significant. In normotensive, SAD animals, the BIPR was 55% of
that observed in buffer nerve–intact rats. In the acute hypertensive
rats, after SAD, the magnitude of the BIPR was identical to that
measured in normotensive, SAD rats. However, because the BIPR in the
buffer nerve–intact, acutely hypertensive rats was enhanced, the BIPR
in acute hypertensive, SAD rats was only 31% of that measured in acute
hypertensive, buffer nerve–intact rats. After SAD in chronically
hypertensive rats, the BIPR was 51% of that observed in buffer
nerve–intact, chronically hypertensive rats. The BIPR measured after
SAD in the chronically hypertensive rats was significantly greater than
the BIPR measured in both normotensive, SAD rats and in acute
hypertensive, SAD rats
(Table 2).
Table 2 also provides the difference between the magnitude
of the BIPR measured in the buffer nerve–intact and the SAD animals
for the 3 groups.
In addition to arterial pressure baroreceptor afferent inputs, it is possible that cardiopulmonary afferent inputs contribute to the BIPR. Therefore, the effects of bilateral vagotomy, in addition to SAD, were examined. Combining vagotomy with SAD did not significantly alter baseline MAP compared with buffer nerve–intact animals or the effects of SAD alone (Table 1). Combining vagotomy with SAD did not significantly alter the magnitude of the BIPR observed in normotensive rats (11±2 mm Hg), acute hypertensive rats (15±1 mm Hg), or chronic hypertensive rats (19±3 mm Hg) compared with the effects of SAD alone (P>0.05 for all 3 comparisons).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
These results confirm our previous observations in this model of hypertension6 and those of Tsukamoto and Sved5 in the SHR and DOCA-salt models of hypertension showing that the BIPR is enhanced in chronic hypertensive rats. The present study extends these observations by finding that the BIPR was enhanced after brief (30-minute) increases in MAP and that the enhanced BIPR observed in chronic hypertensive rats could be reduced to normal levels after brief (30 minutes) reductions in MAP. Furthermore, the present study attempted to estimate the relative contribution of presynaptic and postsynaptic mechanisms to the BIPR by examining the BIPR before and after SAD.
SAD is likely to lead to a number of alterations within the NTS, so interpretation of these data as being the result of solely the removal of afferent input is, no doubt, a simplistic assumption. However, the assumption is not without some merit. Our recent report that describes the responses of NTS neurons to changes in blood pressure found that reductions in blood pressure did not significantly reduce the spontaneous discharge of NTS neurons.9 Although baroreceptor unloading is not strictly analogous to SAD, it does suggest that removal of afferent input does not alter the spontaneous discharge of NTS neurons as dramatically as one might predict. Our present analysis cannot exclude possible presynaptic effects of baclofen on other excitatory inputs to NTS neurons, whether these inputs originate from the periphery or within the central nervous system. Keeping in mind the limitations and assumptions inherent in this analysis, we propose that the BIPR remaining after SAD reflects the postsynaptic component of the BIPR because the SAD removes the presynaptic component. Subtraction of the BIPR after SAD, the postsynaptic component, from the BIPR observed before SAD reveals the presynaptic component of the BIPR (Table 2, far right column, Intact-SAD). It is possible that vagal, cardiopulmonary afferents might also contribute to the BIPR and alter our estimates of the presynaptic versus the postsynaptic components of the response. Therefore, we also performed experiments in rats with combined SAD and vagotomy.
The results suggest that the BIPR observed in normotensive rats is an equal mix of presynaptic and postsynaptic inhibition. They also suggest that in normotensive rats, vagal afferents do not contribute to the BIPR. In acute hypertensive rats, the enhanced BIPR appears to be the result of an increase in the presynaptic component of the BIPR, the calculated postsynaptic component being identical to that observed in normotensive rats (12±1 mm Hg). A likely explanation for this observation is that the pronounced increase in baroreceptor afferent input to the NTS during acute increases in MAP provides a greater substrate for the presynaptic inhibitory effects of baclofen. Vagal afferents do not contribute to the BIPR observed in acute hypertension.
The situation in chronic hypertension is more complex. The results suggest that there is an increase in both the presynaptic and postsynaptic components of the BIPR. Jones and Thoren10 reported that in chronic hypertension, there is an increase in the number of baroreceptor afferents discharging at resting levels of MAP, and our observation of an increased presynaptic component of the BIPR in chronic hypertension is consistent with the interpretation that there is an increased baroreceptor afferent input to the NTS in chronic hypertension. The increase in the calculated presynaptic component is slightly less than that calculated in acute hypertension, and this could reflect some degree of baroreceptor resetting in chronic hypertension.
Of particular interest is the increase in the postsynaptic component of the BIPR in chronic hypertension, that is, the magnitude of the BIPR observed after SAD. We previously reported that the enhanced BIPR observed in chronic hypertension is associated with an increased expression of GABAB mRNA in the NTS,6 and on the basis of the present results, we propose that this increased mRNA results in increased expression of the postsynaptic GABAB receptor. We have found that the sensitivity of NTS neurons to exogenously applied baclofen is enhanced in chronic hypertension (J. Zhang and S.W. Mifflin, unpublished observations), and this is consistent with the interpretation of enhanced expression of the postsynaptic GABAB receptor. The exact stimulus for the increased postsynaptic expression of GABAB receptors remains to be defined, but it could be mediated by neurotransmitters released by baroreceptor afferents or other inputs to NTS neurons, or it could be related to alterations in the levels of a circulating hormone.
The normalization of the BIPR observed in chronic hypertensive rats during acute reductions in blood pressure, produced through intravenous infusion of nitroprusside, was surprising. If the enhanced BIPR observed in chronic hypertensive rats is mediated in part by an increase in the postsynaptic response to baclofen, we predicted that after the acute normalization of MAP, we would still observe an enhanced BIPR. However, we observed a BIPR that was not different from that observed in normotensive rats. The reason for this might be related to the degree to which we lowered MAP with nitroprusside. If there is some degree of receptor resetting in the presynaptic component as discussed earlier, then our decision to reduce MAP to the level observed in normotensive rats may account for this observation. Because there is baroreceptor resetting in chronic hypertension, the level of baroreceptor afferent input may be less in the chronic hypertensive rats than in the normotensive rats compared at the same MAP. This would lead to an overestimate of the presynaptic versus postsynaptic components of the BIPR.
To conclude, these results suggest that there is large degree of plasticity in the cardiovascular responses to the microinjection of baclofen in the NTS. Adaptations occur on an acute (30 minutes) and chronic (4 week) time scale, although we did not precisely define the temporal course of these adaptations. The adaptations appear to result from alterations in peripheral afferent input on a short time scale and alterations in both the level of afferent input and the neurons that integrate these afferent inputs on a longer time scale. It is proposed that these alterations reduce the level of increased excitatory input to NTS neurons due to increased afferent input in hypertension, whether the hypertension is acute or chronic. We have previously reported that the discharge of most NTS neurons is not altered in chronic hypertension11 ; therefore, we propose that this plasticity in the initial processing of cardiovascular afferent information represents an attempt to normalize NTS neuronal discharge in response to increased baroreceptor afferent input and to maintain some degree of reflex buffering capability.
|
Acknowledgments |
|---|
This work was supported by grant HL-56637.The authors gratefully acknowledge the expert technical assistance of Myrna Herrera-Rosales.
Received November 25, 2000; first decision November 28, 2000; accepted December 14, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Brooks PA, Glaum SR. GABAB receptors modulate a tetanus-induced sustained potentiation of monosynaptic inhibitory transmission in the rat nucleus tractus solitarii in vitro. J Auton Nerv Syst. 1995;54:16–26.[Medline] [Order article via Infotrieve]
2. Zhang J, Mifflin SW. Receptor subtype specific effects of GABA agonists on neurons receiving aortic depressor nerve inputs within the nucleus of the solitary tract. J Auton Nerv Syst. 1998;73:170–181.[Medline] [Order article via Infotrieve]
3. Sved JC, Sved AF. Cardiovascular responses elicited by gamma amino butyric acid in the nucleus tractus solitarius: evidence of the action of GABAB receptor. Neuropharmacology. 1989;28:515–520.[Medline] [Order article via Infotrieve]
4. Sved AF, Tsukamoto K. Tonic stimulation of GABAB receptors in the nucleus tractus solitarius modulates the baroreceptor reflex. Brain Res. 1992;592:37–43.[Medline] [Order article via Infotrieve]
5.
Tsukamoto K, Sved
AF. Enhanced -aminobutyric acid-mediated responses in nucleus
tractus solitarius of hypertensive rats.
Hypertension. 1993;22:819–825.
6.
Durgam VR, Vitela
M, Mifflin SW. Enhanced GABA-B receptor agonist responses and mRNA
within the nucleus of the solitary tract in hypertension.
Hypertension. 1999;33:530–536.
7. Grollman A. A simplified procedure for inducing chronic renal hypertension in the mammal. Proc Soc Exp Biol Med. 1944;57:102–104.
8.
Hinojosa-Laborde C,
Guerra P, Haywood JR. Sympathetic nervous system in high sodium
one-kidney, figure-8 renal hypertension.
Hypertension. 1992;20:96–102.
9.
Zhang J, Mifflin
SW. Responses of aortic depressor nerve evoked neurones in rat nucleus
of the solitary tract to changes in blood pressure.
J Physiol. 2000;529:2:431–443.
10. Jones JV, Thoren PN. Characteristics of aortic baroreceptors with non-medullated afferents arising from the aortic arch of rabbits with chronic renovascular hypertension. Acta Physiol Scand. 1977;101:286–293.[Medline] [Order article via Infotrieve]
11.
Zhang J, Mifflin
SW. Integration of aortic nerve inputs in hypertensive rats.
Hypertension. 2000;35:430–436.
This article has been cited by other articles:
 |
|
 |
|
  Q. Zhang, F. Yao, S. T. O'Rourke, S. Y. Qian, and C. Sun Angiotensin II enhances GABAB receptor-mediated responses and expression in nucleus tractus solitarii of rats Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1837 - H1844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Yao, C. Sumners, S. T. O'Rourke, and C. Sun Angiotensin II increases GABAB receptor expression in nucleus tractus solitarii of rats Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2712 - H2720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, M. Herrera-Rosales, and S. Mifflin Chronic Hypertension Enhances the Postsynaptic Effect of Baclofen in the Nucleus Tractus Solitarius Hypertension, March 1, 2007; 49(3): 659 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. G. Teschemacher, S. Wang, T. Lonergan, H. Duale, H. Waki, J. F. R. Paton, and S. Kasparov Targeting specific neuronal populations using adeno- and lentiviral vectors: applications for imaging and studies of cell function Exp Physiol, January 1, 2005; 90(1): 61 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Seagard, C. Dean, S. Patel, D. J. Rademacher, F. A. Hopp, W. T. Schmeling, and C. J. Hillard Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H992 - H1000. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Malpas What sets the long-term level of sympathetic nerve activity: is there a role for arterial baroreceptors? Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2004; 286(1): R1 - R12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Sved and J. C. Sved Plasticity of GABA function in the nucleus tractus solitarius in hypertension Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1272 - R1273. [Full Text] [PDF]
|
|
|
|
|
|
L. Mei, J. Zhang, and S. Mifflin Hypertension alters GABA receptor-mediated inhibition of neurons in the nucleus of the solitary tract Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1276 - R1286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Mifflin What Does the Brain Know About Blood Pressure? Physiology, December 1, 2001; 16(6): 266 - 271. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||