| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR |
1 Henry Wellcome L.I.N.E, University of Bristol, Whitson Street, Bristol BS1 3NY, UK
2 Department of Physiology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 21 June 2006;
accepted after revision 20 July 2006;
first published online 27 July 2006)
Corresponding author J. F. R. Paton: Department of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. Email: julian.f.r.paton{at}bristol.ac.uk
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The PVN contains magnocellular and parvocellular divisions. In the former are found the vasopressin- and oxytocin-containing neurones that project exclusively to the posterior pituitary whereas the latter region contains the ‘autonomic’ neurones that excite the sympathetic nervous system (Swanson & Sawchenko, 1983). Parvocellular neurones project to premotor sympathoexcitatory neurones located either in the rostral ventrolateral medulla (RVLM) and/or the sympathetic preganglionic neurones of the intermediolateral cell column (IML) of the spinal cord. Previous studies have shown that the autonomic parvocellular neurones play a significant role in cardiovascular regulation, particularly in response to homeostatic disturbance including plasma salt loading (Kannan et al. 1989; Weiss et al. 1996; May et al. 2000; Chen & Toney, 2001; Toney et al. 2003). What is not clear is which descending PVN parvocellular pathway(s) (either via the RVLM and/or direct spinal) mediate(s) the sympathoexcitatory response to plasma hyperosmolality, and what is (are) the neurotransmitter substance(s) employed.
Previous studies have demonstrated vasopressinergic terminals within the spinal cord including both the superficial dorsal horn and IML (Buijs, 1978; Nilaver et al. 1980). Vasopressinergic mechanisms at the spinal level have been associated with both the increase in arterial pressure, induced when vasopressin was applied intrathecally, and following direct stimulation of the PVN (Malpas & Coote, 1994; Porter & Brody 1986a,b; Tan & Tsou, 1986). However, what physiological stimulus results in activating the PVN-spinal vasopressinergic pathway has not been revealed. In the present study, we hypothesized that hypothalamic vasopressinergic spinally projecting neurones are activated during hyperosmolality, to release vasopressin within the spinal cord that results in sympathoexcitation. To this end, we developed an in situ decorticate arterially perfused rat preparation containing hypothalamic structures. This preparation was based on those previously described (Paton, 1996; Pickering & Paton, 2006), and allowed very precise and repeatable changes in systemic osmolality without cumulative salt-loading effects. Here, we (1) demonstrate the integrity of the PVN in this in situ preparation; (2) compare hyperosmolality-induced sympathetic responses with those evoked in the conscious in vivo rat; and (3) show that the sympathoexcitatory response to physiological levels of salt loading is mediated, primarily, by the PVN, and depends on the integrity of V1a receptors within the spinal cord.
Part of this study was presented to the Physiological Society in abstract form (Antunes et al. 2005).
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In vivo conscious rat studies
Adult male Wistar rats weighing 300–350 g were obtained from University of Bristol colony and kept at a constant temperature of 22°C and a relative humidity of 50–60% under a controlled light/dark cycle (12/12 h light/dark). Rats had access to normal rat chow and drinking water ad libitum.
Cardiovascular parameter recording. A radiotelemetry system (Data Sciences International, Arden Hills, MN, USA) was used for recording of arterial pressure as previously described (Waki et al. 2003, 2006). The system consists of three basic elements: (1) a transmitter for monitoring arterial pressure (TA11PA-C40); (2) a receiver (RP-1); and (3) an adapter (R11CPA) with an ambient pressure monitor (APR-1) to output analog signals of arterial pressure. The system is calibrated relative to atmospheric pressure. The pulsatile blood pressure signal was recorded and then stored on a computer via an analog input box (IP-810 A, GigaTex Co, Ltd, Miyagi, Japan) connected to an analog I/OPC card(AD 12–8(PM), CONTECCo Ltd, Japan) for digitization.
Transmitter implant surgery. Each transmitter was implanted at least 5 days before any experimental protocol. Rats were anaesthetized with a mixture of ketamine (60 mg kg–1) and medetomidine (250 mg kg–1) injected intramuscularly. The level of anaesthesia was checked frequently by assessing limb withdrawal reflexes to noxious pinching. A midline laparotomy was made in the supine position, and the intestines were reflected to expose the abdominal aorta. The tip of the catheter (outside diameter 0.7 mm, thin-walled thermoplastic membrane) connected to transmitter (diameter 15 mm) was inserted into the abdominal aorta caudal to the root of the left renal artery and held in place with tissue adhesive (Vetbond, 3M). The body of the transmitter was sutured to the ventral wall of the abdominal cavity. After surgery, anaesthesia was reversed with a subcutaneous injection of atipamezole (1 mg kg–1), a prophylactic antibiotic was injected intramuscularly (penicillin, 1000 U), and the rats were returned to their home cages for recovery (typically five days). The day before experiments rats were re-anaesthetized with halothane (5%) and the right femoral vein was cannulated with a catheter (PE-10 connected to PE-50 (Clay Adams, Parsippany, NJ, USA)), tunnelled subcutaneously, and exteriorized through the back of the neck for systemic drugs delivery.
Systemic drugs delivery.
Rats initially received intravenous (I.V.) injections of isotonic saline (154 mM NaCl) in which the plasma osmolality was 289 ± 1 mosmol (kg water)–1. On the following day the same group of rats (n
=
5) received the same volume of hypertonic saline (HS, 3 M NaCl), which resulted in an increased plasma osmolality of 299 ± 1 mosmol (kg water)–1 (peak response) as measured 90 s after injection. In a second group of rats, HS (I.V.) was injected after pre-treatment with prazosin (
1-adrenoceptor antagonist, 1 mg kg–1 ml–1, I.V.). The efficacy of this drug dose was challenged with phenylephrine (15 µg kg–1 ml–1, I.V.), and found sufficient to block the evoked pressor response. In a third group, HS (I.V.) was injected after pretreatment with [ß-mercapto-ß,ß-cyclopentamethylene-propionyl1,O-me-Tyr2,-Arg8]-vasopressin (a V1a receptor antagonist, 10 µg kg–1 ml–1, I.V.). The efficacy of this antagonist was tested by intravenous injection of [Arg8]-vasopressin acetate (AVP, 10 µg kg–1 ml–1). To avoid the cumulative effects of salt loading, HS was administrated once only in each rat. Either HS or isotonic saline was administered at a volume of 0.14 ml 100 (g body weight)–1 and lasted for 40 s.
Plasma osmolality measurements. Blood samples (200 µl) were collected via a venous catheter using heparinized syringes. After collection of each sample, an equal volume of isotonic saline was reintroduced (I.V.) in order to avoid changes to the circulating blood volume. For the first five minutes, blood samples were collected every minute after HS administration (NaCl 3 M, 0.14 ml kg–1, I.V.). Subsequent blood samples were collected every 30, 90, 120, and 180 min post-HS injection. The plasma osmolality was measured using a freezing point depression osmometer (Camlab, Roebling Micro-osmometer, Cambridge, UK).
Data analyses. Telemetry data were acquired using Hey-Presto software (Waki et al. 2006) through an analog input box and I/O PC card (as described above) displayed on the computer screen and stored on the hard drive. The mean arterial pressure (MAP) was exported into Excel (Microsoft Corporation, USA) for further statistical evaluation. The MAP values were averaged over 180 min post-osmolality challenge. The first 5 min of recording immediately after injection of HS (I.V.) were averaged every 30 s, in order to obtain high temporal resolution of the changes in MAP elicited by HS and drugs. For the remaining time, MAP data was sampled every 15 min. Changes in MAP (mmHg) were statistically analysed by two-way ANOVA followed by Bonferroni post hoc test for multiple comparisons, and the differences were considered significant at P < 0.05. All values are expressed as the mean ± standard error of mean (S.E.M.), and n is the number of rats in each group.
Decorticate, unanaesthetized, arterially perfused in situ preparation of rat
Male Wistar rats weighing 70–90 g were anaesthetized deeply with halothane (5%), and the level of anaesthesia was assessed by a failure to respond to a noxious pinch of either a paw or the tail. The stomach, intestines and spleen were ligated and removed via midline laparotomy. The sternum was split and the ribcage retracted to allow access to the mediastinum. The pericardium was removed and the left phrenic nerve was isolated. The animal was submerged in cooled Ringer solution (see below) and the cerebral hemispheres exposed by removal of the parietal bones. The cerebral cortices, hippocampus and thalamic area were removed by gentle aspiration. The preoptic area and its adjacent septal nuclei and hypothalamic areas remained largely intact (see Fig. 7A and B). The preparation was skinned and transferred to the recording chamber. A double-lumen cannula was introduced into the ascending aorta via the left ventricle. The preparation was perfused at flow rates of 28 ± 2 ml min–1 using a roller pump (Watson Marlow 505S) with Ringer solution containing an oncotic agent (Ficoll 70, 1.25%; Sigma, St Louis, USA), gassed with carbogen (95% O2 and 5% CO2), warmed to 32°C and filtered using a nylon screen (pore size: 25 µm). After respiratory-related movements commenced, a neuromuscular blocker (vecuronium bromide, 40 µg ml–1, Norcuron Organon Teknika) was added to the perfusate to stabilize mechanically the preparation. The second lumen of the cannula was used to monitor aortic perfusion pressure. Phrenic nerve activity (PNA) was recorded from its distal end using a glass suction monopolar electrode held in a 3-D micromanipulator. Rhythmic ramping PNA gave a continuous physiological index of preparation viability. The lumbar sympathetic chain (L2–L3) was visualized through a binocular microscope and recordings made from the distal cut end using a bipolar glass suction electrode. Signals were AC-amplified (Neurolog NL104) and band-pass filtered (8 Hz–3 kHz). Sympathetic nerve activity exhibited marked respiratory modulation, and was profoundly attenuated by an increase in perfusion pressure (arterial baroreceptor stimulation).
|
Microinjection studies. The head of the preparation was fixed by ear bars and a nasal clamp. The angle of the head was positioned so that the dorsal surface of the brainstem was horizontal in every experiment; this aided accurate and consistent placement of micropipettes into the PVN. In pilot studies we mapped out the PVN based on co-ordinates relative to the superior colliculus (SC) (i.e. 2.7 mm rostral, 0.3 mm lateral to midline and 3.4 mm below the brain surface). A three-barrelled glass micropipette (external tip diameter 10–30 µm) was placed into the PVN using a 3-D micromanipulator. The volume microinjected (60 nl) was determined by viewing the movement of the meniscus through a binocular microscope fitted with a precalibrated eyepiece reticule. All microinjections were made bilaterally within a period of 60 s, and the osmotic stimulus tested 2, 15 and 30 min post-microinjection.
Intrathecal injections. A retractor attached to the pelvis was used to stretch the spinal cord to facilitate access. Two vertebral processes were removed at the L2–L3 level. A calibrated glass micropipette (Accu-Fill 90, Micropet; Clay Adams, Parsippany, NJ, USA) was positioned into the subarachnoid space using a 3-D micro manipulator and a volume of up to 0.5 µl injected. In order to check the approximate spread of drugs, a 2% solution of Pontamine Sky Blue was injected at the same site at the end of each experiment.
Osmotic stimulus. The in situ preparation was initially perfused with isosmotic Ringer solution containing (mM): NaCl 120, NaHCO3 24, KCl 5, CaCl2 2.5, MgSO4 1.25, KH2PO4 1.25, dextrose 10; pH 7.3 after carbogenation. The osmolality, measured by a freezing point depression osmometer, was 291 ± 1 mosmol (kg water)–1. Hyper- and hyposmotic ionic Ringer solution were prepared by adjusting the final concentration of NaCl. In addition, a non-NaCl osmolyte (mannitol) was used. Hyperosmotic Ringer solution contained either 135 mM NaCl or mannitol (0.5%), and resulted in osmolalities of 321 ± 1 and 320 ± 1 mosmol (kg water)–1, respectively. The hyposmotic solution contained 105 mM resulting in 259 ± 1 mosmol (kg water)–1. Initially the in situ preparation was perfused with isotonic Ringer solution. A second reservoir of either hyper- or hyposmotic Ringer solution was connected to the perfusion system via a three-way tap and circulated for 40 s. This exposure time was established in pilot experiments to produce a submaximal, reversible and highly reproducible increase in sympathetic nerve activity.
Histological analysis. PVN injection sites were marked with 2% Pontamine Sky Blue (60 nl) contained in one barrel of a three-barrel micropipette. The brain was removed and fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Coronal sections (45 µm thick) were cut using a cryostat (CM1900, Leica, Switzerland) and thaw-mounted on gelatin-subbed glass slides. The sections were counterstained with methyl green, and the injection sites documented on predrawn atlas sections from Paxinos & Watson (1996). Only data in which the microinjections were confirmed to be within the PVN were analysed in this study. The histology for the brain transection protocols followed the same procedure described above; however, these brains were cut in the sagittal plane for better visualization of the CVOs (Fig. 7).
Data analyses. All in situ rat data were acquired using a CED 1401 A–D interface (CED, Cambridge Electronic Design, Cambridge, UK) and a computer running Spike 2 software (CED) with custom-written scripts for data acquisition and on- and off-line analyses. Lumbar sympathetic nerve activity (LSNA) was displayed as a moving average (100 ms time constant) and measured as the area under the curve per second, both 40 s before and 40 s after an osmotic stimulus. To standardize the data across preparations, LSNA changes were expressed as a percentage of baseline. Baseline noise was assessed by application of lidocaine (2%) to the sympathetic chain at the end of the experiment. One-way ANOVA for repeated measures followed by Tukey's post hoc test was used, and differences were taken as significant at P < 0.05. All values are expressed as the mean ± standard error of mean (S.E.M.) and n is the number of preparations.
Drugs. The following drugs were either microinjected or given intrathecally: L-glutamate (20 mM), kynurenic acid (glutamatergic antagonist of ionotropic receptors, 100 mM), isoguvacine (selective GABAA agonist, 1 mM), ß-mercapto-ß,ß-cyclopentamethylenepropionyl1,O-me-Tyr2,-Arg8-vasopressin (V1a receptor antagonist, 1 µg ml–1), Phe2,Ile3,Orn8-Vasopressin (V1 receptor agonist, 1 µg ml–1), AVP, pontamine Sky Blue dye (2%). All drugs were purchase from Sigma, UK, with the exception of V1 agonist, which was purchased from Bachem, UK.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Intravenous injections of hyperosmotic saline (HS) increase arterial pressure in conscious rats (n = 15). HS injected intravenously, which produced an increase in plasma osmolality to 299 ± 1 mosmol (kg water)–1 elicited an increase in MAP (120 ± 1 mmHg; P < 0.05; n = 5; Fig. 1) that reached a peak 90 s following the injection. An injection of the same volume of isotonic saline (I.V.) had no significant effect on either MAP (102 ± 2 mmHg; n = 5) or plasma osmolality (289 ± 1 mosmol (kg water)–1). Five minutes after HS injection, MAP started to return to baseline levels and was fully recovered 180 min later (Fig. 1 inset).
|
The intravenous infusion rate of HS (0.14 ml.100 (g body weight)–1 for 40 s) produced neither cardiac arrthymia nor a change in heart rate at any time point when compared to isotonic saline injections (data not shown). In a separate group of rats (n
= 3), we verified that the pressor effect elicited by phenylephrine (
MAP: 54 ± 4 mmHg; 15 µg kg–1, I.V.) was attenuated significantly by prazosin (1 mg kg–1 ml–1; MAP was 3 ± 1 mmHg). Moreover, the efficacy of the V1a antagonist was confirmed by measuring the pressor response to I.V. injection of [Arg8]-vasopressin acetate (AVP, 10 µg–1 ml–1). The pressor responses elicited by AVP (MAP: 57 ± 4 mmHg) were reduced significantly after administration of the V1a antagonist (MAP: 1 ± 0.5 mmHg).
In situ studies
The integrity of the hypothalamus is essential for the sympathoexcitation induced by different osmotically active solutes. To validate the integrity of the in situ decorticate preparation, we switched the isosmotic solution to a second reservoir containing hyperosmotic Ringer solution. We anticipated that this should elevate sympathetic nerve activity as seen in the in vivo rat. Indeed, the hyperosmotic perfusate (135 mM NaCl) induced sympathoexcitation (32 ± 5%; n = 6; Fig. 3). This effect was found to be reproducible eliciting consistent responses during repeated osmotic challenges. Next, we wanted to test whether the sympathetic response was mediated by hypothalamic structures. To this end, we challenged the preparation with a hyperosmotic stimulus (135 mM NaCl) before (intact hypothalamus, Fig. 2A) and after precollicular transection (no hypothalamus, Fig. 2A). Switching from the isosmotic to hyperosmotic perfusate increased the LSNA by 32 ± 5% (Fig. 3), which was attenuated significantly after the transection (i.e. 7 ± 3%, P < 0.01; n = 6; Fig. 3). It is important to note that after precollicular transection the LSNA baseline did not change (Fig. 2A).
|
|
The integrity of the CVOs is essential for the hyperosmolality evoked sympathoexcitation. In order to verify central detection of the hyperosmotic stimulus, we performed a selective ablation of the CVOs sparing the paraventricular nucleus (see Fig. 7B). Figure 2B shows recordings of the integrated and raw LSNA from a group of preparations (n = 4) during isosmotic (120 mM NaCl) and hyperosmotic conditions (135 mM NaCl), before and after transection of the CVOs. The increase in the LSNA by 21 ± 2%; n = 4) was significantly reduced (2 ± 1%; P < 0.001; Fig. 3) after transection of the CVOs. Importantly, this lesion did not change basal LSNA.
Hyposmotic-induced sympathoexcitation is not mediated via the hypothalamus. For comparison, we also challenged the preparation with a hyposmotic stimulus (105 mM NaCl; Fig. 2D) before and after precollicular transection. As with the hyperosmotic stimulus, the hyposmotic perfusate increased LSNA (20 ± 3%, n 6; Fig. 3). However, in stark contrast to hyperosmotic perfusate, the sympathoexcitation persisted after removing the hypothalamus (i.e. 21 ± 3%, n = 6; Fig. 3).
Because the changes in sympathetic activity to a hyperosmotic stimulus (135 mM NaCl) were robust and repeatable it was only this variable that we studied further.
The PVN plays a significant role in mediating the hyperosmotic-induced sympathoexcitation. To further validate the preparation and to demonstrate a role for the PVN in mediating the hyperosmolality-evoked sympathoexcitatory responses, we performed a number of microinjection experiments using two different protocols: (i) a reversible inactivation of the PVN with bilateral microinjections of isoguvacine (a GABAA receptor agonist); (ii) blockade of excitatory amino acid ionotropic receptors with kynurenic acid (a non-selective ionotropic glutamatergic receptor antagonist). Following isoguvacine, baseline LSNA was unperturbed but the hyperosmotic-induced increase in LSNA (25 ± 2%) was attenuated significantly (7 ± 2%; P < 0.01; n 5; Fig. 4A), but recovered to a level not different from control 15 min after the microinjection (i.e. 23 ± 2%). Similarly, the hyperosmotically induced sympathoexcitation (25 ± 2%; Fig. 4B) was attenuated significantly at 2 min (8 ± 3%; P < 0.01) and 15 min (13 ± 4%; P < 0.05) after kynurenic acid microinjections into the PVN in a separate group of preparations (n = 5). By 30 min, the response had recovered fully (i.e. 27 ± 3%). As seen with isoguvacine, bilateral microinjection of kynurenic acid failed to alter basal LSNA.
|
|
|
Brain transection and PVN microinjection sites. Careful histological analysis revealed that the transections effectively dissociated the CVO from the preparation while keeping the PVN intact (Fig. 7A and B), while those made at the pre-collicular level effectively removed all brainstem and spinal inputs from the hypothalamus. As demonstrated by the coronal (Fig. 7C) and schematic diagrams (Fig. 7D), all of the microinjections sites (isoguvacine, kynurenic acid, L-glutamate and hyperosmotic Ringer solution) that provided the data presented here were found within the PVN. In a group of three rats, microinjection sites (isoguvacine and kynurenic acid) were found to be outside the PVN. In these animals the HS-induced sympathoexcitation was not affected, concluding that the effects of isoguvacine or kynurenic acid are highly site-specific and confined to the PVN.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
HS in conscious rats elicited an immediate increase in MAP (Fig. 1) that completely recovered 180 min later. This pressor response was expected since it is well-established that hypertonic saline injected intravenously produces a pressor effect (Garcia-Estan et al. 1989; Akins & Bealer, 1990; Bealer, 2000). Studies performed by Garcia-Estan et al. (1989) have shown that the hypertensive response was due to a sympathetically mediated vasoconstriction. Our results are in agreement with this, since the pretreatment with prazosin blunted the pressor effect elicited by HS (I.V.). After prazosin, we revealed an initial hypotensive response that could be due to a direct effect of HS-induced vasodilatation on some vascular beds (Steenbergen & Bohlen, 1993; Pedrino et al. 2005), which is normally masked by the concomitant sympathetically mediated vasoconstriction. Since the administration of isotonic saline had no effect on basal arterial pressure, we can rule out that the increase in blood pressure was not secondary to volume expansion.
Since peripheral administration of HS increases circulating levels of vasopressin, it has been postulated that it may be responsible for the rise in arterial pressure (Landgraf et al. 1988; Russ et al. 1992). Therefore, we sought to investigate the role of vasopressin on the hyperosmolality-induced pressor responses in conscious rats. Our results have shown that injection of HS produced no significant change in MAP at any time point after pre-treatment with a V1a receptor antagonist (Fig. 1). Therefore, we suggest that the initial increase in MAP with acute salt loading is due to a vasoconstrictor effect of both the sympathetic nervous system (1-adrenoceptor and V1a receptor), whereas the prolonged elevations of MAP following hyperosmolality are mediated by a V1a receptor-sensitive mechanism only.
Taking the results from our in vivo experiments, we hypothesized that the integrity of hypothalamic vasopressinergic spinally projecting neurones was essential for the sympathoexcitation. This was assessed in a in situ decorticate arterially perfused rat preparation that left intact the lamina terminalis areas comprising the median preoptic nucleus (MnPO), subfornical organ (SFO) and organum vasculosum of the lamina termalis (OVLT), as well as the adjacent septal nuclei and hypothalamic areas (Fig. 7A). This preparation avoided the need for anaesthesia, allowed accurate control of the circulating osmolality, and exhibited robust, reproducible sympathetic nerve activity responses using physiologically relevant adjustments in osmolality. We suggest that the absence of anaesthesia helped preserve the sensitivity of the system to osmotic changes that were well within physiological limits. By using separate perfusate we also avoided the compounding problem of cumulative salt loading effects during repeated hyperosmotic challenges as can be the case in vivo.
The hypothalamus plays a critical role in homeostatic adjustments related to fluid balance disturbance (Antunes-Rodrigues et al. 2004). Therefore, we sought to test the importance of hypothalamic structures (particularly the PVN) in mediating the osmolality-induced sympathoexcitation. When the in situ preparation was challenged with an acute hyperosmotic stimulus (135 mM NaCl) of 321 ± 1 mosmol (kg water)–1, for 40 s, we observed an increase in the LSNA before, but not after, removal of the hypothalamus (precollicular transection). Interestingly, we did observe a small residual increase in the LSNA following HS after the precollicular transection (7 ± 3%). This may be a direct effect of excess sodium chloride on the brainstem or spinal cord neurones (see Isawa et al. 2000). We also determined that the preparation was sensitive to another, non-NaCl osmolyte, namely mannitol. Our data show that a hyperosmotic stimulus using mannitol elicited an increase in sympathetic nerve activity, which was practically eliminated after precollicular transection.
Several studies have demonstrated the presence of osmosensitive receptors in neurones of the lamina terminalis, which contains the CVOs (for review see Bourque et al. 1994; Bourque & Oliet, 1997). These brain areas lack a complete blood–brain barrier, and they are intrinsically sensitive to changes in fluid osmolality. As the precollicular transection removes the entire hypothalamus we could not predict exactly which forebrain area could drive the hyperosmotic-induced sympathoexcitation. Hence, we performed a selective ablation of the CVOs, while leaving the PVN intact (Fig. 7A and B). After severing connections from the CVOs, the increase in LSNA elicited by hyperosmotic stimulus was almost abolished indicating that the integrity of CVOs and their neuronal connections to the PVN are essential for osmolality-driven sympathoexcitation. Indeed, the absence of peripheral osmoreceptors in our preparation, such as those located in mesenteric and hepatic portal vasculature (Vallet & Baertschi, 1980; Baertschi & Vallet, 1981; Hosomi & Morita, 1996), further support a role for central osmoreceptors in mediating the sympathoexcitation to salt loading.
We suppose that the hyperosmolality-induced sympathoexcitation involves excitation of PVN neurones by central osmoreceptors (SFO, OVLT and MnPO); the latter are well known to detect changes in osmolality (see: Sawchenko & Swanson, 1983; McKinley et al. 1992; Bains & Ferguson, 1995; Weiss et al. 1996; Toney et al. 2003). This is supported by the absence of sympathoexcitation after: (i) selective CVO lesions; (ii) hyperpolarization of the PVN with isoguvacine; and (iii) blockade of ionotropic glutamatergic transmission in the PVN; the latter emphasizing the importance of glutamate receptors. Moreover, our data do not support the presence of osmoreceptors in the PVN (at least up to 321 ± 1 mosmol (kg water)–1), since hyperosmotic perfusate injected into the PVN had a negligible effect on LSNA (Fig. 5). Finally, we found that the integrity of the PVN is essential for mediating hyperosmolality-induced sympathoexcitation
Interestingly, an acute hyposmotic stimulus (259 ± 1 mosmol) also produced an increase in LSNA that persisted after precollicular transection and is therefore not hypothalamic dependent. Autonomic adjustments to hyposmotic stimulus are controversial. Brooks et al. (2005) showed that intracarotid hypotonic fluid infusion in dehydrated rats evoked a decrease in LSNA. In contrast, Brown et al. (2005) found that water (providing a hyposmotic stimulus), but not saline drinking in human subjects, increased sympathetic activity. In light of our results, we suggest that hyposmotic perfusate may act directly on the brainstem/spinal cord to elicit spontaneous firing of neurones due to an imbalance of sodium chloride (Isawa et al. 2000). This remains to be tested.
It is well established that an osmotic challenge elicits a sympathoexcitation, and parvocellular neurones from the PVN play a pivotal role in mediating this response (Swanson & Kuypers, 1980; Kannan et al. 1989; Badoer, 1996; Toney et al. 2003; Stocker et al. 2004a). Indeed, the PVN contains parvocellular neurones with projections to the intermediolateral cell column of the spinal cord and rostroventrolateral medulla, two major areas regulating sympathetic nerve activity (Cechetto & Saper, 1988; Swanson & Sawchenko, 1983; Coote, 1995; Stern, 2004; Stocker et al. 2004b). While we have demonstrated a role for glutamatergic transmission in the PVN for the hyperosmolality-driven sympathoexcitation, we recognize the importance of circulating (DiBona & Jones, 2001) and CVO-released angiotensin II (ANGII) in the PVN (Li & Ferguson, 1993), as well as its interactions with GABA (Chen & Toney, 2001; Unger et al. 1983). Indeed, GABAA (Decavel & Van den Pol, 1990; Haywood et al. 2001) and ANGII-AT1 receptors (Tsutsumi & Saavedra, 1991) are both expressed in autonomic regions of the PVN. As ANGII excites spinally projecting PVN neurons by attenuating GABAergic synaptic inputs presynaptically (Li et al. 2003), we acknowledge a possible contributing role for ANGII in producing the sympathoexcitation to hyperosmolality in this study.
With regard to PVN efferent pathways, we cannot rule out the glutamatergic projections to the RVLM-spinal projections. Brooks et al. (2004) have reported that antagonism of glutamate receptors in the RVLM reduced the increase in LSNA produced by acute hyperosmolality in water-deprived rats, suggesting a role for the RVLM. This contrasts to our study showing a dominant role for the PVN-spinal neurones. The difference might be explained by the presence of anaesthesia in the sudy of Brooks et al. (2004) (2004). Further, Stocker et al. (2004b) demonstrated that parvocellular PVN neurones projecting to the RVLM and spinal cord were activated (c-Fos immunoreactivity) by water deprivation. Thus, we accept that PVN neurones projecting to the RVLM may also contribute to the hyperosmolality-induced sympathoexcitation (see Stocker et al. 2006).
Projections from the PVN to the brainstem and spinal cord were demonstrated (Saper et al. 1976; Shafton et al. 1998; Pyner & Coote, 1999, 2000), giving an anatomical correlate for the long-known influence of the hypothalamus on the autonomic nervous system (Swanson & Sawchenko, 1983). Fibres immunoreactive for vasopressin make up an abundant terminal network in the spinal cord, both in the superficial dorsal horn and in the intermediolateral cell column (Buijs, 1978; Nilaver et al. 1980), and spinal vasopressin has been associated with sympathoexcitation (Porter & Brody 1986a,b; Malpas & Coote, 1994). Since blockade of vasopressin receptors (subtype V1a) in the spinal cord at the lower thoracic and upper lumbar level attenuated the sympathoexcitation induced by hyperosmotic stimuli, we suggest that it is the vasopressinergic hypothalamic-spinally projecting neurones that mediate the hyperosmolality-induced sympathoexcitation on the preganglionic sympathetic neurones. One possible caveat of the present study is that while we demonstrated that the V1a receptor antagonist used blocked the sympathoexcitation to spinally applied vasopressin, we did not show specificity of action in terms of whether other non-vasopressinergic excitatory responses (reflex or centrally evoked) were affected as a consequence of non-specific drug action. However, in the absence of any change in the ongoing activity (both tonic and respiratory modulated), we are confident that the antagonist was primarily blocking V1a receptors.
Hallbeck & Blomqvist, (1999) have shown that over 40% of the spinally projecting neurones in the PVN express vasopressin mRNA, and demonstrated that these neurones are localized to specific parts of the PVN. Moreover, studies performed by Motawei et al. (1999) have demonstrated that terminals containing vasopressin-like immunoreactivity are similarly distributed to PVN-descending axons, and in addition terminate near to and are closely associated with preganglionic sympathetic neurones. Malpas & Coote (1994) have demonstrated that V1a antagonist administered intrathecally in anaesthetized rats was able to block the increases in the renal sympathetic activity in response to electrical stimulation of the PVN, or to intrathecal vasopressin injection, ensuring the important role of vasopressin in the PVN-spinal pathway. Thus, based on the fact that the main source of vasopressin in the spinal cord arises from parvocellular neurones of the PVN (Sawchenko & Swanson, 1982; Lang et al. 1983; Cechetto & Saper, 1988), and that sympathetic preganglionic neurones express vasopressin V1a receptors (Sermasi et al. 1998), which are directly excited by vasopressin (Ma & Dun, 1985; Sermasi & Coote, 1994), we suggest that PVN vasopressinergic spinally projecting neurones could mediate the hyperosmolality-induced sympathoexcitation reported herein.
In summary, the in situ preparation is a promising model for understanding hypothalamic, brainstem and spinal cord mechanisms at the systems and cellular levels in the homeostatic regulation of blood pressure and volume. We have demonstrated that this preparation exhibits appropriate autonomic responses to repetitive hyperosmotic stimuli within a physiological osmolality range (± 30 mosmol (kg water)–1 from isotonic 291 ± 1 mosmol (kg water)–1), and that this response is dependent upon the integrity of the PVN with involvement of glutamatergic neurotransmission. In conclusion, we state that vasopressin plays a significant role at the level of the spinal cord for relaying the hyperosmolality-induced sympathoexcitation.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Antunes VR, Yao ST, Murphy D, Pickering AE & Paton JFR (2005). An in situ preparation for studying hypothalamic control of sympathetic activity. J Physiol 567P, PC51.
Antunes-Rodrigues J, de Castro M, Elias LL, Valenca MM & McCann SM (2004). Neuroendocrine control of body fluid metabolism. Physiol Rev 84, 169–208.
Badoer E (1996). Cardiovascular role of parvocellular neurones in the paraventricular nucleus of the hypothalamus. News Physiol Sci 11, 43–47.
Baertschi AJ & Vallet PG (1981). Osmosensitivity of the hepatic portal vein area and vasopressin release in rats. J Physiol 315, 217–230.
Bains JS & Ferguson AV (1995). Paraventricular nucleus neurons projecting to the spinal cord receive excitatory input from the subfornical organ. Am J Physiol 268, R625–R633.[Medline]
Bealer SL (2000). Central control of cardiac baroreflex responses during peripheral hyperosmolality. Am J Physiol 278, R1157–R1163.
Bourque CW & Oliet SHR (1997). Osmoreceptors in the central nervous system. Annu Rev Physiol 59, 601–619.[CrossRef][Medline]
Bourque CW, Oliet SH & Richard D (1994). Osmoreceptors, osmoreception, and osmoregulation. Front Neuroendocrinol 15, 231–274.[CrossRef][Medline]
Brooks VL, Freeman KL & O'Donaughy TL (2004). Acute and chronic increases in osmolality increase excitatory amino acid drive of the rostral ventrolateral medulla in rats. Am J Physiol 287, R1359–R1368.[CrossRef]
Brooks VL, Qi Y & O'Donaughy TL (2005). Increased osmolality of conscious water-deprived rats supports arterial pressure and sympathetic activity via a brain action. Am J Physiol 288, R1248–R1255.
Brown CM, Barberini L, Dulloo AG & Montani JP (2005). Cardiovascular responses to water drinking: does osmolality play a role? Am J Physiol Regul Integr Comp Physiol 289, R1687–R1692.
Buijs RM (1978). Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Pathways to the limbic system, medulla oblongata and spinal cord. Cell Tissue Res 192, 423–435.[Medline]
Camacho A & Phillips MI (1981). Horseradish peroxidase study in rat of the neural connections of the organum vasculosum of the lamina terminalis. Neurosci Lett 25, 201–204.[CrossRef][Medline]
Cechetto F & Saper CB (1988). Neurochemical organisation of the hypothalamic projection to the spinal cord in the rat. J Comp Neurol 272, 579–604.[CrossRef][Medline]
Chen QH & Toney GM (2001). AT(1)-receptor blockade in the hypothalamic PVN reduces central hyperosmolality-induced renal sympathoexcitation. Am J Physiol Regul Integr Comp Physiol 281, R1844–R1853.
Coote JH (1995). Cardiovascular function of the paraventricular nucleus of the hypothalamus. Biol Signals 4, 142–149.[Medline]
Decavel C & Van den Pol AN (1990). GABA: a dominant neurotransmitter in the hypothalamus. J Comp Neurol 302, 1019–1037.[CrossRef][Medline]
DiBona GF & Jones SY (2001). Effect of dietary sodium intake on the responses to bicuculline in the paraventricular nucleus of rats. Hypertension 38, 192–197.
Garcia-Estan J, Carbonell LF, Garcia-Salom M, Salazar FJ & Quesada T (1989). Hemodynamic effects of hypertonic saline in the conscious rat. Life Sci 44, 1343–1350.[CrossRef][Medline]
Hallbeck M & Blomqvist A (1999). Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus. J Comp Neurol 411, 201–211.[CrossRef][Medline]
Hatzinikolaou P, Gavras H, Brunner HR & Gavras I (1980). Sodium-induced elevation of blood pressure in the anephric state. Science 209, 935–936.
Hatzinikolaou P, Gavras H, Brunner HR & Gavras I (1981). Role of vasopressin, catecholamines, and plasma Volume in hypertonic saline-induced hypertension. Am J Physiol 240, H827–H831.[Medline]
Haywood JR, Mifflin SW, Craig T, Calderon A, Hensler JG & Hinojosa-Laborde C (2001). -Aminobutyric acid (GABA)-A function and binding in the paraventricular nucleus of the hypothalamus in chronic renal-wrap hypertension. Hypertension 37, 614–618.
Hosomi H & Morita H (1996). Hepatorenal and hepatointestinal reflexes in sodium homeostasis. News Physiol Sci 11, 103–107.
Isawa S, Inone K, Adachi A & Furahashi M (2000). Activity of neurons in the nucleus of the solitaary tract of rats: effect of osmotic and mechanical stimuli. Neurosci Lett 288, 33–36.[CrossRef][Medline]
Johnson AK, Cunningham JT & Thunhorst RL (1996). Integrative role of the lamina terminalis in the regulation of cardiovascular and body fluid homeostasis. Clin Exp Pharmacol Physiol 23, 183–191.[Medline]
Kannan H, Hayashida Y & Yamashita H (1989). Increase in renal sympathetic outflow by paraventricular nucleus stimulation in awake rats. Am J Physiol 256, R1325–R1330.[Medline]
Landgraf R, Neumann I & Schwarzberg H (1988). Central and peripheral release of vasopressin and oxytocin in the conscious rat after osmotic stimulation. Brain Res 457, 219–225.[CrossRef][Medline]
Lang RE, Heil J, Ganten D, Hermann K, Rascher W & Unger T (1983). Effects of lesions in the paraventricular nucleus of the hypothalamus on vasopressin and oxytocin contents in brainstem and spinal cord of rat. Brain Res 260, 326–329.[CrossRef][Medline]
Larsen PJ & Mikkelsen JD (1995). Functional identification of central afferent projections conveying information of acute ‘stress’ to the hypothalamic paraventricular nucleus. J Neurosci 15, 2609–2627.[Abstract]
Li DP, Chen SR & Pan HL (2003). Angiotensin II stimulates spinally projecting paraventricular neurons through presynaptic disinhibition. J Neurosci 23, 5041–5049.
Li Z & Ferguson AV (1993). Subfornical organ efferents to paraventricular nucleus utilize angiotensin as a neurotransmitter. Am J Physiol 265, R302–R309.[Medline]
Ma RC & Dun NJ (1985). Vasopressin depolarises lateral horn cells of neonatal rat spinal cord in vitro. Brain Res 348, 36–43.[CrossRef][Medline]
Malpas SC & Coote JH (1994). Role of vasopressin in sympathetic response to paraventricular nucleus stimulation in anesthetized rats. Am J Physiol 266, R228–R236.[Medline]
May CN, McAllen RM & McKinley MJ (2000). Renal nerve inhibition by central NaCl and ANG II is abolished by lesions of the lamina terminalis. Am J Physiol 279, R1827–R1833.
McKinley MJ, Bicknell RJ, Hards D, McAllen RM, Vivas L, Weisinger RS & Oldfield BJ (1992). Efferent neural pathways of the lamina terminalis subserving osmoregulation. Prog Brain Res 91, 395–402.[Medline]
Motawei K, Pyner S, Ranson RN, Kamel M & Coote JH (1999). Terminals of paraventricular spinal neurones are closely associated with adrenal medullary sympathetic preganglionic neurones: immunocytochemical evidence for vasopressin as a possible neurotransmitter in this pathway. Exp Brain Res 126, 68–76.[CrossRef][Medline]
Nilaver G, Zimmerman EA, Wilkins J, Michaels J, Hoffman D & Silverman AJ (1980). Magnocellular hypothalamic projections to the lower brain stem and spinal cord of the rat. Immunocytochemical evidence for predominance of the oxytocin-neurophysin system compared to the vasopressin-neurophysin system. Neuroendocrinology 30, 150–158.[Medline]
Paton JF (1996). A working heart-brainstem preparation of the mouse. J Neurosci Meth 65, 63–68.[CrossRef][Medline]
Paxinos G & Watson C (1996). The Rat Brain in Stereotaxic Coordinates. Academic Press, New York.
Pedrino GR, Nakagawa Sera CT, Cravo SL & Colombari DS. (2005). Anteroventral third ventricle lesions impair cardiovascular responses to intravenous hypertonic saline infusion. Auton Neurosci 117, 9–16.[CrossRef][Medline]
Pickering AE & Paton JF (2006). A decerebrate, artificially perfused in situ preparation of rat: utility for the study of autonomic and nociceptive processing. J Neurosci Methods February 21; (Epub ahead of print).
Porter JP & Brody MJ (1986a). Spinal vasopressin mechanisms of cardiovascular regulation. Am J Physiol 251, 510–517.
Porter JP & Brody MJ (1986b). The paraventricular nucleus and cardiovascular regulation: role of spinal vasopressinergic mechanisms. J Hypertens Suppl 4, 181–184.[CrossRef]
Pyner S & Coote JH (1999). Identification of an efferent projection from the paraventricular nucleus of the hypothalamus terminating close to spinally projecting rostral ventrolateral medullary neurons. Neuroscience 88, 949–957.[CrossRef][Medline]
Pyner S & Coote JH (2000). Identification of branching paraventricular neurons of the hypothalamus that project to the rostroventrolateral medulla and spinal cord. Neurosci 100, 549–556.[CrossRef][Medline]
Russ RD, Brizzee BL & Walker BR (1992). Role of vasopressin in cardiovascular responses to acute and chronic hyperosmolality. Am J Physiol 262, R25–R32.[Medline]
Saper CB & Levisohn D (1983). Afferent connections of the median preoptic nucleus in the rat: anatomical evidence for a cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region. Brain Res 288, 21–31.[CrossRef][Medline]
Saper CB, Loewy AD, Swanson LW & Cowan WM (1976). Direct hypothalamoautonomic connections. Brain Res 117, 305–312.[CrossRef][Medline]
Sawchenko PE & Swanson LW (1982). Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol 205, 260–272.[CrossRef][Medline]
Sawchenko PE & Swanson LW (1983). The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol 218, 121–144.[CrossRef][Medline]
Scrogin KE, Grygielko ET & Brooks VL (1999). Osmolality: a physiological long-term regulator of lumbar sympathetic nerve activity and arterial pressure. Am J Physiol 276, R1579–R1586.[Medline]
Sermasi E & Coote JH (1994). Oxytocin acts at V1a receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro. Brain Res 647, 323–332.[CrossRef][Medline]
Sermasi E, Howell J, Wheatley M & Coote JH (1998). Localisation of arginine vasopressin V1a receptors on sympatho-adrenal preganglionic neurones. Exp Brain Res 119, 85–91.[CrossRef][Medline]
Shafton AD, Ryan A & Badoer E (1998). Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat. Brain Res 801, 239–243.[CrossRef][Medline]
Share L & Claybaugh JR (1972). Regulation of body fluids. Annu Rev Physiol 34, 235–260.[CrossRef][Medline]
Steenbergen JM & Bohlen HG (1993). Sodium hyperosmolarity of intestinal lymph causes arteriolar vasodilation in part mediated by EDRF. Am J Physiol 265, H323–H328.[Medline]
Stern JE (2004). Nitric oxide and homeostatic control: an intercellular signalling molecule contributing to autonomic and neuroendocrine integration? Prog Biophys Mol Biol 84, 197–215.[CrossRef][Medline]
Stocker SD, Cunningham JT & Toney GM (2004b). Water deprivation increases Fos immunoreactivity in PVN autonomic neurons with projections to the spinal cord and rostral ventrolateral medulla. Am J Physiol 287, R1172–R1183.
Stocker SD, Hunwick KJ & Toney GM (2005a). Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water-deprived rats. J Physiol 563, 249–263.
Stocker SD, Schiltz JC & Sved AF (2004a). Acute increases in arterial blood pressure do not reduce plasma vasopressin levels stimulated by angiotensin II or hyperosmolality in rats. Am J Physiol 287, R127–R137.
Stocker SD, Simmons JR, Stornetta RL, Toney GM & Guyenet PG (2006). Water deprivation activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the rostral ventrolateral medulla. J Comp Neurol 494, 673–685.[CrossRef][Medline]
Stocker SD & Toney GM (2005). Median preoptic neurones projecting to the hypothalamic paraventricular nucleus respond to osmotic, circulating Ang II and baroreceptor input in the rat. J Physiol 568, 599–615.
Swanson LWH & Kuypers GJM (1980). The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence-labeling methods. J Comp Neurol 194, 555–570.[CrossRef]
), kynurenic acid (
) or unilateral microinjections of L-glutamate and hyperosmotic Ringer solution (
) into the PVN. Asterisks (*) represent microinjection sites of isoguvacine and kynurenic acid outside of the PVN, in a group of three rats that were ineffective in altering HS-induced sympathoexcitatory responses. Abbreviations: AC, anterior commissure; SFO, subfornical organ; MnPO, median preoptic nucleus; PVN, paraventricular hypothalamic nucleus; PaDC, paraventricular hypothalamic nucleus, dorsal cap; PaLM, paraventricular hypothalamic nucleus, lateral magnocellular part; PaMP, paraventricular hypothalamic nucleus, medial parvicellular part; PaV, paraventricular hypothalamic nucleus, ventral part; Pe, periventricular hypothalamic nucleus; ME, median eminence; Arc, arcuate hypothalamic nucleus; 3V, third ventricle.
) LSNA during isosmotic (120 mM NaCl) and hyperosmotic stimuli (135 mM NaCl) before (intact hypothalamus) and after precollicular brain transection (no hypothalamus, A) or CVOs transection (B). Recordings in C show the sympathoexcitatory effect of mannitol (0.5%) before and after precollicular transection. A hyposmotic stimulus (105 mM NaCl, D) also produced sympathoexcitation, but this was not affected by precollicular brain transection. **P < 0.01 and ***P < 0.001 compared to before brain transection.
