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Department of 1 Physiology, Universidade Federal de São Paulo – Escola Paulista de Medicina, São Paulo, SP, Brazil 2 Department of Physiology and Pathology, Faculdade de Odontologia, Universidade Estadual Paulista, Araraquara, SP, Brazil
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(Received 1 June 2006;
accepted after revision 15 August 2006; first published online 17 August 2006)
Corresponding author S. L. Cravo: Department of Physiology, Universidade Federal de São Paulo, Rua Botucatu 862, 04023-060 São Paulo, SP, Brazil. Email: sldcravo{at}fcr.epm.br
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Introduction |
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Changes in volume and composition of the extracellular fluid are readily detected by the central nervous system (CNS). Alterations in plasma concentrations of circulating hormones, such as angiotensin II, and signals originating from central or peripheral osmoreceptors, baroreceptors and cardiopulmonary receptors alert the CNS to the change.
Several studies have demonstrated that both acute increases in blood volume and increases in plasma sodium concentration induce responses such as renal vasodilatation (Lovick et al. 1993; Colombari & Cravo, 1999; Colombari et al. 2000; Leonard et al. 2000; Pedrino et al. 2005b), decreased renal sympathetic nerve activity (Morita & Vatner, 1985; Weiss et al. 1996; Nishida et al. 1998; Leonard et al. 2000), release of atrial natriuretic peptide (ANP; Rauch et al. 1990; Durlo et al. 2004; Godino et al. 2005) and oxytocin (OT; Morris & Alexander, 1989; Durlo et al. 2004; Godino et al. 2005), natriuresis and diuresis (Morita & Vatner, 1985; Schoorlemmer et al. 2000). Collectively, these responses help restore blood volume and osmolality.
The activation and co-ordination of these responses result from the integrative actions of the CNS. However, the central pathways involved are not entirely understood.
Several lines of evidence indicate that catecholaminergic A1 cells in the caudal ventrolateral medulla (CVLM) are involved in the control of water and salt balance. A1 Cells receive projections from arterial baroreceptors as well as from vagal cardiopulmonary volume receptors (Day & Sibbald, 1990; Day et al. 1992). A1 Cells also project to several hypothalamic nuclei involved in water and salt homeostasis, including the subfornical organ (SFO), the median preoptic nucleus (MePO), and the magnocellular neurosecretory neurones in the paraventricular (PVN) and supraoptic nucleus (SON; Tucker et al. 1987; Tanaka et al. 2002). Brainstem noradrenergic neurones are activated during several conditions that affect volume and composition of the extracellular compartment (Hochstenbach & Ciriello, 1995; Howe et al. 2004; Godino et al. 2005).
Previously, we demonstrated that denervation of the baroreceptors and electrolytic lesions of the anterioventral wall of the brain third ventricle (AV3V) abolished renal vasodilatation induced by volume expansion (VE) or hypertonic saline infusion (HS; Colombari & Cravo, 1999; Colombari et al. 2000; Pedrino et al. 2005b). Other studies have shown that blockade of the adrenergic transmission in the AV3V reduced ANP release induced by changes in circulating volume (Antunes-Rodrigues et al. 1993; Bealer, 1997). Also, microinjection of noradrenaline into the thrid ventricle or adjacent tissue increases renal sodium excretion (Morris et al. 1976). These findings suggest that A1 cells in the CVLM represent a necessary link in the neural circuits activated during acute changes in the extracellular fluid volume or composition.
A lesion that is highly specific for a discrete population of neurones can reveal a great deal about the function of that population. Recently, an immunotoxin was described that targets dopamine β-hydroxylase (DβH), the enzyme catalysing the conversion of dopamine to noradrenaline. This toxin consists of a monoclonal antibody to DβH conjugated by disulphide linkage to saporin, a ribosome-inactivating protein from seeds of the plant Saponaria officinalis (Picklo et al. 1994). Several studies showed selective destruction of DβH-containing neurones after injection of anti-DβH–saporin into the cerebral ventricles (Wrenn et al. 1996) or into brain nuclei (Madden et al. 1999; Madden & Sved, 2003).
In the present study, we tested the hypothesis that besides modulating hormone release, A1 cells may also be involved in the cardiovascular responses to acute changes in circulating volume and composition. We used anti-DβH–saporin to lesion noradrenergic neurones selectively in the caudal ventrolateral medulla (CVLM) region and measured the effects of these lesions on pressor and renal blood flow responses induced by acute hypernatraemia and blood volume expansion. Some of these results have been presented in abstract form (Pedrino et al. 2005a).
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All experiments were performed in adult male Wistar rats (320–360 g). They were obtained from the central animal house of the Universidade Federal de São Paulo – Escola Paulista de Medicina. All protocols described here were approved by the Medical Ethics Committee at the Universidade Federal de São Paulo.
Microinjections of anti-DβH–saporin or saporin into CVLM
Animals were anaesthetized with halothane (2–3% in 100% O2) and mounted prone in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) with the incisor bar 11 mm below the interaural line. After partial removal of the occipital bone, the meninges covering the dorsal surface of the brainstem were cut and retracted, and the calamus scriptorius was visualized. Microinjections of anti-DβH–saporin (6.3 ng in 60 nl; Advanced Targeting Systems, San Diego, CA, USA) or an equimolar dose of saporin (1.3 ng in 60 nl; Advanced Targeting Systems) were made bilaterally at two levels of the CVLM. For all microinjections into CVLM, a glass micropipette was positioned as follows: 0.0 and 0.5 mm caudal to the calamus scriptorius, 2.0 mm lateral to the mid-line and 2.0 mm ventral to the dorsal surface. These co-ordinates are based on the region of the CVLM comprising the A1 group (Tucker et al. 1987). After microinjection, the micropipette was left in place for 3–5 min. Then the incision was closed and the animals were placed on a heated pad to maintain body temperature during recovery. A prophylactic antibiotic dose (penicillin, 60 000 IU kg–1, i.m. Sigma, St Louis, MO, USA) was injected after the surgery.
Surgical procedures
Animals were studied 15–25 days after microinjections into the CVLM. On the day of the experiments, rats were anaesthetized with 2–3% halothane in 100% O2. The right femoral artery was cannulated for blood pressure recording. The right femoral vein was cannulated for drug administration (urethane; 1.2 g kg–1, I.V.) and blood sampling. The right jugular vein was exposed, and a catheter was advanced to the right atrium for infusion of HS or for isotonic VE. The trachea was cannulated to reduce airway resistance, and the rats were mounted prone in a stereotaxic apparatus (David Kopf Instruments) with the bite bar set 3.4 mm below the interaural line.
Body temperature was maintained at 37 ± 0.5°C with a thermostatically controlled heated table. Miniature ultrasonic transit-time flow probes (Transonic Systems, Inc., Ithaca, NY, USA) were placed around the left renal artery.
Recording of arterial pressure, heart rate and blood flow
To record arterial pressure, the arterial catheter was connected to a pressure transducer attached to a bridge amplifier (ETH-200, ETH-200, iwork/CB Sciences, Dover, NH, USA). Pulsatile pressure was recorded continuously with a PowerLab system (AD Instruments). Mean arterial pressure (MAP) and heart rate (HR) were determined from the pulsatile signal with Chart software (AD Instruments). To measure blood flow, flow probes were connected to an ultrasonic transit-time flowmeter (Transonic Sytems, Inc.). The mean blood flow signal was recorded with PowerLab Systems.
Acute changes in circulating volume
To increase plasma sodium concentration, we infused HS through the jugular vein cannula (3 M NaCl, 0.18 ml (100 g body weight)–1, over 1 min). Isotonic VE was obtained by infusion of 4% Ficoll (1% body weight, 0.4 ml min–1; Sigma).
Blood sampling and analysis
Blood samples (0.20 ml each) were withdrawn from the femoral vein cannula 5 min before and 10, 30 and 60 min after HS or VE. After the sample was collected, we injected 0.20 ml sterile 0.15 M NaCl through the cannula to reduce changes in extracellular fluid volume brought about by sampling. Blood haemoglobin concentration was measured immediately with a kit from Sigma (Drabkin's reagent, kit 525). The rest of the sample was centrifuged for 5 min at 6000g. The plasma was removed and stored at –20°C. Plasma sodium concentration was measured with a flame photometer (model DM6, Digimed, São Paulo, Brazil).
Perfusion, fixation and tissue collection
At the end of the experiments, the animals were perfused through the heart with saline (0.15 M NaCl) followed by a solution of 4% paraformaldehyde (Sigma) in 0.1 M sodium phosphate buffer (500 ml at pH 7.4). The brain was removed and postfixed in 4% paraformaldehyde solution for 1–2 h, then cryoprotected in 30% sucrose solution. Coronal sections of 40 µm of the brainstem were collected (in 4 serially adjacent sets) and stored in 0.02 M potassium phosphate-buffered saline (KPBS, Sigma; pH 7.4) at 4°C until immunohistochemical staining.
Immunohistochemistry
Each fourth brainstem section was processed for immunohistochemical detection of tyrosine hydroxylase (TH). The sections were pre-incubated for 15 min in 0.5% hydrogen peroxide in 0.02 M KPBS (Sigma) followed by a 30 min incubation in 3.5% normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA) in 0.02 M KPBS. The sections were incubated overnight at 4°C with mouse monoclonal antibody (1:2000 dilution, catalogue no. 22941, ImmunoStar Inc., Hudson, WI, USA) with 1.5% normal horse serum and 0.2% Triton X-100, followed by a 1 h incubation with biotinylated horse antimouse IgG (Vector Laboratories Inc.; 1:200 dilution with 0.1% Triton X-100 for 1 h). After these incubations, the sections were processed with the avidin–biotin procedure, using Elite Vectastain reagents (Vector Laboratories Inc.). Diaminobenzidine (DAB) was used to produce a brown cytoplasmic TH reaction product. Sections were mounted on slides, dehydrated in a series of alcohols, cleared in xylene, and coverslipped.
Cell counting and imaging
Counts of labelled neurones were performed in every fourth medulla oblongata section (40 of each 160 µm). All immunolabelled neuronal perikarya in the ventrolateral medulla (VLM; A1/C1) and nucleus tractus solitarius (NTS; A2/C2) were counted bilaterally in order to quantify the extent of the anti-DβH–saporin-induced lesion. Neurones were counted at x200 magnification with a Nikon light microscope.
Data analysis
The changes in renal blood flow were expressed as a percentage of control values. Renal vascular conductance (RVC) was calculated by dividing the renal blood flow (in ml min–1) by the arterial pressure, and the changes were expressed as a percentage of control values. Results are presented as means ± S.E.M. The data were analysed by two-way analysis of variance followed by the Fisher LSD test. A value of P < 0.05 was considered to denote a significant difference. The effects of treatment with anti-DβH–saporin or saporin on the number of catecholaminergic medullary neurones are presented as means ± S.E.M. Groups were compared by one-way ANOVA. When group means differed significantly, the Newman–Keuls post hoc test was used to detect pairwise differences.
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Tyrosine hydroxylase-positive cells were found within the VLM and the NTS from approximately 3 mm caudal to 2 mm rostral to the obex (Figs 1 and 2). In rats treated with bilateral microinjections of anti-DβH–saporin into the CVLM (n = 14), the number of TH-positive cells in this area was markedly reduced. In the region between 300 and 1700 µm caudal to the obex, which encompasses the area of noradrenergic neurones (A1 group) that project to the MePO and the PVN (Tucker et al. 1987), the number of TH-positive neurones was reduced by 79% compared to saporin treated animals (sham animals) (P < 0.001; Figs 1 and 2). The region of loss of TH-positive neurones appeared to extend rostrally from the injection site to the C1 adrenergic cell group. Within the region located from 300 µm caudal to 2100 µm rostral to the obex (the region encompassing the C1 cell group), a reduction of approximately 29% in the number of TH-immunopositive cells was observed (P < 0.001; Figs 1 and 2). Microinjection of anti-DβH–saporin into the CVLM slightly reduced the number of TH-positive cells in the A2 and C2 cell groups in the NTS. In the region between 2400 µm caudal to 2080 µm rostral to the obex, TH-immunopositive cell counts were approximately 15% less than in sham animals (Figs 1 and 2).
Effects of destruction of the medullary catecholaminergic neurones on the changes in MAP, HR, RBF and RVC induced by HS infusion (Fig. 3)
Body weight and baseline MAP, HR and RBF levels are shown in Table 1. These variables were similar in sham and anti-DβH–saporin-treated rats.
After HS infusion in sham animals (n = 9), we observed small increases in MAP that peaked at 10 min (9 ± 2 mmHg increase). Mean arterial pressure returned to basal levels 30 min after HS infusion (4 ± 2 mmHg change from baseline values). A transitory bradycardic response was seen in the first 20 min after HS infusion (peaking at 10 min, –18 ± 7 beats min–1 change from baseline). After 20 min of HS infusion, a sustained increase in RBF and RVC (to 155 ± 7 and 145 ± 6%, respectively, of baseline values) was observed. Renal blood flow and RVC remained elevated throughout the experimental period (RBF and RVC at 60 min: 148 ± 8 and 147 ± 9%, respectively, of baseline values). In anti-DβH–saporin-treated animals (n = 7), the pressor response to HS was augmented when compared with the sham group (an increase of 16 ± 3 versus 9 ± 2 mmHg above baseline, respectively, at 10 min). In lesioned rats, RBF responses to HS were blunted (125 ± 6%) and VC increases were abolished (108 ± 5%) 20 min after acute HS. Similar results (RBF, 120 ± 5%; RVC, 106 ± 6%) were obtained 60 min after HS infusion (Fig. 3). Heart rate did not fall significantly after HS in lesioned rats (12 ± 7 beats min–1, at 20 min).
Effects of destruction of the medullary catecholaminergic neurones on the changes in MAP, HR, RBF and RVC induced by blood volume expansion (VE; Fig. 4)
In sham animals (n = 6), VE invariably increased MAP. The peak increase in MAP occurred within 10 min (8 ± 5 mmHg), and blood pressure returned to baseline values 30 min after VE (3 ± 2 mmHg change from baseline). In sham rats, 20 min after VE, RBF and RVC increased to 149 ± 10 and 145 ± 12%, respectively, and remained elevated until 60 min (145 ± 11 and 140 ± 13%). Heart rate did not change after VE (–3 ± 7 beats min–1, at 20 min). In anti-DβH–saporin-treated animals (n = 7), the increases in RBF and RVC induced by VE were significantly smaller at 20 min (132 ± 6 and 126 ± 5%, respectively) and at 60 min (121 ± 10 and 117 ± 9%). Pressor (peak at 10 min; 7 ± 3 mmHg) and HR responses to VE (–9 ± 5 beats min–1, at 20 min after VE) were similar to those observed in sham animals (Fig. 4).
Effects of HS infusion and VE on plasma sodium and blood haemoglobin concentrations
Pre-infusion plasma sodium concentrations were similar in sham (142 ± 0.9 mM, n = 3) and anti-DβH–saporin-treated animals (144 ± 2.0 mM, n = 4). Plasma sodium increased after HS infusion in both groups (149 ± 1.0 and 151 +1.6 mM, respectively, 10 min after infusion), and remained at these levels throughout the experimental period. Blood haemoglobin concentration fell by 2 ± 1.6% in sham and 1 ± 1.2% in anti-DβH–saporin-treated animals (10 min after HS infusion), indicating that blood volume tended to increase slightly in both groups.
Plasma sodium concentration was not affected by volume expansion in sham (from 140 ± 0.7 to 140 ± 0.7 mM, 10 min after VE, n = 3) or anti-DβH–saporin-treated animals (from 142 ± 1.6 to 140 ± 0.5 mM, 10 min after VE, n = 3). Blood haemoglobin concentration fell similarly after VE in sham and treated animals (12 ± 0.7 and 11 ± 1.1%, respectively).
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Recently, an immunotoxin (anti-DβH–saporin) was developed as a tool to selectively destroy neurones that express DβH on their plasma membrane (noradrenergic and adrenergic neurones). This toxin consists of the ribosomal toxin saporin conjugated to an anti-DβH antibody (Picklo et al. 1994). Dopamine β-hydroxylase is a vesicular enzyme that is exposed on the extracellular side of the cell membrane upon release of noradrenaline and thus allows the targeting of these cells with anti-DβH–saporin. Once inside the cell, saporin irreversibly inactivates the 60S ribosomal subunit, thus blocking protein synthesis and eventually killing the cell. Previous studies have shown selective destruction of DβH-containing neurones when the anti-DβH–saporin complex was microinjected into the cerebral ventricles (Wrenn et al. 1996) in regions containing catecholaminergic nerve terminals (Schreihofer & Guyenet, 2000) or even in regions containing adrenergic or noradrenergic cell bodies (Madden et al. 1999; Madden & Sved, 2003). In contrast, unconjugated saporin does not seem to enter the cell and is thought to be harmless.
The present study demonstrates that intraparenchymal microinjections of anti-DβH–saporin into the CVLM produce an extensive depletion of the catecholaminergic cell population. Our data indicate that bilateral microinjections of anti-DβH–saporin induced a marked loss (79%) of TH-containing neurones within 2 mm caudal to the obex. Previous studies indicated that caudal to the obex the vast majority of TH-positive cells are non-phenylethanolamine-N-methyltransferase (PNMT)-positive cells, so they are assumed to belong to the A1 catecholaminergic cell group (Tucker et al. 1987). With increasing distance from the injection site, the number of TH-positive cells increases and approaches the number observed in control animals treated with unconjugated saporin. The estimated loss of TH-positive cells in the rostral ventrolateral medulla (RVLM) region was approximately 14%.
Previous reports support the assertion that the loss of immunoreactivity is indicative of neuronal death (Madden et al. 1999; Schreihofer & Guyenet, 2000; Wiley & Kline, 2000). It is unlikely that the noradrenergic neurones survived but failed to produce TH, since saporin acts by blocking ribosomal function. The effect of saporin seems permanent; no TH or DβH was detected 9 months after intracerebroventricular injections of anti-DβH–saporin (Wrenn et al. 1996). Also, a prominent gliosis within the area of the toxin injection was consistently observed in all animals receiving anti-DβH–saporin, also suggesting neuronal death. Overall, these results support the conclusion that the toxin destroyed A1 cells. These results are in agreement with recent studies in which microinjection of anti-DβH–saporin into the RVLM depleted 85–90% of PNMT neurones within the RVLM (Madden et al. 1999; Madden & Sved, 2003).
Another relevant aspect to be considered is whether microinjections of anti-DβH–saporin into the CVLM destroyed non-A1 neurones. The mechanism of specific internalization of the conjugated anti-DβH–saporin determines that only cells synthesizing DβH can take up the conjugated toxin. Furthermore, our results indicate that the distribution of TH-immunoreactive neurones in the CVLM of sham animals that received microinjections of unconjugated saporin agrees closely with previous quantitative reports on the distribution of TH-immunopositive cells along the rostrocaudal axis of the ventrolateral medulla (Tucker et al. 1987; Ruggiero et al. 1994). Similary, previous reports (Madden et al. 1999; Schreihofer & Guyenet, 2000; Madden & Sved, 2003) using anti-DβH–saporin demonstrated that rats treated with IgG–saporin, saporin or vehicle showed no depletion of catecholaminergic cell profiles at any medullary level examined. Once more, the distribution of TH-positive cells observed in control groups of these studies agrees closely with that observed in control groups in the present study. Since unconjugated saporin did not destroy A1 cells, it is likely that neither unconjugated saporin nor saporin conjugated to DβH entered non-A1 cells.
A further important aspect in evaluating this method was to find out whether the anti-DβH–saporin injection into the CVLM destroys DβH-containing cells outside the A1 region. Our data indicate reductions of about 15% in TH-immunopositive cells within the A2 and C2 cell groups and of 29% in the C1 group. It is likely that these reductions result either from diffusion of the DβH–saporin complex from the injection sites or from uptake of DβH–saporin by catecholaminergic terminals from these cells groups.
Madden & Sved (2003) demonstrated that lesions less than 80% of the C1 area produced by anti-DβH—saporin were ineffective in altering the sympathoexcitatory responses induced by baroreceptor unloading or chemoreceptor activation. Therefore, the lack of or reduction in the cardiovascular responses in the A1-lesioned rats in our study cannot be attributed to the small lesions in the C1 area. Similarly, it is unlikely that the 15% reduction of TH-positive cells in the A2/C2 area is responsible for the reductions in the cardiovascular responses observed in our study.
Consequences of the destruction of A1 neurones on cardiovascular responses induced by acute changes in circulating volume
Results obtained in the present study demonstrate that A1 noradrenergic neurones located in the CVLM are important for the cardiovascular responses evoked by hyperosmolality and volume expansion. Depletion of these neurones prevented renal vasodilatation induced by increases in plasma sodium concentration and reduced the response to isotonic VE. Baseline plasma sodium concentrations and increases induced by HS were similar in sham and anti-DβH–saporin-treated animals. Similarly, the increase in blood volume induced by VE was equivalent in both groups. Therefore, the results obtained are not due to differences in the stimulation in these groups. In addition, the cardiovascular adjustments induced by HS infusion in both groups appear to be related to an increase in plasma sodium and not to a volume expansion induced by intravenous HS infusion, since blood haemoglobin did not change after HS. This is in line with results from other authors who found no difference in haematocrit after identical HS infusions (Rauch et al. 1990; Pedrino et al. 2005b).
Hypertonic saline infusion and VE in sham animals produced a pattern of cardiovascular adjustments characterized by a discrete and transient hypertension and bradycardia, simultaneously with marked and sustained increases in renal blood flow and vascular conductance. These adjustments are comparable to those observed in previous studies using volume expansion or HS infusion (Nishida et al. 1998; Colombari & Cravo, 1999; Colombari et al. 2000; Pedrino et al. 2005b). It is accepted that these adjustments are part of the animal's integrated response to a sudden increase in plasma sodium concentration or circulating volume. Increases in circulating volume activate several mechanisms resulting in natriuresis and diuresis until normal sodium and volume levels are resumed (Morita & Vatner, 1985; Schoorlemmer et al. 2000). Renal vasodilatation is a significant factor contributing to the increase in renal excretion of sodium and water.
Similar to other studies (Lovick et al. 1993; Colombari & Cravo, 1999), we observed that acute VE induced a transient (< 10 min) hypertension and did not significantly affect HR in sham or A1-lesioned rats. In contrast, some studies did not find this transient hypertension (Badoer et al. 1997; Howe et al. 2004; Godino et al. 2005). These differences may be caused by use of different species, methods of blood volume expansion (saline, blood or macromolecules), and the duration and magnitude of volume expansion.
Hypertonic saline infusion increased blood pressure. The lesion increased both duration and magnitude of the pressor response. The pressor response seems to involve neural and humoral components. Changes in the symphathetic vasoconstrictor tone induced by HS infusion are complex, involving distinct components depending on the magnitude and duration of the osmotic disturbance. A recent model proposes that a sudden increase in osmolality initially inhibits renal nerve activity to reduce renal vascular conductance and sodium reabsorption, leading to sodium loss (Toney et al. 2003). If this initial component fails to reduce osmolality, a generalized increase in sympathetic activity increases arterial pressure, leading to increased renal perfusion pressure, glomerular filtration rate and sodium loss. We suggest that the increased pressor response in A1-lesioned animals may result from a failure of the initial component of the response, which consists of increased renal vascular conductance.
Recently, we demonstrated that VE and HS infusion induced a sustained renal vasodilatation (Colombari & Cravo, 1999; Colombari et al. 2000; Pedrino et al. 2005b). This response appears to involve both neural and hormonal mechanisms. We also demonstrated that renal vasodilatation in response to VE was blocked by baroreceptor denervation, but was not affected by bilateral vagotomy (Colombari et al. 2000). These results suggest that baroreceptors but not cardiopulmonary receptors represent the main afferent pathway triggering these responses. Various lines of evidence suggest that release of ANP and OT and reduction in renal sympathetic nerve activity, induced by acute changes in circulating volume, may contribute to this response (Morris & Alexander, 1989; Weiss et al. 1996; Nishida et al. 1998; Godino et al. 2005).
Numerous studies have demonstrated the importance of hypothalamic structures in cardiovascular, humoral and behavioural responses induced by changes in circulating volume. Recently, we demonstrated that integrity of the periventricular tissue surrounding the AV3V region is essential for the renal vasodilatation that follows acute changes in extracellular fluid compartment composition and volume (Colombari & Cravo, 1999; Pedrino et al. 2005b).
The AV3V region contains neurones that have direct connections with the PVN and supraoptic nuclei (Weiss & Hatton, 1990). These connections appear to be important to responses induced by changes in the body fluid volume or composition (McKinley et al. 2004). Indeed, recent studies have shown an important role for the PVN in the reduction of renal nerve activity and renal vasodilatation elicited by increases in plasma osmolality and blood volume (Lovick et al. 1993; Haselton et al. 1994; Badoer et al. 2003). Moreover, other studies have shown that electrolytic lesions in the AV3V region inhibited both volume- and osmotic-induced increases in plasma ANP (Rauch et al. 1990; Antunes-Rodrigues et al. 1991).
Neuroanatomical studies have shown that medullary noradrenergic neurones project to hypothalamic areas including the AV3V, the PVN and SON (Tucker et al. 1987; Fernandez-Galaz et al. 1994). In fact, A1 cells may represent the major source of catecholaminergic afferents to these areas. A previous study demonstrated that about 80% of the noradrenergic cells in the A1 group project to the PVN and AV3V (Tucker et al. 1987). A1 Neurones make direct synaptic contacts with neurones in the MePO that project to the PVN (Kawano & Masuko, 1999).
Similarly, previous studies suggested that noradrenergic neurotransmission in the AV3V region is necessary to produce the responses to hypervolaemia and hyperosmolality (Morris et al. 1976; Antunes-Rodrigues et al. 1993; Bealer, 1997). Acute pharmacological blockade of 
1-adrenoceptors in the AV3V reduced ANP release induced by changes in circulating volume (Antunes-Rodrigues et al. 1993; Bealer, 1997). Conversely, microinjection of noradrenaline into the thrid ventricle or adjacent tissue increases renal sodium excretion and ANP release (Morris et al. 1976). Therefore, activation of A1 cells in the ventrolateral medulla may represent an essential step in the neural pathways involved in cardiovascular responses to volume expansion and HS infusion. In animals with A1 lesions, the information necessary for the cardiovascular responses is lost.
A functional relationship between A1 cells and increases in blood volume and hyperosmolality was demonstrated previously in studies employing early gene expression (Hochstenbach & Ciriello, 1995, 1996; Howe et al. 2004; Godino et al. 2005). Subcutaneous, intraperitoneal or intravenous administration of HS and VE increases Fos and c-Fos mRNA expression in the VLM and NTS (Hochstenbach & Ciriello, 1995; Howe et al. 2004; Godino et al. 2005). Hochstenbach & Ciriello (1995) demonstrated that intravenous infusions of HS increased Fos-like immunoreactivity in the the A1 group and to a lesser extent in the A2 group. Fos expression in the CVLM after HS was not changed by AV3V lesions, suggesting that these neurones are activated by peripheral rather than central pathways (Hochstenbach & Ciriello, 1996).
It has to be pointed out that A1 cells are also activated by acute reductions in central blood volume (Head et al. 1987; Buller et al. 1999; Potts et al. 2000). Buller et al. (1999) found that A1 lesions reduced the number of Fos-positive neurosecretory vasopressin cells in the SON and PVN induced by hypotensive haemorrhage. Similarly, other studies demonstrated that A1 lesions reduced the vasopressin secretion induced by decreased circulating volume (Head et al. 1987; Renaud, 1996).
The new finding of this study is that catecholaminergic cells in the CVLM participate in the cardiovascular responses induced by acute increases in central blood volume and plasma osmolality. Previous studies have clearly demonstrated the involvement of A1 cells in cardiovascular responses induced by acute reductions in circulating volume and unloading of arterial baroreceptors. Our results indicate that these cells are also important for responses induced by increases in circulating volume and by hypernatraemia. More specifically, the lesion of catecholaminergic cells in the CVLM prevented renal vasodilatation induced by increases in plasma sodium and reduced this response induced by volume expansion.
Our results are compatible with the hypothesis that A1 cells are activated upon stimulation of peripheral baroreceptor and cardiopulmonary afferents, engaging efferent pathways that increase renal vascular conductance, and that catecholaminergic projections from the A1 group to the AV3V area may be one of these pathways. Since A1 neurones affect circulating volume and peripheral resistance simultaneously, they may be key neurones for our understanding of volume-induced forms of hypertension.
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P < 0.05 different from sham lesion.