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Fos expression in spinally projecting neurons after hypotension in the conscious rabbit
Autonomic Neuroscience: Basic and Clinical 100 (2002) 10 – 20 www.elsevier.com/locate/autneu
Fos expression in spinally projecting neurons after hypotension in the conscious rabbit Jaimie W. Polson, Steven Mrljak, Patrick D. Potts, Roger A.L. Dampney * Department of Physiology and Institute for Biomedical Research, University of Sydney, Sydney, NSW 2006, Australia Received 8 February 2002; received in revised form 23 May 2002; accepted 12 June 2002
Abstract Hypotension produces a reflex increase in the activity of sympathetic vasomotor nerves. Studies in anaesthetised animals have established that neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic vasomotor preganglionic neurons in the spinal cord are a critical component of the central pathways mediating this reflex response. There are also neurons in supramedullary regions (the A5 area in the pons and the paraventricular nucleus (PVN) in the hypothalamus), however, that project directly to the sympathetic vasomotor outflow. The aim of this study was to identify and map neurons within the A5 area and PVN, as well as in the RVLM, which may contribute to the reflex sympathoexcitatory response to a hypotensive challenge in conscious rabbits. In a preliminary operation, a retrogradely transported tracer was injected into a site centred on the intermediolateral cell column in the upper lumbar spinal cord. After a waiting period of at least 1 week, a moderate hypotension (decrease in arterial pressure of approximately 20 mm Hg) was induced in conscious rabbits for 60 min by continuous infusion of sodium nitroprusside. In confirmation of previous studies, hypotension resulted in the expression of Fos in the RVLM, the A5 area and PVN. There were also retrogradely labelled neurons in all these regions. In both the RVLM and A5 area, approximately 40% of the retrogradely labelled neurons were also immunoreactive for Fos. In contrast, in the PVN the proportion of retrogradely labelled neurons that were also Fos-positive was much less (approximately 6%). This study has demonstrated that, in the conscious rabbit, a significant proportion of spinally projecting neurons within discrete regions in the RVLM and A5 area are activated by hypotension (as indicated by Fos expression). In the PVN, only a very small proportion of spinally projecting neurons are activated by hypotension, and thus these neurons appear to be regulated primarily by inputs other than baroreceptor inputs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Baroreceptor reflex; Central cardiovascular pathways; Rostral ventrolateral medulla; A5 area; Paraventricular nucleus; Immediate early genes
1. Introduction It is well established that the rostral ventrolateral medulla (RVLM) contains neurons that provide a direct excitatory input to sympathetic cardiac and vasomotor preganglionic neurons in the spinal cord, which are a critical component of the central pathways mediating the baroreceptor and other cardiovascular reflexes (for review, see Guyenet, 1990 or Dampney, 1994). In particular, studies in anaesthetised animals have shown that such presympathetic neurons are powerfully inhibited by baroreceptor inputs (Barman and Gebber, 1985; Sun and Guyenet, 1985; McAllen, 1986). Consistent with these observations, unloading of barorecep-
*
Corresponding author. Department of Physiology F13, The University of Sydney, NSW 2006, Australia. Fax: +61-2-93516470. E-mail address: [email protected] (R.A.L. Dampney).
tors by induced hypotension in conscious animals results in activation of neurons in the RVLM, as indicated by the expression of Fos, the protein product of the immediate early gene c-fos (Chan and Sawchenko, 1994; Li and Dampney, 1994; Graham et al., 1995; Horiuchi et al., 1999), an effect which is prevented by denervation of carotid sinus and aortic baroreceptors (Potts et al., 1997). Apart from the RVLM, presympathetic neurons are also located in other medullary and supramedullary regions, including the medullary raphe nuclei, the A5 area in the pons and the paraventricular nucleus (PVN) in the hypothalamus (Strack et al., 1989). The observation that bilateral blockade of GABA receptors in the RVLM abolishes the baroreceptor-sympathetic reflex (Sun and Guyenet, 1987), however, suggests that presympathetic neurons in these other regions play little or no role in this reflex, at least in anaesthetised animals. On the other hand, previous studies in our laboratory have shown that induced hypotension in
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 1 4 3 - 1
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conscious rabbits result in a significant increase in Fos expression above baseline levels in the A5 area and PVN, although not in the medullary raphe nuclei (Li and Dampney, 1994; Horiuchi et al., 1999). In conscious rabbits with denervated sinoaortic baroreceptors, however, hypotension resulted in no increase in Fos expression above baseline levels in the A5 area, while in the PVN there was an increase in Fos expression but it was much less than that observed in baroreceptor intact animals (Potts et al., 1997). These previous observations indicate that there are neurons in the A5 area and PVN that are activated by hypotension, primarily by signals arising from baroreceptors. It is not clear, however, what function such activated neurons may have. One possibility is that these neurons are presympathetic, and are involved in the baroreceptor reflex regulation of sympathetic nerve activity. Alternatively, they may convey baroreceptor signals to other target nuclei. The PVN, for example, contains several different populations of neurons that project to different targets, including the median eminence, posterior pituitary, nucleus tractus solitarius (NTS) and RVLM as well as the spinal cord (Swanson and Kuypers, 1980; Shafton et al., 1998). Similarly, neurons in the A5 area have extensive projections to a number of cardiovascular regulatory nuclei in the brainstem and forebrain (Byrum and Guyenet, 1987). The aim of this study was to identify spinally projecting neurons in the RVLM, A5 area and PVN that are activated by a hypotensive challenge in the conscious state. For this purpose, we have mapped the distribution of neurons within these three brain regions that express Fos in response to a period of induced hypotension in conscious rabbits, and which were also retrogradely labelled from an upper lumbar segment of the spinal cord. The upper lumbar part of the spinal cord was selected because it contains a high density of sympathetic preganglionic neurons regulating vasomotor activity in the hindlimbs and abdominal viscera (McLachlan et al., 1985; Strack et al., 1988; Li et al., 1992a). Although this method results in retrograde labelling only of a small sample of the neurons in the RVLM, A5 area and PVN that project to the spinal cord, double-labelling for the presence of Fos in response to a hypotensive challenge in the conscious animal allows a determination of the location of putative cardiovascular presympathetic neurons in these three brain regions. Parts of this study have been published previously in abstract form (Polson et al., 2000).
2. Materials and methods 2.1. General procedures Experiments were performed on six adult rabbits of either sex (two New Zealand White and four crossbred from Baker Medical Research Institute, Melbourne, Australia; 2.5 – 3.5 kg). All experiments were carried out in accordance with the Guidelines for Animal Experimentation
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of the National Health and Medical Research Council of Australia. 2.2. Injection of tracers Rabbits were premedicated with the opioid analgesic buprenorphine hydrochloride (Temgesic, Reckitt and Coleman; 0.03 mg/kg i.m.) for the purpose of preemptive analgesia. General anaesthesia was then induced with a mixture of alphaxalone and alphadolone (Saffan, PittmanMoore, initial dose 5 mg total steroids/kg i.v., followed by a continuous infusion of 8 – 16 mg/kg/h i.v.) and intubated with a perinatal endotracheal tube (Mallinkrodt Lo-Pro, 3.0mm i.d.). The rabbit was then artificially ventilated with 35 – 45% oxygen in room air, at a rate that maintained endtidal CO2 in the range 3.5 – 4.5%. Body temperature was maintained at approximately 39 jC using a thermostatically regulated heating lamp connected to a rectal temperature probe. The rabbit was positioned in an atraumatic stereotaxic frame, and the vertebral column at the level of T12 to L2 was exposed. The spinous process of vertebra T12 was clamped and fixed to the stereotaxic frame to reduce movement, and the tissue overlying either the L1 or L2 vertebra was retracted to expose the vertebral laminae. After ensuring that the level of general anaesthesia was adequate, by the absence of a withdrawal response to toe pinch, a bolus injection of the neuromuscular blocker alcuronium bromide (Alloferin, Roche; 0.1 –0.2 mg/kg i.v.) was administered to prevent movements that might arise from mechanical stimulation of the spinal cord. The spinal cord and overlying dura mater was then exposed via a laminectomy. A small incision was made in the dura mater and a glass micropipette, containing either rhodamine- or fluorescein-labelled microspheres (n = 5; Lumafluor) or cholera toxin b-subunit (n = 1; List Biological Laboratories), was inserted into the spinal cord, approximately 0.7 mm lateral to the midline, at a depth of 1.0 – 1.5 mm. The volume of the injectate containing fluorescent microspheres was 480 –910 nl, and of cholera toxin b-subunit was 135 nl. A single unilateral injection was made slowly, over a period of approximately 30 min. Following the injection, Gelfoam powder (Upjohn) was placed over the exposed spinal cord, and the overlying muscles and skin sutured in layers. Benzylpenicillin (Commonwealth Serum Laboratories) was applied both topically to the wound (approximately 60 mg) and intramuscularly (30 mg/kg). Upon completion of the surgery and recovery from the neuromuscular block, the infusion of alphaxalone and alphadolone was terminated. The rabbit was closely observed during the period of recovery from general anaesthesia and during the postoperative period. Analgesia was maintained with buprenorphine (0.03 mg/kg/12 h) and the nonsteroidal anti-inflammatory agent ketoprofen (2.0 mg/kg/24 h) for a minimum of 3 days, and antibiotic prophylaxis (benzylpenicillin, 30 mg/kg/24 h) for a minimum of 4 days. No animals showed any signs of postoperative infection.
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2.3. Induction of hypotension Following a recovery period of 1– 3 weeks, a 60-min period of hypotension was induced in the conscious unrestrained rabbits, according to the procedure described previously (Li and Dampney, 1994). Briefly, arterial pressure was measured via a cannula placed in a central ear artery, and sodium nitroprusside was infused intravenously, via a marginal ear vein, at a rate (0.2 – 1.0 mg/kg/h, adjusted continuously) that produced a fall in arterial pressure of approximately 20 mm Hg, for a period of 60 min. It has been shown previously that a fall in arterial pressure of 20 mm Hg produces close to the maximum baroreflex change in sympathetic activity and heart rate in the conscious rabbit (Undesser et al., 1985; Weinstock et al., 1988), while the duration of the stimulus (60 min) has been shown to be optimal for producing Fos expression in activated neurons (Li and Dampney, 1994). Following the 60-min period of hypotension, the infusion was stopped and arterial pressure and heart rate monitored for a further 30 min. The rabbit was then anaesthetised with sodium pentobarbitone (60 mg/kg i.v.) and perfused transcardially with 1 l of heparinized saline, followed by 2 l of 4% paraformaldehyde in 0.1 M phosphatebuffered saline, pH 7.3. The brain and the region of spinal cord surrounding the injection site were removed and placed in 30% sucrose in 0.1 M phosphate buffer. Five series of sections (40-Am thick) were cut on a freezing microtome for immunohistochemical processing and analysis. No additional control experiments were performed in this study because such experiments have previously been carried out in this laboratory (Li and Dampney, 1994; Polson et al., 1995). These previous studies demonstrated clearly that in rabbits undergoing the same procedures and using the same antibody as in the present study, but with an infusion of the saline vehicle instead of sodium nitroprusside, there is no significant change in arterial pressure and also very low levels of Fos expression in central neurons (Li and Dampney, 1994; Polson et al., 1995). 2.4. Immunohistochemical procedures One or two series of free-floating sections were processed immunohistochemically for Fos immunoreactivity using an avidin– biotin horseradish peroxidase procedure, as described previously (Polson et al., 1995; Hirooka et al., 1997). This reaction procedure has been shown previously not to affect the quality of the fluorescence observed in the rhodamine- or fluorescein-labelled microspheres (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). In one rabbit, in which cholera toxin b-subunit (CTB) was used as a retrograde tracer, an immunohistochemical reaction was also performed, using polyclonal sheep anticholera toxin b-subunit IgG (1:10,000, List Biological Laboratories), to detect the presence of the CTB in neurons. This procedure was similar to that used to detect Fos immunoreactivity, with the exception that nickel ammonium
sulphate was not added to the final reaction solution. In addition, in two rabbits, a series of sections that had been stained for Fos immunoreactivity was also processed for tyrosine hydroxylase immunoreactivity, using mouse monoclonal anti-tyrosine hydroxylase IgG (1:4000, Incstar), and also for vasopressin immunoreactivity, using a sheep polyclonal anti-vasopressin antibody (1:2000, gift from Dr Michael McKinley, Howard Florey Institute, Australia). Upon completion of the immunohistochemical reactions, the sections were mounted onto slides, dried and coverslipped with Fluoromount (Gurr). 2.5. Microscopic analysis Sections though the medulla, pons and hypothalamus were examined with an Olympus BH2 microscope fitted with a BH2-RFC fluorescence attachment, to identify neurons labelled for Fos-like immunoreactivity (Fos-LI), retrograde tracer, or both, as described previously (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). Within the RVLM, A5 area and PVN, systematic mapping of sections 200 Am (RVLM and A5 area) or 400 Am (PVN) apart was performed over the extent of the region containing labelled neurons, using a computer-assisted procedure (Magellan Programme, Halasz and Martin, 1985). These maps were then used for the purpose of quantitative analysis. For each region in each experiment, the mean number of single- and double-labelled neurons per section was determined, bilaterally. The proportion of retrogradely labelled neurons in each region that were double-labelled was then calculated. Data were expressed as mean F 1 standard error of the mean (S.E.M.).
3. Results 3.1. Injection sites In all six animals, the injection sites were located between the rostral rootlets of the L1 segment and the middle portion of the L2 segment. In each animal, the injection site extended rostrocaudally over a maximum
Fig. 1. Transverse section through the upper lumbar spinal cord showing the area encompassed by fluorescent microsphere injection sites from five experiments. The different shadings indicate different injection sites. In each experiment, a single injection was made on one side only.
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Table 1 Fos-LI, retrogradely labelled and double-labelled neurons in different brain regions Region
Fos-LI neurons (number/section)
Retrogradely labelled neurons (number/section)
Double-labelled neurons (number/section)
Double-labelled neurons (% retrogradely neurons)
RVLM A5 area PVN
52.8 F 3.0 16.6 F 3.5 238.6 F 54.5
4.3 F 0.7 3.2 F 0.8 20.1 F 4.4
1.8 F 0.4 1.4 F 0.5 1.0 F 0.3
42 F 7 40 F 5 6F1
Values are mean F S.E.M. Labelled cells were counted bilaterally in each brain region. RVLM, rostral ventrolateral medulla; PVN, paraventricular nucleus.
distance of approximately one quarter of a segment in length. In transverse sections (Fig. 1), the injection sites in all cases included the intermediolateral cell column, but also extended into the dorsal horn and the medial part of the lateral funiculus.
88 F 5 bpm, compared to the preinfusion values. We have previously shown that in rabbits infused with the vehicle solution alone, there is no significant change in arterial pressure or heart rate (Li and Dampney, 1994; Polson et al., 1995).
3.2. Cardiovascular effects of sodium nitroprusside infusion
3.3. Overall distribution of labelled neurons
The resting mean arterial pressure and heart rate before sodium nitroprusside infusion were 79 F 3 mm Hg and 224 F 10 bpm, respectively. During the infusion period, the mean arterial pressure was decreased, on average, by 18 F 3 mm Hg, and the average heart rate was increased by
The number and distribution of Fos-LI neurons in the RVLM, A5 area and PVN, following a period of hypotension, were similar to that determined in previous studies from our laboratory (Li and Dampney, 1994; Horiuchi et al., 1999). The distribution of retrogradely labelled neurons in
Fig. 2. (A) Low-power photomicrograph showing Fos-LI neurons in the RVLM following a hypotensive stimulus. (B) High power photomicrograph of the area encompassed in the box in A. (C) Fluorescent illumination of the same field as B, showing two retrogradely labelled neurons (indicated by arrows), both of which were also Fos-positive. The scale bar is 100 Am in each case.
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these regions was also similar to that described in previous studies from our laboratory in which horseradish peroxidase was used as the retrograde tracer (Blessing et al., 1981; Farlow et al., 1984), although the number of retrogradely labelled neurons was much less in the present study (probably because the volume of tracer was much smaller and the injection site much more restricted than in these previous studies). Although there was some variability between experiments in the number of retrogradely labelled neurons in each region, there was no significant correlation between the number of retrogradely labelled neurons and the proportion of these that were also double-labelled for Fos immunoreactivity in these regions (r = 0.3, P>0.3). The distribution and number of retrogradely labelled neurons in the experiment in which CTB was used as the retrograde tracer was similar to that in the other experiments in which fluorescent-labelled microspheres were used. The mean numbers of Fos-LI, retrogradely labelled and doublelabelled neurons per section in the RVLM, A5 area and PVN are shown in Table 1. The following description will focus primarily on the distribution of double-labelled neurons within each of these three regions.
3.5. Double-labelled neurons in the A5 area In the pons, retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally in the A5 area just lateral to the superior olive as well as in the region medial to the dorsal part of the superior olive (Fig. 4A,B). Fos-LI cells were also found in A5 area lateral to the superior olive, but
3.4. Double-labelled neurons in the RVLM In the RVLM, retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally, in confirmation of a previous study in the rabbit (Blessing et al., 1981). Similarly, as previously reported (Li and Dampney, 1994; Horiuchi et al., 1999), hypotension induced a high degree of Fos expression within the RVLM (Figs. 2A,B and 3A,B). Small numbers of Fos-LI cells were also located medial to the nucleus ambiguus and dorsal to the inferior olive, and in the midline raphe region (Fig. 3B). However, neurons that were both Fos-LI and retrogradely labelled were virtually confined to the RVLM, except for the occasional doublelabelled neuron just dorsal to the inferior olive or in the midline raphe (Fig. 3C). Within the RVLM, double-labelled neurons were found mainly in the region just ventrolateral and ventral to the compact part of the nucleus ambiguus (Fig. 3C). Examples of double-labelled neurons are shown in Fig. 2C. Double-labelled neurons represented approximately 40% of all retrogradely labelled neurons within the RVLM (Table 1). Both Fos-LI and retrogradely labelled neurons were observed also in the NTS (Fig. 3A), but none of these were double-labelled. In two experiments, sections through the RVLM were also processed for tyrosine hydroxylase (TH) immunoreactivity. Although no attempt was made to quantify the number of TH-immunoreactive neurons, it was noted that more than half of the Fos-LI neurons in this region were also immunoreactive for TH, in confirmation of a previous study (Li and Dampney, 1994). In particular, some triple-labelled neurons (i.e. neurons which contained retrograde tracer, Fos-LI and TH immunoreactivity) were also observed in the RVLM region described above.
Fig. 3. (A) Distribution of labelled neurons in one section through the rostral medulla, in one experiment. (B) Labelled neurons in seven equidistant sections (200 Am apart) through the RVLM in one experiment, drawn on to a single section, on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the RVLM in six experiments, drawn on a single representative section, using the nucleus ambiguus (NA), inferior olivary nucleus (IO) and ventrolateral surface as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neurons; small filled circles, Fos-positive neurons; open circle with filled centre, double-labelled neurons. Other abbreviations: TS, nucleus tractus solitarius; Vsp, spinal trigeminal nucleus.
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Fig. 4. (A) Distribution of labelled neurons in 10 equidistant sections (200 Am apart) through the A5 area in the pons in one experiment, drawn on to a single section. (B) Magnification of the area within the rectangle bounded by the dashed lines in A, which is on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the A5 area in five experiments, drawn on a single representative section, using the superior olivary nucleus (SON) and ventrolateral surface as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neuron; small filled circles, Fospositive neuron; open circle with filled centre, double-labelled neuron. Other abbreviations: 7n, facial nerve.
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Fig. 5. (A) Distribution of labelled neurons in four equidistant sections (400 Am apart) through the hypothalamus at the level of the paraventricular nucleus (PVN), in one experiment, drawn on to a single section. (B) Magnification of the area within the rectangle bounded by the dashed lines in A, which is on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the PVN in six experiments, drawn on a single representative section, using the third ventricle (3V) and fornix (F) as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neuron; small filled circles, Fos-positive neuron; open circle with filled centre, double-labelled neuron.
very few were located in the region dorsomedial to the superior olive (Fig. 4A,B). Double-labelled neurons in the A5 area were nearly all confined to the discrete region just
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lateral to the superior olive, although an occasional doublelabelled neuron was also observed in the regions ventral or medial to the superior olive (Fig. 4C). As in the RVLM, double-labelled neurons represented approximately 40% of all retrogradely labelled neurons within the A5 area (Table 1). In the two experiments in which sections were also processed for TH immunoreactivity, it was found that the large majority of the Fos-LI neurons in the A5 area, including the subgroup that also contained retrograde tracer, were immunoreactive for TH. 3.6. Double-labelled neurons in the PVN Retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally in the PVN (Fig. 5). There were many more retrogradely labelled neurons per section in the PVN than in the RVLM or A5 area (Table 1), consistent with a previous study in the rabbit (Farlow et al., 1984). However, the proportion of retrogradely labelled neurons in the PVN that were double-labelled with Fos immunoreactivity (6%) was much less than in the other two regions (Table 1). In two experiments, approximately one third of the Fos-LI neurons in the PVN were observed to be immunoreactive for vasopressin, as described previously (Li and Dampney, 1994), but the Fos-LI/vasopressin-containing neurons were not also retrogradely labelled.
4. Discussion In this study, performed in the conscious rabbit, approximately 40% of neurons within the RVLM and A5 area that projected to the upper lumbar spinal cord were activated by hypotension, as indicated by the expression of Fos, a marker of neuronal activation. Thus, although studies in anaesthetised animals indicate that spinally projecting neurons in the RVLM play a critical role in the baroreceptor reflex control of sympathetic vasomotor activity (Dampney, 1994), the results of the present study demonstrate that, in the conscious state, a significant number of spinally projecting neurons in the A5 area may also contribute significantly to the reflex sympathoexcitation evoked by a hypotensive challenge. In contrast, a much smaller proportion of spinally projecting neurons in the PVN are activated by hypotension, indicating that these neurons are regulated primarily by nonbaroreceptor inputs. 4.1. Methodological considerations The method of using Fos immunohistochemistry in combination with retrograde tracing to identify central pathways mediating cardiovascular reflexes has been discussed previously (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). The main advantage of this technique is that the study can be performed in conscious animals, thus avoiding the problem of anaesthesia perturb-
ing the function of central cardiovascular neurons (Dorward et al., 1985). As in previous studies from our laboratory (Li and Dampney, 1994; Horiuchi et al., 1999), the magnitude of the induced hypotension (fall in arterial pressure of approximately 20 mm Hg) was chosen in this study because this produces close to the maximum baroreflex change in sympathetic activity and heart rate in the conscious rabbit (Undesser et al., 1985; Weinstock et al., 1988), while the duration of the stimulus (60 min) has been shown to be optimal for producing Fos expression in activated neurons (Li and Dampney, 1994). We have shown in previous control experiments that under identical experimental conditions, and using the same antibodies and immunohistochemical procedures to those in the present study, infusion of the vehicle solution alone results in a very low level of Fos expression (Li and Dampney, 1994). In another study, we observed that the baseline level of Fos expression was also very low in rabbits in which a retrograde tracer had been injected into the brainstem in a preliminary operation under general anaesthesia performed a week or more prior to the experiment (Polson et al., 1995). These previous studies therefore indicate that neither the experimental conditions nor the prior surgical procedures result in a significant baseline level of Fos expression. Although injections of the retrograde tracer were centred on the intermediolateral cell column, they extended into surrounding regions, particularly the dorsal horn. It is therefore likely that the retrogradely labelled neurons included neurons with axons projecting to targets other than sympathetic preganglionic neurons. However, spinally projecting neurons in the RVLM, A5 area and PVN project primarily, if not exclusively, to sympathetic preganglionic nuclei (Loewy et al., 1979; Ross et al., 1984; Dampney et al., 1987; Hosoya et al., 1991; Zagon and Smith, 1993; Ranson et al., 1998). Further, it seems unlikely that spinally projecting neurons projecting to targets other than sympathetic preganglionic neurons would be strongly activated by moderate hypotension. Therefore, neurons within these regions that are both spinally projecting and activated by hypotension can be regarded as putative cardiovascular presympathetic neurons. The retrograde tracer was injected into the L1 – L2 segments of the spinal cord, which contain sympathetic preganglionic neurons controlling the hindlimb vasculature, pelvic organs, and the kidney and adrenal gland (McLachlan et al., 1985; Strack et al., 1988; Li et al., 1992a; Michaelis et al., 1993). Therefore, the retrogradely labelled neurons identified in this study would not have included neurons projecting to sympathetic preganglionic neurons in thoracic segments, including those that regulate the heart. Furthermore, the tracer injection sites were highly restricted in the rostrocaudal direction, and thus covered only part of the intermediolateral cell column within the L1 –L2 segments. Thus, the retrogradely labelled neurons are likely to represent only a small fraction of the total number of brain
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neurons that project to the L1 –L2 segments. In fact, in a previous study in the rabbit (Blessing et al., 1981), in which a much larger volume of horseradish peroxidase (5 Al) was injected unilaterally into an upper lumbar segment, the number of retrogradely labelled neurons per section in the RVLM was approximately 10 times greater than the number counted in the RVLM in the present study. Nevertheless, the distribution of retrogradely labelled neurons in the RVLM in the present study was very similar to that previously described (Blessing et al., 1981), suggesting that the retrogradely labelled neurons in the present study were a representative sample of neurons in the RVLM as well as the A5 area and PVN that project to the upper lumbar spinal cord. There was also no significant correlation between the number of retrogradely labeled neurons and the proportion of these that were double-labelled in the RVLM, A5 area or PVN, indicating also that the double-labelled neurons within each of these regions are also a representative sample of the putative presympathetic neurons in these regions that are activated by hypotension. Thus, the results provide new information about the distribution of putative presympathetic neurons in the RVLM, A5 area and PVN that are activated by a hypotensive challenge in the conscious state. 4.2. Nature of the stimulus We have previously discussed in detail the factors that lead to increased Fos expression in central neurons in response to sustained hypotension in conscious rabbits (Li and Dampney, 1994; Potts et al., 1997), and so this issue will be considered only briefly here. We have shown previously that sinoaortic denervation greatly reduces the increase in Fos expression in the RVLM, A5 area and PVN evoked by hypotension in the conscious rabbit (Potts et al., 1997). Sinoaortic denervation eliminates afferent inputs from arterial chemoreceptors as well as baroreceptors, and so it could be argued that, apart from unloading of arterial baroreceptors, the hypotension-induced activation of central neurons may be due partly to stimulation of peripheral chemoreceptors, either as a consequence of the nitroprusside-induced hypotension, or by the release of cyanide, a metabolite of sodium nitroprusside, into the circulation. We have shown previously, however, that stimulation of arterial chemoreceptors by hypoxia in the conscious rabbit produces a different pattern of Fos expression in the brainstem than that resulting from sodium nitroprusside-induced hypotension (Hirooka et al., 1997; Horiuchi et al., 1999). Secondly, infusion of sodium nitroprusside at a rate similar to that used in the present study together with concurrent infusion of phenylephrine, such that arterial pressure was maintained at resting levels, results in a low level of Fos expression in the brain, similar to that observed when vehicle alone is infused (Li and Dampney, 1994), indicating that sodium nitroprusside infusion at these concentrations does not produce any
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significant direct neural effects. Thus, although a contribution from arterial chemoreceptors to the hypotensioninduced activation of neurons in the RVLM, A5 area and PVN cannot be ruled out, it appears that any such contribution would be small compared to that due to unloading of arterial baroreceptors. Another possible factor contributing to activation of central neurons, particularly in the PVN, is an increase in the level of circulating angiotensin II as a consequence of hypotension-induced renin release (Reid et al., 1978). We have shown previously that Fos expression in the PVN is increased following angiotensin II infusion in conscious barodenervated rabbits (Potts et al., 1999), and also that hypotension-induced Fos expression in the PVN is reduced by blockade of angiotensin II receptors (Potts and Dampney, unpublished observations). Thus, although baroreceptor unloading appears to be the principal factor leading to activation of PVN neurons in response to hypotension (Potts et al., 1997), an increase in the level of circulating angiotensin II (which stimulates neurons in the circumventricular organs that in turn project to the PVN; Oldfield et al., 1994) also appears to be a contributory factor. However, an electrophysiological study in the rat indicated that PVN neurons activated in this way do not include those that project to the spinal cord (Bains and Ferguson, 1995). In summary, we suggest that the activation of spinally projecting neurons in the RVLM, A5 area and PVN in response to hypotension is due principally to unloading of arterial baroreceptors, although the possibility that other factors such as stimulation of arterial chemoreceptors or changes in the levels of angiotensin II or other hormones may also contribute cannot be entirely excluded. 4.3. Spinally projecting neurons in the RVLM activated by hypotension Within the medulla, virtually all double-labelled neurons were confined to a discrete region ventral and ventrolateral to the compact formation of the nucleus ambiguus. The distribution of these neurons corresponds very closely to the distribution of barosensitive spinally projecting neurons as determined from electrophysiological studies in the anaesthetised rabbit (Terui et al., 1986; Li et al., 1991). Only approximately 40% of the retrogradely labelled neurons in the RVLM were Fos-positive. The remaining retrogradely labelled neurons may include neurons that project to targets other than sympathetic preganglionic neurons, as suggested above, and thus would not be expected to be activated by baroreceptor unloading. Alternatively, some of these neurons may not have been activated to a sufficient degree to express Fos. RVLM presympathetic neurons consist of subgroups that regulate different vascular beds (Dampney, 1994) and some of these, such as those regulating the sympathetic outflow to cutaneous blood vessels, are relatively insensitive to baroreceptor inputs (Michaelis et al., 1993).
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4.4. Spinally projecting neurons in the A5 area activated by hypotension A surprising observation was that the proportion (approximately 40%) of spinally projecting neurons in the A5 area that expressed Fos in response to hypotension was very similar to that in the RVLM. Baroreceptor signals may reach A5 presympathetic neurons either via a direct projection from the NTS (Byrum and Guyenet, 1987) or indirectly via interneurons in other regions such as the caudal ventrolateral medulla, parabrachial complex or hypothalamic PVN, all of which receive baroreceptor inputs and which also project to the A5 area (Byrum and Guyenet, 1987; Li et al., 1992b; Dampney, 1994). Unlike the RVLM, inhibition of neurons in the A5 area does not abolish the baroreceptor reflex (Maiorov et al., 2000). In fact, bilateral inhibition of neurons in the A5 area in conscious rabbits has been shown to increase the range of the renal sympathetic nerve response to falls in arterial pressure, suggesting that these neurons may act to limit the magnitude of the sympathetic response to baroreceptor unloading (Maiorov et al., 2000). The results of the present study therefore suggest that this modulatory action by A5 neurons may occur, at least in part, at the level of the spinal cord. Consistent with this conclusion, previous studies have shown that noradrenaline (which is believed to be released from the axon terminals of A5 neurons) can alter the responsiveness of sympathetic preganglionic neurons, via changes in K + conductance (Inokuchi et al., 1992; Coote and Lewis, 1995). Furthermore, noradrenaline has been shown to reduce glutamatergic evoked excitatory postsynaptic currents in sympathetic preganglionic neurons, via a presynaptic inhibitory action (Miyazaki et al., 1988). 4.5. Spinally projecting neurons in the PVN activated by hypotension Although there were many more retrogradely labelled neurons in the PVN than in the RVLM or A5 area, the proportion of these that also expressed Fos in response to hypotension was much lower than in the other two regions (6% vs. approximately 40%). This low proportion was not due to a relatively low level of hypotension-induced Fos expression in the PVN, because as in previous studies from our laboratory in conscious rabbits, the number of Fos-LI neurons in the PVN following hypotension was much greater than in the RVLM or A5 area (Li and Dampney, 1994; Horiuchi et al., 1999). Thus, it appears that the vast majority of spinally projecting PVN neurons are not involved in the regulation of sympathetic activity in response to hypotension. Similarly, Badoer et al. (1993) found that the number of spinally projecting PVN neurons that express Fos following either hypotensive or nonhypotensive hemorrhage is also quite low (approximately 12 – 14%). Thus, our results, together with those of Badoer et al. (1993), indicate that many PVN neurons projecting to the
spinal cord may regulate the sympathetic outflow in response to inputs other than those from baroreceptors or cardiopulmonary receptors, such as signals arising from higher centres or circumventricular organs (Bains and Ferguson, 1995). Nevertheless, the results showed that some spinally projecting neurons in the PVN (although only a very small proportion) did express Fos in response to hypotension, and thus may play a role in the baroreceptor reflex. In anaesthetised animals, however, lesions of the PVN do not attenuate the baroreflex control of sympathetic activity (Patel and Schmid, 1988), whereas lesions or inhibition of RVLM neurons completely abolish this reflex (Dampney, 1994). It is possible that baroreflex control of sympathetic activity by PVN presympathetic neurons is suppressed by anaesthesia, or alternatively that it becomes significant only under conditions where hypotension is sustained (as in the current experiments) rather than transient. Our results therefore suggest that in conscious animals a small proportion of PVN presympathetic neurons may potentially contribute to the baroreflex control of sympathetic activity, at least under some conditions. At the same time, both the present study and several previous studies (Li and Dampney, 1994; Horiuchi et al., 1999) have shown that many PVN neurons are activated by hypotension. This effect is largely dependent on inputs from peripheral baroreceptors (Potts et al., 1997). If these neurons are not primarily involved in the reflex control of sympathetic activity, what are their main functions? A previous study in our laboratory showed that approximately 40% of PVN neurons activated by hypotension contain vasopressin (Li and Dampney, 1994), which is consistent with studies that show that a fall in blood pressure produces an increase in the circulating levels of vasopressin via activation of magnocellular neurons in the PVN and supraoptic nucleus, (for review, see Day, 1989). In addition, hypotension induces a large increase in adrenocorticotropin secretion (Raff et al., 1988), which in turn is dependent upon neurons in the PVN (McDonald et al., 1988). It seems very likely, therefore, that many of the PVN neurons that express Fos in response to hypotension in conscious rabbits regulate the secretion of vasopressin or adrenocorticotropin from the pituitary.
5. Conclusions This study has demonstrated that, in the conscious rabbit, a significant proportion of spinally projecting neurons within discrete regions in the RVLM and A5 area are activated by hypotension, and are thus putative presympathetic neurons whose activity is reflexly regulated by baroreceptor inputs. In the PVN, only a very small proportion of spinally projecting neurons are activated by hypotension, and thus, these neurons appear to be regulated primarily by inputs other than baroreceptor inputs.
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Acknowledgements This work was supported by the National Health and Medical Research Council of Australia. References Badoer, E., McKinley, M.J., Oldfield, B.J., McAllen, R.M., 1993. A comparison of hypotensive and non-hypotensive hemorrhage on fos expression in spinally projecting neurons of the paraventricular nucleus and rostral ventrolateral medulla. Brain Res. 610, 216 – 223. Bains, J.S., Ferguson, A.V., 1995. Paraventricular nucleus neurons projecting to the spinal cord receive excitatory input from the subfornical organ. Am. J. Physiol. 268, R625 – R633. Barman, S.M., Gebber, G.L., 1985. Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons. J. Neurophysiol. 53, 1551 – 1566. Blessing, W.W., Goodchild, A.K., Dampney, R.A.L., Chalmers, J.P., 1981. Cell groups in the lower brain stem of the rabbit projecting to the spinal cord, with special reference to catecholamine-containing cells. Brain Res. 221, 35 – 55. Byrum, C.E., Guyenet, P.G., 1987. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J. Comp. Neurol. 261, 529 – 542. Chan, R.K.W., Sawchenko, P.E., 1994. Spatially and temporally differentiated patterns of c-fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J. Comp. Neurol. 348, 433 – 460. Coote, J.H., Lewis, D.I., 1995. Bulbospinal catecholamine neurones and sympathetic pattern generation. J. Physiol. Pharmacol. 46, 259 – 271. Dampney, R.A.L., 1994. Functional organization of central pathways regulating the cardiovascular system. Physiol. Rev. 74, 323 – 364. Dampney, R.A.L., Czachurski, J., Dembowsky, K., Goodchild, A.K., Seller, H., 1987. Afferent connections and spinal projections of the pressor region in the rostral ventrolateral medulla of the cat. J. Auton. Nerv. Syst. 20, 73 – 86. Day, T., 1989. Control of neurosecretory vasopressin cells by noradrenergic projections of the caudal ventrolateral medulla. In: Ciriello, J., Caverson, M.M., Polosa, C. (Eds.), Central Neural Organization of Cardiovascular Control. Progress in Brain Research, vol. 81. Elsevier, Amsterdam, pp. 303 – 317. Dorward, P.K., Riedel, W., Burke, S.L., Gipps, J., Korner, P.I., 1985. The renal sympathetic baroreflex in the rabbit. Arterial and cardiac baroreceptor influences, resetting, and effect of anesthesia. Circ. Res. 57, 618 – 633. Farlow, D.M., Goodchild, A.K., Dampney, R.A.L., 1984. Evidence that vasomotor neurons in the rostral ventrolateral medulla project to the spinal sympathetic outflow via the dorsomedial pressor area. Brain Res. 298, 313 – 320. Graham, J.C., Hoffman, G.E., Sved, A.F., 1995. c-Fos expression in brain in response to hypotension and hypertension in conscious rats. J. Auton. Nerv. Syst. 55, 92 – 104. Guyenet, P.G., 1990. Role of the ventral medulla oblongata in blood pressure regulation. In: Loewy, A.D., Spyer, K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford Univ. Press, New York, pp. 145 – 167. Halasz, P., Martin, P., 1985. Magellan, a Programme for the Quantitative Analysis of Histological Sections. P. Halasz and P. Martin, Sydney. Hirooka, Y., Polson, J.W., Potts, P.D., Dampney, R.A.L., 1997. Hypoxiainduced fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla. Neuroscience 80, 1209 – 1224. Horiuchi, J., Potts, P.D., Polson, J.W., Dampney, R.A.L., 1999. Distribution of neurons projecting to the rostral ventrolateral medullary pressor region that are activated by sustained hypotension. Neuroscience 89, 1319 – 1329.
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Sun, M.K., Guyenet, P.G., 1985. GABA-mediated baroreceptor inhibition of reticulospinal neurons. Am. J. Physiol. 249, R672 – R680. Sun, M.K., Guyenet, P.G., 1987. Arterial baroreceptor and vagal inputs to sympathoexcitatory neurons in rat medulla. Am. J. Physiol. 252, R699 – R709. Swanson, L.W., Kuypers, H.G.Y.M., 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 double-labelling methods. J. Comp. Neurol. 194, 555 – 570. Terui, N., Saeki, Y., Kuwaki, T., Kumada, M., 1986. Barosensory neurons in the ventrolateral medulla in rabbits and their responses to various afferent inputs from peripheral and central sources. Jpn. J. Physiol. 36, 1141 – 1164. Undesser, K.P., Jing-Yun, P., Lynn, M.P., Bishop, V.S., 1985. Baroreflex control of sympathetic nerve activity after elevations of pressure in conscious rabbits. Am. J. Physiol. 248, H827 – H834. Weinstock, M., Korner, P.I., Head, G.A., Dorward, P.K., 1988. Differentiation of cardiac baroreflex properties by cuff and drug methods in two rabbit strains. Am. J. Physiol. 255, R654 – R664. Zagon, A., Smith, A.D., 1993. Monosynaptic projections from the rostral ventrolateral medulla-oblongata to identified sympathetic preganglionic neurons. Neuroscience 54, 729 – 743.
Fos expression in spinally projecting neurons after hypotension in the conscious rabbit Jaimie W. Polson, Steven Mrljak, Patrick D. Potts, Roger A.L. Dampney * Department of Physiology and Institute for Biomedical Research, University of Sydney, Sydney, NSW 2006, Australia Received 8 February 2002; received in revised form 23 May 2002; accepted 12 June 2002
Abstract Hypotension produces a reflex increase in the activity of sympathetic vasomotor nerves. Studies in anaesthetised animals have established that neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic vasomotor preganglionic neurons in the spinal cord are a critical component of the central pathways mediating this reflex response. There are also neurons in supramedullary regions (the A5 area in the pons and the paraventricular nucleus (PVN) in the hypothalamus), however, that project directly to the sympathetic vasomotor outflow. The aim of this study was to identify and map neurons within the A5 area and PVN, as well as in the RVLM, which may contribute to the reflex sympathoexcitatory response to a hypotensive challenge in conscious rabbits. In a preliminary operation, a retrogradely transported tracer was injected into a site centred on the intermediolateral cell column in the upper lumbar spinal cord. After a waiting period of at least 1 week, a moderate hypotension (decrease in arterial pressure of approximately 20 mm Hg) was induced in conscious rabbits for 60 min by continuous infusion of sodium nitroprusside. In confirmation of previous studies, hypotension resulted in the expression of Fos in the RVLM, the A5 area and PVN. There were also retrogradely labelled neurons in all these regions. In both the RVLM and A5 area, approximately 40% of the retrogradely labelled neurons were also immunoreactive for Fos. In contrast, in the PVN the proportion of retrogradely labelled neurons that were also Fos-positive was much less (approximately 6%). This study has demonstrated that, in the conscious rabbit, a significant proportion of spinally projecting neurons within discrete regions in the RVLM and A5 area are activated by hypotension (as indicated by Fos expression). In the PVN, only a very small proportion of spinally projecting neurons are activated by hypotension, and thus these neurons appear to be regulated primarily by inputs other than baroreceptor inputs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Baroreceptor reflex; Central cardiovascular pathways; Rostral ventrolateral medulla; A5 area; Paraventricular nucleus; Immediate early genes
1. Introduction It is well established that the rostral ventrolateral medulla (RVLM) contains neurons that provide a direct excitatory input to sympathetic cardiac and vasomotor preganglionic neurons in the spinal cord, which are a critical component of the central pathways mediating the baroreceptor and other cardiovascular reflexes (for review, see Guyenet, 1990 or Dampney, 1994). In particular, studies in anaesthetised animals have shown that such presympathetic neurons are powerfully inhibited by baroreceptor inputs (Barman and Gebber, 1985; Sun and Guyenet, 1985; McAllen, 1986). Consistent with these observations, unloading of barorecep-
*
Corresponding author. Department of Physiology F13, The University of Sydney, NSW 2006, Australia. Fax: +61-2-93516470. E-mail address: [email protected] (R.A.L. Dampney).
tors by induced hypotension in conscious animals results in activation of neurons in the RVLM, as indicated by the expression of Fos, the protein product of the immediate early gene c-fos (Chan and Sawchenko, 1994; Li and Dampney, 1994; Graham et al., 1995; Horiuchi et al., 1999), an effect which is prevented by denervation of carotid sinus and aortic baroreceptors (Potts et al., 1997). Apart from the RVLM, presympathetic neurons are also located in other medullary and supramedullary regions, including the medullary raphe nuclei, the A5 area in the pons and the paraventricular nucleus (PVN) in the hypothalamus (Strack et al., 1989). The observation that bilateral blockade of GABA receptors in the RVLM abolishes the baroreceptor-sympathetic reflex (Sun and Guyenet, 1987), however, suggests that presympathetic neurons in these other regions play little or no role in this reflex, at least in anaesthetised animals. On the other hand, previous studies in our laboratory have shown that induced hypotension in
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 1 4 3 - 1
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conscious rabbits result in a significant increase in Fos expression above baseline levels in the A5 area and PVN, although not in the medullary raphe nuclei (Li and Dampney, 1994; Horiuchi et al., 1999). In conscious rabbits with denervated sinoaortic baroreceptors, however, hypotension resulted in no increase in Fos expression above baseline levels in the A5 area, while in the PVN there was an increase in Fos expression but it was much less than that observed in baroreceptor intact animals (Potts et al., 1997). These previous observations indicate that there are neurons in the A5 area and PVN that are activated by hypotension, primarily by signals arising from baroreceptors. It is not clear, however, what function such activated neurons may have. One possibility is that these neurons are presympathetic, and are involved in the baroreceptor reflex regulation of sympathetic nerve activity. Alternatively, they may convey baroreceptor signals to other target nuclei. The PVN, for example, contains several different populations of neurons that project to different targets, including the median eminence, posterior pituitary, nucleus tractus solitarius (NTS) and RVLM as well as the spinal cord (Swanson and Kuypers, 1980; Shafton et al., 1998). Similarly, neurons in the A5 area have extensive projections to a number of cardiovascular regulatory nuclei in the brainstem and forebrain (Byrum and Guyenet, 1987). The aim of this study was to identify spinally projecting neurons in the RVLM, A5 area and PVN that are activated by a hypotensive challenge in the conscious state. For this purpose, we have mapped the distribution of neurons within these three brain regions that express Fos in response to a period of induced hypotension in conscious rabbits, and which were also retrogradely labelled from an upper lumbar segment of the spinal cord. The upper lumbar part of the spinal cord was selected because it contains a high density of sympathetic preganglionic neurons regulating vasomotor activity in the hindlimbs and abdominal viscera (McLachlan et al., 1985; Strack et al., 1988; Li et al., 1992a). Although this method results in retrograde labelling only of a small sample of the neurons in the RVLM, A5 area and PVN that project to the spinal cord, double-labelling for the presence of Fos in response to a hypotensive challenge in the conscious animal allows a determination of the location of putative cardiovascular presympathetic neurons in these three brain regions. Parts of this study have been published previously in abstract form (Polson et al., 2000).
2. Materials and methods 2.1. General procedures Experiments were performed on six adult rabbits of either sex (two New Zealand White and four crossbred from Baker Medical Research Institute, Melbourne, Australia; 2.5 – 3.5 kg). All experiments were carried out in accordance with the Guidelines for Animal Experimentation
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of the National Health and Medical Research Council of Australia. 2.2. Injection of tracers Rabbits were premedicated with the opioid analgesic buprenorphine hydrochloride (Temgesic, Reckitt and Coleman; 0.03 mg/kg i.m.) for the purpose of preemptive analgesia. General anaesthesia was then induced with a mixture of alphaxalone and alphadolone (Saffan, PittmanMoore, initial dose 5 mg total steroids/kg i.v., followed by a continuous infusion of 8 – 16 mg/kg/h i.v.) and intubated with a perinatal endotracheal tube (Mallinkrodt Lo-Pro, 3.0mm i.d.). The rabbit was then artificially ventilated with 35 – 45% oxygen in room air, at a rate that maintained endtidal CO2 in the range 3.5 – 4.5%. Body temperature was maintained at approximately 39 jC using a thermostatically regulated heating lamp connected to a rectal temperature probe. The rabbit was positioned in an atraumatic stereotaxic frame, and the vertebral column at the level of T12 to L2 was exposed. The spinous process of vertebra T12 was clamped and fixed to the stereotaxic frame to reduce movement, and the tissue overlying either the L1 or L2 vertebra was retracted to expose the vertebral laminae. After ensuring that the level of general anaesthesia was adequate, by the absence of a withdrawal response to toe pinch, a bolus injection of the neuromuscular blocker alcuronium bromide (Alloferin, Roche; 0.1 –0.2 mg/kg i.v.) was administered to prevent movements that might arise from mechanical stimulation of the spinal cord. The spinal cord and overlying dura mater was then exposed via a laminectomy. A small incision was made in the dura mater and a glass micropipette, containing either rhodamine- or fluorescein-labelled microspheres (n = 5; Lumafluor) or cholera toxin b-subunit (n = 1; List Biological Laboratories), was inserted into the spinal cord, approximately 0.7 mm lateral to the midline, at a depth of 1.0 – 1.5 mm. The volume of the injectate containing fluorescent microspheres was 480 –910 nl, and of cholera toxin b-subunit was 135 nl. A single unilateral injection was made slowly, over a period of approximately 30 min. Following the injection, Gelfoam powder (Upjohn) was placed over the exposed spinal cord, and the overlying muscles and skin sutured in layers. Benzylpenicillin (Commonwealth Serum Laboratories) was applied both topically to the wound (approximately 60 mg) and intramuscularly (30 mg/kg). Upon completion of the surgery and recovery from the neuromuscular block, the infusion of alphaxalone and alphadolone was terminated. The rabbit was closely observed during the period of recovery from general anaesthesia and during the postoperative period. Analgesia was maintained with buprenorphine (0.03 mg/kg/12 h) and the nonsteroidal anti-inflammatory agent ketoprofen (2.0 mg/kg/24 h) for a minimum of 3 days, and antibiotic prophylaxis (benzylpenicillin, 30 mg/kg/24 h) for a minimum of 4 days. No animals showed any signs of postoperative infection.
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2.3. Induction of hypotension Following a recovery period of 1– 3 weeks, a 60-min period of hypotension was induced in the conscious unrestrained rabbits, according to the procedure described previously (Li and Dampney, 1994). Briefly, arterial pressure was measured via a cannula placed in a central ear artery, and sodium nitroprusside was infused intravenously, via a marginal ear vein, at a rate (0.2 – 1.0 mg/kg/h, adjusted continuously) that produced a fall in arterial pressure of approximately 20 mm Hg, for a period of 60 min. It has been shown previously that a fall in arterial pressure of 20 mm Hg produces close to the maximum baroreflex change in sympathetic activity and heart rate in the conscious rabbit (Undesser et al., 1985; Weinstock et al., 1988), while the duration of the stimulus (60 min) has been shown to be optimal for producing Fos expression in activated neurons (Li and Dampney, 1994). Following the 60-min period of hypotension, the infusion was stopped and arterial pressure and heart rate monitored for a further 30 min. The rabbit was then anaesthetised with sodium pentobarbitone (60 mg/kg i.v.) and perfused transcardially with 1 l of heparinized saline, followed by 2 l of 4% paraformaldehyde in 0.1 M phosphatebuffered saline, pH 7.3. The brain and the region of spinal cord surrounding the injection site were removed and placed in 30% sucrose in 0.1 M phosphate buffer. Five series of sections (40-Am thick) were cut on a freezing microtome for immunohistochemical processing and analysis. No additional control experiments were performed in this study because such experiments have previously been carried out in this laboratory (Li and Dampney, 1994; Polson et al., 1995). These previous studies demonstrated clearly that in rabbits undergoing the same procedures and using the same antibody as in the present study, but with an infusion of the saline vehicle instead of sodium nitroprusside, there is no significant change in arterial pressure and also very low levels of Fos expression in central neurons (Li and Dampney, 1994; Polson et al., 1995). 2.4. Immunohistochemical procedures One or two series of free-floating sections were processed immunohistochemically for Fos immunoreactivity using an avidin– biotin horseradish peroxidase procedure, as described previously (Polson et al., 1995; Hirooka et al., 1997). This reaction procedure has been shown previously not to affect the quality of the fluorescence observed in the rhodamine- or fluorescein-labelled microspheres (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). In one rabbit, in which cholera toxin b-subunit (CTB) was used as a retrograde tracer, an immunohistochemical reaction was also performed, using polyclonal sheep anticholera toxin b-subunit IgG (1:10,000, List Biological Laboratories), to detect the presence of the CTB in neurons. This procedure was similar to that used to detect Fos immunoreactivity, with the exception that nickel ammonium
sulphate was not added to the final reaction solution. In addition, in two rabbits, a series of sections that had been stained for Fos immunoreactivity was also processed for tyrosine hydroxylase immunoreactivity, using mouse monoclonal anti-tyrosine hydroxylase IgG (1:4000, Incstar), and also for vasopressin immunoreactivity, using a sheep polyclonal anti-vasopressin antibody (1:2000, gift from Dr Michael McKinley, Howard Florey Institute, Australia). Upon completion of the immunohistochemical reactions, the sections were mounted onto slides, dried and coverslipped with Fluoromount (Gurr). 2.5. Microscopic analysis Sections though the medulla, pons and hypothalamus were examined with an Olympus BH2 microscope fitted with a BH2-RFC fluorescence attachment, to identify neurons labelled for Fos-like immunoreactivity (Fos-LI), retrograde tracer, or both, as described previously (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). Within the RVLM, A5 area and PVN, systematic mapping of sections 200 Am (RVLM and A5 area) or 400 Am (PVN) apart was performed over the extent of the region containing labelled neurons, using a computer-assisted procedure (Magellan Programme, Halasz and Martin, 1985). These maps were then used for the purpose of quantitative analysis. For each region in each experiment, the mean number of single- and double-labelled neurons per section was determined, bilaterally. The proportion of retrogradely labelled neurons in each region that were double-labelled was then calculated. Data were expressed as mean F 1 standard error of the mean (S.E.M.).
3. Results 3.1. Injection sites In all six animals, the injection sites were located between the rostral rootlets of the L1 segment and the middle portion of the L2 segment. In each animal, the injection site extended rostrocaudally over a maximum
Fig. 1. Transverse section through the upper lumbar spinal cord showing the area encompassed by fluorescent microsphere injection sites from five experiments. The different shadings indicate different injection sites. In each experiment, a single injection was made on one side only.
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Table 1 Fos-LI, retrogradely labelled and double-labelled neurons in different brain regions Region
Fos-LI neurons (number/section)
Retrogradely labelled neurons (number/section)
Double-labelled neurons (number/section)
Double-labelled neurons (% retrogradely neurons)
RVLM A5 area PVN
52.8 F 3.0 16.6 F 3.5 238.6 F 54.5
4.3 F 0.7 3.2 F 0.8 20.1 F 4.4
1.8 F 0.4 1.4 F 0.5 1.0 F 0.3
42 F 7 40 F 5 6F1
Values are mean F S.E.M. Labelled cells were counted bilaterally in each brain region. RVLM, rostral ventrolateral medulla; PVN, paraventricular nucleus.
distance of approximately one quarter of a segment in length. In transverse sections (Fig. 1), the injection sites in all cases included the intermediolateral cell column, but also extended into the dorsal horn and the medial part of the lateral funiculus.
88 F 5 bpm, compared to the preinfusion values. We have previously shown that in rabbits infused with the vehicle solution alone, there is no significant change in arterial pressure or heart rate (Li and Dampney, 1994; Polson et al., 1995).
3.2. Cardiovascular effects of sodium nitroprusside infusion
3.3. Overall distribution of labelled neurons
The resting mean arterial pressure and heart rate before sodium nitroprusside infusion were 79 F 3 mm Hg and 224 F 10 bpm, respectively. During the infusion period, the mean arterial pressure was decreased, on average, by 18 F 3 mm Hg, and the average heart rate was increased by
The number and distribution of Fos-LI neurons in the RVLM, A5 area and PVN, following a period of hypotension, were similar to that determined in previous studies from our laboratory (Li and Dampney, 1994; Horiuchi et al., 1999). The distribution of retrogradely labelled neurons in
Fig. 2. (A) Low-power photomicrograph showing Fos-LI neurons in the RVLM following a hypotensive stimulus. (B) High power photomicrograph of the area encompassed in the box in A. (C) Fluorescent illumination of the same field as B, showing two retrogradely labelled neurons (indicated by arrows), both of which were also Fos-positive. The scale bar is 100 Am in each case.
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these regions was also similar to that described in previous studies from our laboratory in which horseradish peroxidase was used as the retrograde tracer (Blessing et al., 1981; Farlow et al., 1984), although the number of retrogradely labelled neurons was much less in the present study (probably because the volume of tracer was much smaller and the injection site much more restricted than in these previous studies). Although there was some variability between experiments in the number of retrogradely labelled neurons in each region, there was no significant correlation between the number of retrogradely labelled neurons and the proportion of these that were also double-labelled for Fos immunoreactivity in these regions (r = 0.3, P>0.3). The distribution and number of retrogradely labelled neurons in the experiment in which CTB was used as the retrograde tracer was similar to that in the other experiments in which fluorescent-labelled microspheres were used. The mean numbers of Fos-LI, retrogradely labelled and doublelabelled neurons per section in the RVLM, A5 area and PVN are shown in Table 1. The following description will focus primarily on the distribution of double-labelled neurons within each of these three regions.
3.5. Double-labelled neurons in the A5 area In the pons, retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally in the A5 area just lateral to the superior olive as well as in the region medial to the dorsal part of the superior olive (Fig. 4A,B). Fos-LI cells were also found in A5 area lateral to the superior olive, but
3.4. Double-labelled neurons in the RVLM In the RVLM, retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally, in confirmation of a previous study in the rabbit (Blessing et al., 1981). Similarly, as previously reported (Li and Dampney, 1994; Horiuchi et al., 1999), hypotension induced a high degree of Fos expression within the RVLM (Figs. 2A,B and 3A,B). Small numbers of Fos-LI cells were also located medial to the nucleus ambiguus and dorsal to the inferior olive, and in the midline raphe region (Fig. 3B). However, neurons that were both Fos-LI and retrogradely labelled were virtually confined to the RVLM, except for the occasional doublelabelled neuron just dorsal to the inferior olive or in the midline raphe (Fig. 3C). Within the RVLM, double-labelled neurons were found mainly in the region just ventrolateral and ventral to the compact part of the nucleus ambiguus (Fig. 3C). Examples of double-labelled neurons are shown in Fig. 2C. Double-labelled neurons represented approximately 40% of all retrogradely labelled neurons within the RVLM (Table 1). Both Fos-LI and retrogradely labelled neurons were observed also in the NTS (Fig. 3A), but none of these were double-labelled. In two experiments, sections through the RVLM were also processed for tyrosine hydroxylase (TH) immunoreactivity. Although no attempt was made to quantify the number of TH-immunoreactive neurons, it was noted that more than half of the Fos-LI neurons in this region were also immunoreactive for TH, in confirmation of a previous study (Li and Dampney, 1994). In particular, some triple-labelled neurons (i.e. neurons which contained retrograde tracer, Fos-LI and TH immunoreactivity) were also observed in the RVLM region described above.
Fig. 3. (A) Distribution of labelled neurons in one section through the rostral medulla, in one experiment. (B) Labelled neurons in seven equidistant sections (200 Am apart) through the RVLM in one experiment, drawn on to a single section, on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the RVLM in six experiments, drawn on a single representative section, using the nucleus ambiguus (NA), inferior olivary nucleus (IO) and ventrolateral surface as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neurons; small filled circles, Fos-positive neurons; open circle with filled centre, double-labelled neurons. Other abbreviations: TS, nucleus tractus solitarius; Vsp, spinal trigeminal nucleus.
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Fig. 4. (A) Distribution of labelled neurons in 10 equidistant sections (200 Am apart) through the A5 area in the pons in one experiment, drawn on to a single section. (B) Magnification of the area within the rectangle bounded by the dashed lines in A, which is on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the A5 area in five experiments, drawn on a single representative section, using the superior olivary nucleus (SON) and ventrolateral surface as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neuron; small filled circles, Fospositive neuron; open circle with filled centre, double-labelled neuron. Other abbreviations: 7n, facial nerve.
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Fig. 5. (A) Distribution of labelled neurons in four equidistant sections (400 Am apart) through the hypothalamus at the level of the paraventricular nucleus (PVN), in one experiment, drawn on to a single section. (B) Magnification of the area within the rectangle bounded by the dashed lines in A, which is on the side ipsilateral to the injection site in the spinal cord. (C) Distribution of all double-labelled neurons in the PVN in six experiments, drawn on a single representative section, using the third ventricle (3V) and fornix (F) as anatomical landmarks, on the side ipsilateral to the injection site in the spinal cord. Shaded larger circles, retrogradely labelled neuron; small filled circles, Fos-positive neuron; open circle with filled centre, double-labelled neuron.
very few were located in the region dorsomedial to the superior olive (Fig. 4A,B). Double-labelled neurons in the A5 area were nearly all confined to the discrete region just
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lateral to the superior olive, although an occasional doublelabelled neuron was also observed in the regions ventral or medial to the superior olive (Fig. 4C). As in the RVLM, double-labelled neurons represented approximately 40% of all retrogradely labelled neurons within the A5 area (Table 1). In the two experiments in which sections were also processed for TH immunoreactivity, it was found that the large majority of the Fos-LI neurons in the A5 area, including the subgroup that also contained retrograde tracer, were immunoreactive for TH. 3.6. Double-labelled neurons in the PVN Retrogradely labelled neurons were located bilaterally but predominantly ipsilaterally in the PVN (Fig. 5). There were many more retrogradely labelled neurons per section in the PVN than in the RVLM or A5 area (Table 1), consistent with a previous study in the rabbit (Farlow et al., 1984). However, the proportion of retrogradely labelled neurons in the PVN that were double-labelled with Fos immunoreactivity (6%) was much less than in the other two regions (Table 1). In two experiments, approximately one third of the Fos-LI neurons in the PVN were observed to be immunoreactive for vasopressin, as described previously (Li and Dampney, 1994), but the Fos-LI/vasopressin-containing neurons were not also retrogradely labelled.
4. Discussion In this study, performed in the conscious rabbit, approximately 40% of neurons within the RVLM and A5 area that projected to the upper lumbar spinal cord were activated by hypotension, as indicated by the expression of Fos, a marker of neuronal activation. Thus, although studies in anaesthetised animals indicate that spinally projecting neurons in the RVLM play a critical role in the baroreceptor reflex control of sympathetic vasomotor activity (Dampney, 1994), the results of the present study demonstrate that, in the conscious state, a significant number of spinally projecting neurons in the A5 area may also contribute significantly to the reflex sympathoexcitation evoked by a hypotensive challenge. In contrast, a much smaller proportion of spinally projecting neurons in the PVN are activated by hypotension, indicating that these neurons are regulated primarily by nonbaroreceptor inputs. 4.1. Methodological considerations The method of using Fos immunohistochemistry in combination with retrograde tracing to identify central pathways mediating cardiovascular reflexes has been discussed previously (Polson et al., 1995; Hirooka et al., 1997; Horiuchi et al., 1999). The main advantage of this technique is that the study can be performed in conscious animals, thus avoiding the problem of anaesthesia perturb-
ing the function of central cardiovascular neurons (Dorward et al., 1985). As in previous studies from our laboratory (Li and Dampney, 1994; Horiuchi et al., 1999), the magnitude of the induced hypotension (fall in arterial pressure of approximately 20 mm Hg) was chosen in this study because this produces close to the maximum baroreflex change in sympathetic activity and heart rate in the conscious rabbit (Undesser et al., 1985; Weinstock et al., 1988), while the duration of the stimulus (60 min) has been shown to be optimal for producing Fos expression in activated neurons (Li and Dampney, 1994). We have shown in previous control experiments that under identical experimental conditions, and using the same antibodies and immunohistochemical procedures to those in the present study, infusion of the vehicle solution alone results in a very low level of Fos expression (Li and Dampney, 1994). In another study, we observed that the baseline level of Fos expression was also very low in rabbits in which a retrograde tracer had been injected into the brainstem in a preliminary operation under general anaesthesia performed a week or more prior to the experiment (Polson et al., 1995). These previous studies therefore indicate that neither the experimental conditions nor the prior surgical procedures result in a significant baseline level of Fos expression. Although injections of the retrograde tracer were centred on the intermediolateral cell column, they extended into surrounding regions, particularly the dorsal horn. It is therefore likely that the retrogradely labelled neurons included neurons with axons projecting to targets other than sympathetic preganglionic neurons. However, spinally projecting neurons in the RVLM, A5 area and PVN project primarily, if not exclusively, to sympathetic preganglionic nuclei (Loewy et al., 1979; Ross et al., 1984; Dampney et al., 1987; Hosoya et al., 1991; Zagon and Smith, 1993; Ranson et al., 1998). Further, it seems unlikely that spinally projecting neurons projecting to targets other than sympathetic preganglionic neurons would be strongly activated by moderate hypotension. Therefore, neurons within these regions that are both spinally projecting and activated by hypotension can be regarded as putative cardiovascular presympathetic neurons. The retrograde tracer was injected into the L1 – L2 segments of the spinal cord, which contain sympathetic preganglionic neurons controlling the hindlimb vasculature, pelvic organs, and the kidney and adrenal gland (McLachlan et al., 1985; Strack et al., 1988; Li et al., 1992a; Michaelis et al., 1993). Therefore, the retrogradely labelled neurons identified in this study would not have included neurons projecting to sympathetic preganglionic neurons in thoracic segments, including those that regulate the heart. Furthermore, the tracer injection sites were highly restricted in the rostrocaudal direction, and thus covered only part of the intermediolateral cell column within the L1 –L2 segments. Thus, the retrogradely labelled neurons are likely to represent only a small fraction of the total number of brain
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neurons that project to the L1 –L2 segments. In fact, in a previous study in the rabbit (Blessing et al., 1981), in which a much larger volume of horseradish peroxidase (5 Al) was injected unilaterally into an upper lumbar segment, the number of retrogradely labelled neurons per section in the RVLM was approximately 10 times greater than the number counted in the RVLM in the present study. Nevertheless, the distribution of retrogradely labelled neurons in the RVLM in the present study was very similar to that previously described (Blessing et al., 1981), suggesting that the retrogradely labelled neurons in the present study were a representative sample of neurons in the RVLM as well as the A5 area and PVN that project to the upper lumbar spinal cord. There was also no significant correlation between the number of retrogradely labeled neurons and the proportion of these that were double-labelled in the RVLM, A5 area or PVN, indicating also that the double-labelled neurons within each of these regions are also a representative sample of the putative presympathetic neurons in these regions that are activated by hypotension. Thus, the results provide new information about the distribution of putative presympathetic neurons in the RVLM, A5 area and PVN that are activated by a hypotensive challenge in the conscious state. 4.2. Nature of the stimulus We have previously discussed in detail the factors that lead to increased Fos expression in central neurons in response to sustained hypotension in conscious rabbits (Li and Dampney, 1994; Potts et al., 1997), and so this issue will be considered only briefly here. We have shown previously that sinoaortic denervation greatly reduces the increase in Fos expression in the RVLM, A5 area and PVN evoked by hypotension in the conscious rabbit (Potts et al., 1997). Sinoaortic denervation eliminates afferent inputs from arterial chemoreceptors as well as baroreceptors, and so it could be argued that, apart from unloading of arterial baroreceptors, the hypotension-induced activation of central neurons may be due partly to stimulation of peripheral chemoreceptors, either as a consequence of the nitroprusside-induced hypotension, or by the release of cyanide, a metabolite of sodium nitroprusside, into the circulation. We have shown previously, however, that stimulation of arterial chemoreceptors by hypoxia in the conscious rabbit produces a different pattern of Fos expression in the brainstem than that resulting from sodium nitroprusside-induced hypotension (Hirooka et al., 1997; Horiuchi et al., 1999). Secondly, infusion of sodium nitroprusside at a rate similar to that used in the present study together with concurrent infusion of phenylephrine, such that arterial pressure was maintained at resting levels, results in a low level of Fos expression in the brain, similar to that observed when vehicle alone is infused (Li and Dampney, 1994), indicating that sodium nitroprusside infusion at these concentrations does not produce any
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significant direct neural effects. Thus, although a contribution from arterial chemoreceptors to the hypotensioninduced activation of neurons in the RVLM, A5 area and PVN cannot be ruled out, it appears that any such contribution would be small compared to that due to unloading of arterial baroreceptors. Another possible factor contributing to activation of central neurons, particularly in the PVN, is an increase in the level of circulating angiotensin II as a consequence of hypotension-induced renin release (Reid et al., 1978). We have shown previously that Fos expression in the PVN is increased following angiotensin II infusion in conscious barodenervated rabbits (Potts et al., 1999), and also that hypotension-induced Fos expression in the PVN is reduced by blockade of angiotensin II receptors (Potts and Dampney, unpublished observations). Thus, although baroreceptor unloading appears to be the principal factor leading to activation of PVN neurons in response to hypotension (Potts et al., 1997), an increase in the level of circulating angiotensin II (which stimulates neurons in the circumventricular organs that in turn project to the PVN; Oldfield et al., 1994) also appears to be a contributory factor. However, an electrophysiological study in the rat indicated that PVN neurons activated in this way do not include those that project to the spinal cord (Bains and Ferguson, 1995). In summary, we suggest that the activation of spinally projecting neurons in the RVLM, A5 area and PVN in response to hypotension is due principally to unloading of arterial baroreceptors, although the possibility that other factors such as stimulation of arterial chemoreceptors or changes in the levels of angiotensin II or other hormones may also contribute cannot be entirely excluded. 4.3. Spinally projecting neurons in the RVLM activated by hypotension Within the medulla, virtually all double-labelled neurons were confined to a discrete region ventral and ventrolateral to the compact formation of the nucleus ambiguus. The distribution of these neurons corresponds very closely to the distribution of barosensitive spinally projecting neurons as determined from electrophysiological studies in the anaesthetised rabbit (Terui et al., 1986; Li et al., 1991). Only approximately 40% of the retrogradely labelled neurons in the RVLM were Fos-positive. The remaining retrogradely labelled neurons may include neurons that project to targets other than sympathetic preganglionic neurons, as suggested above, and thus would not be expected to be activated by baroreceptor unloading. Alternatively, some of these neurons may not have been activated to a sufficient degree to express Fos. RVLM presympathetic neurons consist of subgroups that regulate different vascular beds (Dampney, 1994) and some of these, such as those regulating the sympathetic outflow to cutaneous blood vessels, are relatively insensitive to baroreceptor inputs (Michaelis et al., 1993).
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4.4. Spinally projecting neurons in the A5 area activated by hypotension A surprising observation was that the proportion (approximately 40%) of spinally projecting neurons in the A5 area that expressed Fos in response to hypotension was very similar to that in the RVLM. Baroreceptor signals may reach A5 presympathetic neurons either via a direct projection from the NTS (Byrum and Guyenet, 1987) or indirectly via interneurons in other regions such as the caudal ventrolateral medulla, parabrachial complex or hypothalamic PVN, all of which receive baroreceptor inputs and which also project to the A5 area (Byrum and Guyenet, 1987; Li et al., 1992b; Dampney, 1994). Unlike the RVLM, inhibition of neurons in the A5 area does not abolish the baroreceptor reflex (Maiorov et al., 2000). In fact, bilateral inhibition of neurons in the A5 area in conscious rabbits has been shown to increase the range of the renal sympathetic nerve response to falls in arterial pressure, suggesting that these neurons may act to limit the magnitude of the sympathetic response to baroreceptor unloading (Maiorov et al., 2000). The results of the present study therefore suggest that this modulatory action by A5 neurons may occur, at least in part, at the level of the spinal cord. Consistent with this conclusion, previous studies have shown that noradrenaline (which is believed to be released from the axon terminals of A5 neurons) can alter the responsiveness of sympathetic preganglionic neurons, via changes in K + conductance (Inokuchi et al., 1992; Coote and Lewis, 1995). Furthermore, noradrenaline has been shown to reduce glutamatergic evoked excitatory postsynaptic currents in sympathetic preganglionic neurons, via a presynaptic inhibitory action (Miyazaki et al., 1988). 4.5. Spinally projecting neurons in the PVN activated by hypotension Although there were many more retrogradely labelled neurons in the PVN than in the RVLM or A5 area, the proportion of these that also expressed Fos in response to hypotension was much lower than in the other two regions (6% vs. approximately 40%). This low proportion was not due to a relatively low level of hypotension-induced Fos expression in the PVN, because as in previous studies from our laboratory in conscious rabbits, the number of Fos-LI neurons in the PVN following hypotension was much greater than in the RVLM or A5 area (Li and Dampney, 1994; Horiuchi et al., 1999). Thus, it appears that the vast majority of spinally projecting PVN neurons are not involved in the regulation of sympathetic activity in response to hypotension. Similarly, Badoer et al. (1993) found that the number of spinally projecting PVN neurons that express Fos following either hypotensive or nonhypotensive hemorrhage is also quite low (approximately 12 – 14%). Thus, our results, together with those of Badoer et al. (1993), indicate that many PVN neurons projecting to the
spinal cord may regulate the sympathetic outflow in response to inputs other than those from baroreceptors or cardiopulmonary receptors, such as signals arising from higher centres or circumventricular organs (Bains and Ferguson, 1995). Nevertheless, the results showed that some spinally projecting neurons in the PVN (although only a very small proportion) did express Fos in response to hypotension, and thus may play a role in the baroreceptor reflex. In anaesthetised animals, however, lesions of the PVN do not attenuate the baroreflex control of sympathetic activity (Patel and Schmid, 1988), whereas lesions or inhibition of RVLM neurons completely abolish this reflex (Dampney, 1994). It is possible that baroreflex control of sympathetic activity by PVN presympathetic neurons is suppressed by anaesthesia, or alternatively that it becomes significant only under conditions where hypotension is sustained (as in the current experiments) rather than transient. Our results therefore suggest that in conscious animals a small proportion of PVN presympathetic neurons may potentially contribute to the baroreflex control of sympathetic activity, at least under some conditions. At the same time, both the present study and several previous studies (Li and Dampney, 1994; Horiuchi et al., 1999) have shown that many PVN neurons are activated by hypotension. This effect is largely dependent on inputs from peripheral baroreceptors (Potts et al., 1997). If these neurons are not primarily involved in the reflex control of sympathetic activity, what are their main functions? A previous study in our laboratory showed that approximately 40% of PVN neurons activated by hypotension contain vasopressin (Li and Dampney, 1994), which is consistent with studies that show that a fall in blood pressure produces an increase in the circulating levels of vasopressin via activation of magnocellular neurons in the PVN and supraoptic nucleus, (for review, see Day, 1989). In addition, hypotension induces a large increase in adrenocorticotropin secretion (Raff et al., 1988), which in turn is dependent upon neurons in the PVN (McDonald et al., 1988). It seems very likely, therefore, that many of the PVN neurons that express Fos in response to hypotension in conscious rabbits regulate the secretion of vasopressin or adrenocorticotropin from the pituitary.
5. Conclusions This study has demonstrated that, in the conscious rabbit, a significant proportion of spinally projecting neurons within discrete regions in the RVLM and A5 area are activated by hypotension, and are thus putative presympathetic neurons whose activity is reflexly regulated by baroreceptor inputs. In the PVN, only a very small proportion of spinally projecting neurons are activated by hypotension, and thus, these neurons appear to be regulated primarily by inputs other than baroreceptor inputs.
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