| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 15 July 2007;
accepted after revision 5 September 2007; first published online 7 September 2007)
Corresponding author E. Badoer: School of Medical Sciences, RMIT University, PO Box 71, Bundoora 3083, Melbourne, Victoria, Australia. Email: emilio.badoer{at}rmit.edu.au
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Exposure to a hot environment elicits reflex responses that promote heat loss (Kanosue et al. 1991, 1994; Scammell et al. 1993; Zhang et al. 1997; Kazuyuki et al. 1998; Nagashima et al. 2000; Morrison, 2001; Owens et al. 2002). Such responses include an increase in heart rate, increased respiration rate, vasodilatation of the skin vasculature, vasoconstriction of the visceral vasculature, sweating in humans and increased salivary secretion and vasodilatation in the tail of rodents (Kanosue et al. 1994; Kazuyuki et al. 1998), all of which are designed to counteract an increase in body temperature. The redistribution of blood flow from the viscera to the skin is critical for the dissipation of heat and this involves autonomic cardiovascular responses which are mediated by the central nervous system (CNS) through the regulation of sympathetic nerve activity (Kanosue et al. 1991; Scammell et al. 1993; Nagashima et al. 2000; Morrison, 2001; Owens et al. 2002).
Exposure to a hot environment activates several nuclei throughout the brain. These include forebrain nuclei such as the preoptic area, known to contain warm-sensitive neurones, and areas in the hypothalamus and brainstem that contain important autonomic nuclei (Kiyohara et al. 1995; McKitrick, 2000; Bachtell et al. 2003; Harikai et al. 2003; Bratincsak & Palkovits, 2004). One important autonomic nucleus is the hypothalamic PVN. This nucleus is of particular interest because we have shown recently that exposure to a hot environment strongly activates neurones in the PVN (Cham et al. 2006), including so-called premotor neurones that send projections to the intermediolateral cell column (IML) of the thoracolumbar spinal cord, where sympathetic preganglionic motor neurones are located. That finding suggests the PVN may be an important site for integration of the peripheral neural involvement in the thermoregulatory responses.
The hypothalamic PVN is composed of functionally different subgroups of neurones such as the parvocellular neurones that project to important autonomic targets that, in addition to the spinally projecting neurones, also include neurones that project to the pressor region of the rostral ventrolateral medulla (RVLM; Swanson & Kuypers, 1980; Shafton et al. 1998; Pyner & Coote, 2001). The RVLM is a critical autonomic region that projects directly to the sympathetic preganglionic motor neurones in the IML and is critical for the tonic regulation of sympathetic nerve activity (Ross et al. 1984; Reis et al. 1988; Guyenet, 1989; Dampney, 1994). Destruction or neuronal inhibition of the RVLM produces dramatic reductions in sympathetic nerve activity, whilst activation of the RVLM elicits marked increases in sympathetic nerve activity to the vasculature (Ross et al. 1984; Reis et al. 1988). Stimulating the PVN elicits marked changes in sympathetic nerve activity, and the anatomical projections from the PVN to the spinal cord or to the RVLM undoubtedly contribute to those changes (Tagawa & Dampney, 1999). As indicated earlier, we have previously shown that spinally projecting neurones are activated in response to exposure to a hot environment; however, whether neurones in the PVN that send projections to the RVLM are also part of the central pathways activated by exposure to a hot environment has not been examined to date. Therefore, the primary aim of the present study was to determine whether RVLM-projecting neurones in the PVN are activated by placing conscious rats into a hot environment.
Nitric oxide (NO) is an important neurotransmitter both in the periphery and in the central nervous system. Nitric oxide, unlike conventional neurotransmitters, does not act on membrane-bound receptors and, since it is easily diffusible through cell membranes, its actions can be widespread by acting on neurones within its diffusion range (Garthwaite, 1991; Snyder, 1992). Nitric oxide production occurs in areas known to have a thermoregulatory function such as the preoptic area, the dorsal horn of the spinal cord and in the hypothalamus and ventrolateral medulla (Vincent & Kimura, 1992; Iadecola et al. 1993). There is growing evidence that nitric oxide plays a role in thermoregulation but the exact function of nitric oxide may depend on its specific sites of action (Gerstberger, 1999). However, microinjection of nitric oxide donors into the third ventricle induces an integrated response involving a reduction in core body temperature and a rise in skin temperature, suggesting an important role in heat dissipation in the rabbit (Eriksson et al. 1997). Conversely, inhibition of nitric oxide production attenuates heat loss and results in an increase in body temperature in rats (Mathai et al. 2004). Additionally, inhibition of NO production reduces saliva production during body warming (Damas, 1994); hyperthermia induces enhanced secretion of saliva, which is spread on the fur to promote heat loss in rats (Kanosue et al. 1991; Damas, 1994). Thus, current evidence suggests that NO in the central nervous system is important in the thermoregulatory pathways mediating heat dissipation (Gourine, 1995; Eriksson et al. 1997; Mathai et al. 1997; Schmid et al. 1998; Simon, 1998; Gerstberger, 1999).
There is a dense concentration of neurones containing nitric oxide synthase (NOS), the enzyme responsible for the production of NO, in the PVN. We have previously shown that exposure to a hot environment activates nitrergic neurones in the PVN and that some of these neurones also project to the spinal cord (Cham et al. 2006). Thus, in the present study, in addition to our primary goal to investigate whether RVLM-projecting neurones in the PVN were activated following exposure to a hot environment, we also determined whether any of those neurones were also nitrergic.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
All experimental protocols used in this study were performed in accordance with the Australian Prevention of Cruelty to Animals Act 1986 and conform to the Guiding Principles for Research Involving Animals and Human Beings (American Physiological Society, 2002) and to the guidelines set out by the National Health and Medical Research Council of Australia and were approved by the RMIT University Animal Ethics committee. Every attempt was made to reduce animal suffering and discomfort and to reduce the number of animals needed to obtain reliable results. Male Sprague–Dawley rats (obtained from Monash University Animal Services, Victoria, Australia) weighing 200–250 g were housed in the Animal Facility (RMIT University, Victoria, Australia) with free access to rat chow and tap water at a room temperature of 22 ± 1°C with a 12 h–12 h light–dark regimen. Prior to the experimental day, animals were handled on a daily basis to minimize stress.
All surgical procedures were performed under general anaesthesia (sodium pentobarbitone 60 mg kg–1 I.P.; Boehringer Ingelheim, Sydney, NSW, Australia). Buscopan Compositum® (0.03 ml kg–1 S.C.; consisting of a mixture of hyoscine-N-butyl bromide (12.5 mg kg–1) and dipyrone (0.1 mg kg–1); Boehringer Ingelheim) was also administered prior to surgery to minimize salivary secretions. Oxytetracycline (200 mg kg–1 S.C., Terramycin®; Provet, Melbourne, Victoria, Australia) was administered to prevent infection and buprenorphine hydrochloride (15 µg I.P., Temgesic®; Reckitt and Colman Pharmaceuticals, Sydney, NSW, Australia) was administered for analgesia after surgical procedures.
Microinjections of retrogradely transported tracer into the RVLM
Under general anaesthesia, the right femoral artery was cannulated to enable blood pressure monitoring. The animals were placed prone and their head was mounted in a Kopf stereotaxic frame such that both bregma and lambda were positioned on the same horizontal plane. A burr hole, approximately 4–5 mm in diameter, was drilled into the occipital bone on the left-hand side of the skull approximately 2 mm lateral to the mid-sagittal suture and 3 mm caudal to the lambdoid suture. The pressor region of the RVLM was identified functionally by microinjection of 25–50 nl of L-glutamate (0.1 M) using a fine glass micropipette (tip diameter of 50–70 µm), which elicited a minimal increase of 20 mmHg in arterial pressure. The precise location of the microinjections was verified histologically at the end of the experiment (Fig. 1). Only animals in which the injected tracer covered the RVLM were used in this study. Typically, the co-ordinates of the pressor area of the RVLM were 1.8–2.2 mm lateral to the mid-sagittal suture, 2.5–3.5 mm caudal to the lambdoid suture and 8.9 mm ventral to the cerebellar surface. After locating the pressor region, the pipette was carefully withdrawn, filled with the neuronal retrogradely transported tracer, rhodamine-tagged microspheres (1:1 dilution with 0.9% sterile saline, LumaFluor, Naples, FL, USA), and re-inserted into the pressor region of the RVLM. The microspheres were then pressure injected in a volume of 250 nl over approximately 5 min. After the injection, the micropipette was left in place for 10 min prior to its removal to reduce tracer spread along the route of the micropipette. After the micropipette was removed, the skin overlying the skull was sutured and the incision closed. Finally, the arterial cannula was carefully removed from the femoral artery to minimize blood loss and the incision was sutured closed. The animal was subsequently given antibiotic and then analgesic and allowed to recover.
Experimental day
Two weeks elapsed after the microinjection of the tracer to allow for its transport. The rats were then placed into the experimental room 24 h preceding the experimental day. On the day of the temperature challenge, animals were randomly assigned to either a heated (n = 6) or control group (n = 6) and transferred, in their home cages, to the temperature chamber (Plexiglass box measuring 75 cm x 60 cm x 55 cm with a metal mesh stage in the bottom). Rats were placed into the heating chamber (ambient temperature 38.9 ± 0.1°C) for a duration of 1 h. Control animals underwent similar procedures except that the temperature chamber was maintained at room temperature (ambient temperature 23 ± 1°C).
Immediately after the temperature challenge, the rats were removed from the chamber and kept at room temperature for 1 h before being deeply anaesthetized with sodium pentobarbitone and transcardially perfused with approximately 350–400 ml of phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). The perfusion pressure was maintained at about 100–120 mmHg. The brains were then carefully removed and stored in the fixative solution for at least 2 h before being transferred into PB containing 20% sucrose solution and left overnight.
Detection of Fos by immunohistochemistry
Serial sections of the hypothalamic PVN (40 µm thick) were cut on a cryostat and one in three serial sections were collected for processing. To identify activated neurones, immunohistochemistry to detect Fos was performed on the sections incubated at room temperature and processed using standard immunohistochemical procedures as previously described (Kantzides & Badoer, 2003; Cham et al. 2006). Briefly, the free-floating sections underwent washes in PB prior to incubation with 10% normal horse serum (NHS) in PB for 1 h at room temperature. This was followed by an overnight incubation with a primary antibody raised in rabbits against a conserved region of human Fos (Ab5, 1:20 000; Oncogene Research Products, Cambridge, MA, USA) containing 2% NHS (JRH Biosciences, Melbourne, Victoria, Australia) and 0.3% Triton X-100 (Sigma Aldrich, Sydney, NSW, Australia). After three 5 min washes in PB, the sections were incubated for 1 h with biotinylated antirabbit secondary antibody (diluted to 1:600 in PB, Sigma Aldrich) that was raised in goats. Following a further three 5 min washes in PB, the sections were incubated for 1 h using Extravidin (Sigma Aldrich) diluted to 1:400 in PB. Subsequently, the sections were then washed in Tris buffer (0.05 M, pH 7.6) and incubated for 10 min in 0.05% 3,3'-diaminobenzidine hydrochloride (DAB; Sigma Aldrich) in 0.05 M Tris buffer. The reaction was initiated by the addition of 5 µl of 17.5% hydrogen peroxide (H2O2; Biotech Pharm Pty Ltd, Sydney, NSW, Australia) and terminated by washes with fresh Tris buffer.
Nicotine adenine dinucleotide phosphate-diaphorase (NADPH-d) staining
Staining for NADPH-d was used as a marker of nitric oxide synthase in cells. Immediately after the immunohistochemistry procedure to detect Fos, the sections were incubated in a mixture of 2.5 mg Nitroblue Tetrazolium (Sigma Aldrich), 10 mg β-NADPH (Sigma Aldrich) and 0.2% Triton X-100 in 10 ml of 0.05 M Tris buffer. The reaction was then allowed to proceed for 30–40 min at room temperature in the dark. The intensity of staining was examined prior to its termination with Tris buffer washes.
Sections were mounted onto gelatine-subbed slides and allowed to dry before another brief wash in water, and re-drying. The slides were then dipped in xylene (Analar, Merck Pty Ltd, Sydney, NSW, Australia) before being coverslipped using DePex mounting medium (BDH, Poole, UK).
Analysis
Both Fos-positive cell nuclei and NADPH-d-positive neurones were identified under normal bright-field illumination. Retrogradely labelled neurones were detected by using a fluorescent light source on a Leica DMLB microscope fitted with a rhodamine filter. Double-labelled neurones, containing retrogradely transported tracer and either a Fos-positive nucleus or NADPH-d-positive cytoplasm, and triple-labelled neurones were detected by rapidly switching between the two light sources. Double-labelled neurones containing both a Fos-positive nucleus and NADPH-d-positive cytoplasm were detected under normal bright-field illumination.
In each rat, labelled neurones were counted unilaterally on the side of the PVN ipsilateral to the injection site (using x200 magnification), in sections which were grouped to represent five different levels encompassing the entire rostral–caudal extent of the PVN. Each level, in each rat, consisted of three sections, two of which were used for quantification. The data were expressed as the average number per section at each level. Within each group of animals, the overall means of Fos-positive cell nuclei, NADPH-d-positive neurones and retrogradely labelled neurones for each group of animals were calculated and compared between the heated and control groups. The overall means of multiple-labelled neurones were also calculated. Each animal contributed the same number of sections to each level for the analysis.
Statistical analysis
The heated and control groups of rats were compared using two-way ANOVA with repeated measures. If there was a significant difference overall then comparisons of each of the five different levels of the PVN were made between the groups using Student's unpaired t test and applying Bonferroni's modification to compensate for multiple comparisons. The statistical software package used was GB-STAT® version 7.0 (Dynamic Microsystems Inc., Silver Spring, MD, USA), and the level of significance was set at P < 0.05.
Mapping
For illustration of the distribution of labelled neurones in the different levels of the PVN, maps were drawn from representative sections in each of the five rostral to caudal levels. The digital files were generated using the software package MD Plot® (version 4.0) and a MD3 microscope digitizer stage (Minnesota Datametric Corporation, Shoreview, MN, USA) attached to a Leica DMLB microscope. The individual maps were subsequently imported into CorelDRAW®9 to assemble the final figures presented.
Photomicroscopy
Images were acquired using a digital SPOT camera mounted on an Olympus BX60 microscope. The digital images obtained were imported into Adobe Photoshop® (version CS, Adobe Systems Inc., San Jose, CA, USA), and only the contrast and brightness were modified for presentation purposes.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Following exposure to a hot environment, the total number of Fos-positive nuclei within the PVN in the heated group of animals (1290 ± 21, unilateral counts) was significantly elevated, by 12-fold, compared with the control group of animals (105 ± 17; F1,10 = 2289.74, P < 0.0001). This increase in the production of Fos occurred throughout the rostral–caudal levels of the PVN examined, with the maximum number of Fos-positive nuclei found predominantly in the middle to caudal levels of the PVN (Figs 2 and 3). Whilst Fos-positive cell nuclei were present in both magnocellular and parvocellular regions, they were only quantified in the parvocellular region of the PVN. Within this region of the PVN, Fos-positive cells were distributed in the dorsal, medial and lateral subnuclei of the PVN (Fig. 3).
By contrast, only a small number of Fos-positive neurones were observed in the control group of animals and these were evenly distributed throughout the rostral–caudal extent of the PVN (Fig. 2).
Distribution of RVLM-projecting neurones in the PVN
RVLM-projecting neurones were observed in all the rostral–caudal levels of the parvocellular PVN (Figs 2 and 3). The distribution pattern of these RVLM-projecting neurones was similar between the control and the heated groups (Fig. 2). The maximum numbers of retrogradely labelled neurones were found in the middle to caudal levels of the PVN (Figs 2 and 3). On average, a total of 155 ± 5 neurones projecting to the RVLM were counted in the PVN of the heated group, which was not significantly different from the control group of animals (145 ± 5, unilateral counts; Fig. 2).
Distribution of RVLM-projecting neurones that also contained Fos
After exposure of the animals to the hot environment, there was a significant increase in the number of RVLM-projecting neurones that also contained a Fos-positive nucleus (13 ± 1; F1,10 = 109.64, P < 0.0001, compared with the control group, 2 ± 1; Fig. 2). These double-labelled neurones represented about 8.2 ± 0.6% of the RVLM-projecting neurones counted in the PVN and were found predominantly in the middle levels of the PVN (Figs 2 and 3). In the control group, there were very few RVLM-projecting neurones that contained a Fos-positive nucleus. These double-labelled neurones represented approximately 1.5% of the RVLM-projecting neurones in the PVN.
Distribution of neurones containing NADPH-d
Neurones positive for NADPH-d were observed throughout the rostral–caudal extent of the PVN (Figs 3 and 4). The distribution profiles of NADPH-d-positive neurones in both the control and the heated group of animals were similar (Fig. 4). On average, a total of 513 ± 10 NADPH-d-positive neurones were counted unilaterally in the heated group, which was not significantly different from that of the control group (486 ± 15).
Distribution of neurones containing NADPH-d and Fos
In the heated group, the average number of neurones in the PVN of each animal that contained both NADPH-d and Fos (157 ± 6) was significantly elevated, by 16-fold, compared with the control group, in which there was on average a total of only 10 ± 0.5 NADPH-d-positive neurones that exhibited a Fos-positive nucleus (F1,10 = 667.68, P < 0.0001; Fig. 4). In the heated group of animals, these double-labelled neurones represented approximately 31% of all the NADPH-d-positive neurones counted in the PVN. This increase occurred throughout the rostral–caudal extent of the PVN, with the maximum number found predominantly in the middle to caudal levels of the PVN (Fig. 4).
Distribution of RVLM-projecting neurones containing NADPH-d
After exposure of the animals to a heated environment, the number of RVLM-projecting neurones containing NADPH-d (6 ± 1) represented approximately 4% of the RVLM-projecting neurones in the PVN. These double-labelled neurones were found primarily in the middle to caudal levels of the PVN (Fig. 4 and 5). A similar distribution profile was also observed with the control group of animals (Fig. 4). The number of double-labelled neurones in the control group (6 ± 1) was not significantly different from that in the heated group of animals (Fig. 4).
Distribution of RVLM-projecting neurones that contained Fos and NADPH-d
In the heated group of animals, triple-labelled neurones were rare and the average number of triple-labelled cells in the entire PVN was 1 ± 0.5. In the control group of animals, no triple-labelled cells were observed at any levels of the PVN exained.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, we also found that following heat exposure, in conscious rats, there was a marked increase in the number of Fos-positive cell nuclei observed in all subdivisions of the PVN. The number of activated neurones peaked in the mid to caudal levels of the parvocellular PVN, which is in agreement with findings from our previous work (Cham et al. 2006). This increased production of Fos following heat exposure is also in agreement with earlier studies emanating from other laboratories (Kiyohara et al. 1995; McKitrick, 2000; Bachtell et al. 2003; Harikai et al. 2003; Bratincsak & Palkovits, 2004). The present and previous studies support the view that the PVN contributes to reflex thermoregulatory responses. Further circumstantial evidence suggesting that the PVN may contribute to the cardiovascular changes elicited by the disturbances in body temperature include: (i) thermosensitive neurones are present in the PVN (Inenaga et al. 1987); and (ii) PVN neurones project to autonomic nuclei in the CNS that influence sympathetic nerve activity to important thermoregulatory effector organs such as the brown adipose tissue, the vasculature of the rat tail, salivary gland, kidney and gut (Morrison, 1999; Hubschle et al. 2001; Oldfield et al. 2002).
There have also been previous reports that have not detected an increase in Fos production in the PVN following an elevated body temperature (Patronas et al. 1998). The discrepancies may be attributed to the differences in species used, the duration of heat exposure, different heating regimes and the degree to which body temperature was elevated. In general, studies that have elicited marked activation of the neurones in the parvocellular PVN have used a higher environmental temperature (Harikai et al. 2003; Bratincsak & Palkovits, 2004). In the present study we used an environmental temperature of 39°C. This is equivalent to a hot summer's day. We have experience with the protocol used in the present study (Mathai et al. 2004; Cham et al. 2006). Keeping rats at this environmental temperature for 1 h increases core body temperature by approximately 3.5°C and elicits the normal behavioural responses characteristic of an elevated body temperature (Mathai et al. 2004; Cham et al. 2006). The stress induced by hyperthermia may increase Fos production; however, we believe the stress is an integral component of the response to exposure to the hot environment rather than a non-specific stimulus to Fos production.
One of the novel findings of the present study is the activation of PVN neurones projecting to the RVLM. We found that approximately 8% of the RVLM-projecting neurones in the PVN were activated following exposure to a hot environment. What is the physiological relevance of these neurones? It could be argued that the proportion activated is small and therefore the physiological relevance of this pathway may be questionable. Certainly, we have found that a similar heating stimulus activates a considerably greater proportion (approximately 22%) of the spinally projecting neurones in the PVN (Cham et al. 2006). Indeed, that proportion is greater than any stimulus reported to date, including severe haemorrhage, which activates the PVN more strongly than elevated body temperature. Thus, the present study illustrates that the activated neurones in the PVN following heat exposure include neurones with efferent connections to the RVLM. However, these may make a smaller contribution than the spinally projecting neurones in the PVN to the cardiovascular responses initiated by heat.
Although the proportion of activated RVLM-projecting neurones may represent a small proportion of the population of identified RVLM-projecting neurones in the PVN, one cannot exclude the possibility that their influence is greater than the proportion would suggest. The RVLM is a brain nucleus that is critical in the tonic regulation of sympathetic nerve activity (Ross et al. 1984; Reis et al. 1988; Guyenet, 1989; Dampney, 1994). Since activation of the PVN can elicit sympatho-excitatory effects and elevations in blood pressure that are, in part, mediated through the RVLM (Coote et al. 1998; Tagawa & Dampney, 1999), it is possible that activation of the neurones in the PVN that project to the RVLM could mediate the increase in sympathetic nerve activity to the visceral vasculature, including the mesenteric and renal beds, which would contribute to the redistribution of blood flow to the peripheral vasculature to enable heat to dissipate when body temperature rises. Similarly, heart rate is increased in response to heating, and activation of an excitatory pathway from the PVN to the RVLM could contribute to the tachycardia. Therefore, further studies are needed to explore the physiological relevance of the pathway from the PVN to the RVLM in response to hyperthermia.
Given that approximately 8% of PVN neurones projecting to the RVLM were activated by the heat stimulus, the results suggest that there is a large population of RVLM-projecting neurones that are not activated. Thus, perhaps, the pathway from the PVN to the RVLM may have a greater involvement in responses to other stimuli. Indeed, there is evidence indicating that the pathway from the PVN to the RVLM mediates reflex responses induced by haemorrhage, dehydration and simulated volume expansion (Badoer et al. 1993; Kantzides & Badoer, 2003; Stocker et al. 2004).
In the present work we used an ambient temperature of 39°C, which is known to elevate body temperature and plasma osmolality and to reduce body fluid (Mathai et al. 2000). Since an elevation in plasma osmolality is known to activate neurones in the PVN (Oldfield et al. 1991; Kantzides & Badoer, 2003; Stocker et al. 2004), one could argue that this stimulus is responsible for the activation of the RVLM-projecting neurones in the PVN. However, we have previously shown that an intravenous infusion of hypertonic saline does not activate RVLM-projecting neurones in the PVN (Kantzides & Badoer, 2003). Thus, it is unlikely that the increase in osmolality that accompanies exposure to a hot environment could account for the increased activation of RVLM-projecting PVN neurones. Whether the reduction in body fluid per se contributes to the response is difficult to determine. Haemorrhage activates a population of PVN neurones that project to the RVLM, but since hypotension was also induced, it is not possible to differentiate the responsible stimulus (Badoer & Merolli, 1998). However, we have previously shown that replacing lost fluid during the heating stimulus does not reduce the number of activated neurones in the PVN, indicating that an elevation in body temperature without fluid loss can activate PVN neurones (Cham et al. 2006).
The PVN is amongst the brain nuclei that have the highest concentration of neurones that contain NOS, the enzyme responsible for the production of the neurotransmitter NO. In the present study there was a significant increase in the number of activated neurones that were also NADPH-d positive (a marker for NOS) in the PVN following exposure to the hot environment. These activated nitrergic neurones represented approximately one-third (31%) of the nitrergic neurones in the parvocellular PVN. This suggests that exposure to a hot environment increases the production of NO within the PVN.
Nitric oxide in the CNS is important in heat dissipation (Schmid et al. 1998; Gerstberger, 1999); thus the PVN may be a potential site of action within the CNS through which NO may influence the redistribution of blood flow to facilitate heat dissipation. Given that NO easily diffuses through membranes, its effects within the PVN may be quite extensive. Nitric oxide within the PVN appears to be tonically active, since blockade of its production elicits marked cardiovascular effects (Zhang et al. 1997). The cardiovascular effects of NO acting within the PVN are mediated via GABA, thus any role of NO in the PVN during elevations in core body temperature are likely to be complex. Perhaps, the role of NO is to act: (i) as a negative feedback system that prevents activated neurones from becoming overactive; and/or (ii) as an inhibitor of neurones which contributes to a complex mechanism that tunes the activity of neurones to facilitate an appropriate integrated response. Thus, the contribution of NO within the PVN in the responses elicited during a temperature challenge warrants further investigation.
Another finding of the present study was the lack of a marked activation of PVN neurones projecting to the RVLM that were also NADPH-d positive (approximately 0.5% of the RVLM-projecting neurones) following heat exposure. Thus, although there was a marked significant increase in the number of activated nitrergic neurones following heat exposure, only very rarely do they project to the RVLM. This suggests that nitrergic RVLM-projecting neurones in the PVN do not play an important role during heat exposure. Nitrergic PVN neurones projecting to the RVLM are, however, activated by simulated volume expansion (Kantzides et al. 2005), suggesting that RVLM-projecting nitrergic neurones may be differentially activated by specific stimuli. Furthermore, since nitrergic spinally projecting neurones but not nitrergic RVLM-projecting neurones are activated following exposure to a hot environment (Cham et al. 2006), it appears that specific stimuli can activate specific subpopulations of nitrergic neurones in the parvocellular PVN.
The neurochemical nature of the PVN neurones projecting to the RVLM that are activated by heating requires further study; however, angiotensin II and vasopressin are possible candidates, since the sympatho-excitatory and hypertensive effects of activation of the PVN can be attenuated by the blockade of angiotensin II receptors in the RVLM (Tagawa & Dampney, 1999) and vasopressin antagonists can reduce the excitatory effects on RVLM neurones induced by stimulation of the PVN (Yang et al. 2001). Furthermore, it is particularly interesting to note that central angiotensin II has been found to be important in the cardiovascular response to hyperthermia (Kregel et al. 1994), and there is a dense concentration of angiotensin II receptors in the PVN (Allen et al. 1992).
Conclusions
The rostral forebrain has long been recognized as a critical site in thermoregulation. The hypothalamic PVN, however, has been largely ignored, despite anatomical and electrophysiological evidence suggesting that it could contribute to the central pathways mediating thermoregulation. The present study provides evidence that neurones that project to the RVLM from the PVN may contribute to the central pathways activated by exposure to a hot environment. Since the RVLM is critical in the tonic maintenance of sympathetic nerve activity, it is possible that the RVLM-projecting neurones in the PVN make a contribution to the cardiovascular responses elicited by exposure to a high environmental temperature. The PVN is known to contribute to the anatomical framework that influences the autonomic nervous system innervating important thermoregulatory organs such as the brown adipose tissue, heart, blood vessels in the skin, kidney and gut, as well as salivary glands and the rat tail (Morrison, 1999; Hubschle et al. 2001; Oldfield et al. 2002; Cano et al. 2003). Neurones in the PVN that contribute to this anatomical framework include neurones that project directly: (i) to the IML of the spinal cord, where sympathetic preganglionic motor neurones are located; (ii) to the RVLM; and (iii) to both those regions via collaterals (Shafton et al. 1998; Pyner & Coote, 2001). We hypothesize that the RVLM-projecting neurones may contribute to the cardiovascular responses elicited by an acute exposure to a hot environment. The degree of contribution, however, requires investigation.
The role of NO in thermoregulation is now emerging as a major focus of investigation. Current evidence suggests that NO within the central nervous system can play a critical role in heat dissipation (Eriksson et al. 1997; Schmid et al. 1998; Gerstberger, 1999). One of the highest concentrations of NOS-containing neurones within the hypothalamus occurs in the PVN, and the present findings highlight that approximately 31% of the nitrergic neurones in the parvocellular PVN are activated by heat exposure. Since NO can diffuse easily across cell membranes, its production within the PVN may have widespread actions during heat exposure. Interestingly, activated nitrergic RVLM-projecting neurones in the PVN were rarely observed following the heating stimulus, suggesting that these neurones do not influence sympathetic nerve activity that contributes to the cardiovascular effects elicited by exposure to a hot environment.
|
|
|
|
|
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
American Physiological Society (2002). Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283, R281–R283.
Bachtell RK, Tsivkovskaia NO & Ryabinin AE (2003). Identification of temperature-sensitive neural circuits in mice using c-Fos expression mapping. Brain Res 960, 157–164.[CrossRef][Medline]
Badoer E (1996). Cardiovascular role of parvocellular neurones in the paraventricular nucleus of the hypothalamus. News Physiol Sci 11, 43–47.
Badoer E, McKinley MJ, Oldfield BJ & McAllen RM (1993). A comparison of non-hypotensive and hypotensive hemorrhage on Fos expression in spinally-projecting neurons of the paraventricular nucleus and rostral ventrolateral medulla. Brain Res 610, 216–223.[CrossRef][Medline]
Badoer E & Merolli J (1998). Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are activated by haemorrhage. Brain Res 791, 317–320.[CrossRef][Medline]
Bratincsak A & Palkovits M (2004). Activation of brain areas in rat following warm and cold ambient exposure. Neuroscience 127, 385–397.[CrossRef][Medline]
Cano G, Passerin AM, Schiltz JC, Card JP, Morrison SF & Sved AF (2003). Anatomical substrates for the central control of sympathetic outflow to interscapular adipose tissue during cold exposure. J Comp Neurol 460, 303–326.[CrossRef][Medline]
Cham JL, Klein R, Owens NC, Mathai ML, McKinley MJ & Badoer E (2006). Activation of spinally-projecting and nitrergic neurons in the PVN following heat exposure. Am J Physiol Regul Integr Comp Physiol 291, R91–R101.
Coote JH, Yang Z, Pyner S & Deering J (1998). Control of sympathetic outflows by the hypothalamic paraventricular nucleus. Clin Exp Pharmacol Physiol 25, 461–463.[Medline]
Damas J (1994). Kallikrein, nitric oxide and the vascular responses of the submaxillary glands in rats exposed to heat. Arch Int Physiol Biochim Biophys 102, 139–146.[Medline]
Dampney RAL (1994). Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74, 323–364.
Eriksson S, Hjelmqvist H, Keil R & Gerstberger R (1997). Central application of a nitric oxide donor activates heat defense in the rabbit. Brain Res 774, 269–273.[CrossRef][Medline]
Garthwaite J (1991). Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci 14, 60–67.[CrossRef][Medline]
Gerstberger R (1999). Nitric oxide and body temperature control. News Physiol Sci 14, 30–36.
Gourine AV (1995). Pharmacological evidence that nitric oxide can act as an endogenous antipyretic factor in endotoxin-induced fever in rabbits. Gen Pharmacol 26, 835–841.[CrossRef][Medline]
Guyenet PG (1989). Role of the ventral medulla oblongata in blood pressure regulation. In Central Regulation of Autonomic Functions, ed. Loewy A & Spyer KM, pp. 145–167. Oxford University Press, Oxford.
Harikai N, Tomogane K, Sugawara T & Tashiro S (2003). Differences in hypothalamic Fos expression between two heat stress conditions in conscious mice. Brain Res Bull 61, 617–626.[CrossRef][Medline]
Hubschle T, Mathai M, McKinley M & Oldfield B (2001). Multisynaptic neuronal pathways from the submandibular and sublingual glands to the lamina terminalis in the rat: a model for the role of the lamina terminalis in the control of osmo- and thermoregulatory behaviour. Clin Exp Pharmacol Physiol 28, 558–569.[CrossRef][Medline]
Iadecola C, Faris PL, Hartman BK & Xu XH (1993). Localization of NADPH diaphorase in neurons of the rostral ventral medulla: possible role of nitric oxide in central autonomic regulation and oxygen chemoreception. Brain Res 603, 173–179.[CrossRef][Medline]
Inenaga K, Osaka T & Yamashita H (1987). Thermosensitivity of neurons in the paraventricular nucleus of the rat slice preparation. Brain Res 424, 126–132.[CrossRef][Medline]
Kanosue K, Niwa K, Andrew PD, Yasuda H, Yanase M, Tanaka H & Matsumura K (1991). Lateral distribution of hypothalamic signals controlling thermoregulatory vasomotor activity and shivering in rats. Am J Physiol Regul Integr Comp Physiol 260, R486–R493.
Kanosue K, Yanase-Fujiwara M & Hosono T (1994). Hypothalamic network for thermoregulatory vasomotor control. Am J Physiol Regul Integr Comp Physiol 267, R283–R288.
Kantzides A & Badoer E (2003). Fos, RVLM-projecting neurons, and spinally projecting neurons in the PVN following hypertonic saline infusion. Am J Physiol Regul Integr Comp Physiol 284, R945–R953.
Kantzides A, Owens NC, De Matteo R & Badoer E (2005). Right atrial stretch activates neurons in the autonomic brain regions that project to the rostral ventrolateral medulla. Neuroscience 133, 775–786.[CrossRef][Medline]
Kazuyuki K, Hosono T, Zhang YH & Chen XM (1998). Neuronal networks controlling thermoregulatory effectors. Prog Brain Res 115, 49–62.[Medline]
Kiyohara T, Miyata S, Nakamura T, Shido O, Nakashima T & Shibata M (1995). Differences in Fos expression in the rat brains between cold and warm ambient exposures. Brain Res Bull 38, 193–201.[CrossRef][Medline]
Kregel KC, Stauss H & Unger T (1994). Modulation of autonomic nervous system adjustments to heat stress by central ANG II receptor antagonism. Am J Physiol Regul Integr Comp Physiol 266, R1985–R1991.
McKitrick DJ (2000). Expression of Fos in the hypothalamus of rats exposed to warm and cold temperatures. Brain Res Bull 53, 307–315.[CrossRef][Medline]
Mathai ML, Arnold I, Febbraio MA & McKinley MJ (2004). Central blockade of nitric oxide synthesis induces hyperthermia that is prevented by indomethacin in rats. J Thermal Biol 29, 401–405.[CrossRef]
Mathai ML, Hjelmqvist H, Keil R & Gerstberger R (1997). Nitric oxide increases cutaneous and respiratory heat dissipation in conscious rabbits. Am J Physiol Regul Integr Comp Physiol 272, R1691–R1697.
Mathai ML, Hubschle T & McKinley MJ (2000). Central angiotensin receptor blockade impairs thermolytic and dipsogenic responses to heat exposure in rats. Am J Physiol Regul Integr Comp Physiol 279, R1821–R1826.
Morrison SF (1999). RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue. Am J Physiol Regul Integr Comp Physiol 276, R962–R973.
Morrison SF (2001). Differential regulation of sympathetic outflows to vasoconstrictor and thermoregulatory effectors. Ann N Y Acad Sci 940, 286–298.
Murakami N & Morimoto A (1982). Metabolic mapping of the rat brain involved in thermoregulatory responses using the [14C]2-deoxyglucose technique. Brain Res 246, 137–140.[CrossRef][Medline]
Nagashima K, Nakai S, Tanaka M & Kanosue K (2000). Neuronal circuitries involved in thermoregulation. Auton Neurosci 85, 18–25.[CrossRef][Medline]
Oldfield BJ, Bicknell RJ, McAllen RM, Weisinger RS & McKinley MJ (1991). Intravenous hypertonic saline induces Fos immunoreactivity in neurons throughout the lamina terminalis. Brain Res 561, 151–156.[CrossRef][Medline]
Oldfield B, Giles ME, Watson A, Anderson CR, Colvill L & McKinley MJ (2002). The neurochemical characterisation of hypothalamic pathways projecting to brown adipose tissue in the rat. Neuroscience 110, 515–526.[CrossRef][Medline]
Owens NC, Ootsuka Y, Kanosue K & McAllen RM (2002). Thermoregulatory control of sympathetic fibres supplying the rat's tail. J Physiol 543, 849–858.
Patronas P, Horowitz M, Simon E & Gerstberger R (1998). Differential stimulation of c-fos expression in hypothalamic nuclei of the rat brain during short-term heat acclimation and mild dehydration. Brain Res 798, 127–139.[CrossRef][Medline]
Pyner S & Coote JH (2001). Identification of branching paraventricular neurons of the hypothalamus that project to the rostroventrolateral medulla and spinal cord. Neuroscience 100, 549–556.[CrossRef]
Reis DJ, Morrison S & Ruggiero DA (1988). The C1 area of the brainstem in tonic and reflex control of blood pressure. Hypertension 11 (Suppl. I), 8–13.[Medline]
Ross CA, Ruggiero DA, Joh TH, Park DH & Reis DJ (1984). Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons. J Comp Neurol 228, 168–185.[CrossRef][Medline]
Scammell TE, Price KJ & Sagar SM (1993). Hyperthermia induces c-fos expression in the preoptic area. Brain Res 618, 303–307.[CrossRef][Medline]
Schmid HA, Riedel W & Simon E (1998). Role of nitric oxide in temperature regulation. Prog Brain Res 115, 87–110.[Medline]
Shafton AD, Ryan AR & Badoer E (1998). Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostal ventrolateral medulla in the rat. Brain Res 801, 239–243.[CrossRef][Medline]
Simon E (1998). Nitric oxide as a peripheral and central mediator in temperature regulation. Amino Acids 14, 87–93.[CrossRef][Medline]
Snyder SH (1992). Nitric oxide: first in a new class of neurotransmitters? Science 257, 494–496.
Stocker SD, Cunningham JT & Toney GM (2004). Water deprivation increases Fos immunoreactivity in PVN autonomic neurons with projections to the spinal cord and rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 287, R1172–R1183.
Swanson LW & Kuypers HGJM (1980). The paraventricular nucleus of the hypothalamus: Cytoarchitectonic subdivisions and organisation of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194, 555–570.[CrossRef][Medline]
Tagawa T & Dampney RAL (1999). AT1 receptors mediate excitatory inputs to rostral ventrolateral medulla pressor neurons from hypothalamus. Hypertension 34, 1301–1307.
Vincent SR & Kimura H (1992). Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755–784.[CrossRef][Medline]
Yang Z, Bertram D & Coote JH (2001). The role of glutamate and vasopressin in the excitation of RVL neurones by paraventricular neurones. Brain Res 908, 99–103.[CrossRef][Medline]
Zhang K, Mayhan WG & Patel KP (1997). Nitric oxide within the paraventricular nucleus mediates changes in renal sympathetic nerve activity. Am J Physiol Regul Integr Comp Physiol 273, R864–R872.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
