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1 School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia
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Abstract |
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(Received 19 December 2006;
accepted after revision 16 February 2007; first published online 28 February 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
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Introduction |
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It is well established that the CNS is essential in the regulation of body temperature, and there are several brain regions that contribute to the CNS pathways that mediate the thermoregulatory responses. Following an elevation in body temperature, several forebrain areas are activated, including the preoptic area, anterior hypothalamus, paraventricular nucleus of the hypothalamus and the periaqueductal grey matter (Boulant, 1981, 1998; Murakami & Morimoto, 1982; Morimoto & Murakami, 1985; Schmid & Pierau, 1993; Scammell et al. 1993; Kiyohara et al. 1995; Hori et al. 1999; Bachtell et al. 2003; Maruyama et al. 2003; Bratincsak & Palkovits, 2004).
Whilst the major integrative centres for temperature regulation are in the hypothalamus and basal forebrain, specific areas of the lower brainstem have recently been identified as key regions mediating the cardiovascular responses following hypothermia. In particular, the medullary raphe in the mid-line and parapyramidal regions appear to be critical for the vasoconstriction in the rat's tail in response to hypothermia (Owens et al. 2002; Nakamura et al. 2004). By contrast, only a few studies have reported an activation of neurones in regions of the lower brainstem following heat exposure (Kiyohara et al. 1995; Bratincsak & Palkovits, 2004). Interestingly, the areas of the lower brainstem that are activated following heat exposure also appear to coincide with regions containing nitrergic neurones (Vincent & Kimura, 1992).
Current evidence suggests that nitric oxide (NO) in the central nervous system is important in the thermoregulatory pathways mediating heat dissipation (Gourine, 1995; Eriksson et al. 1997; Schmid et al. 1998; Simon, 1998; Gerstberger, 1999). For example, blockade of central NO production has recently been found to elevate core body temperature in the rat (Mathai et al. 2004), as well as to augment the febrile response elicited by endotoxin and lipopolysaccharide (Gourine, 1995; Steiner et al. 2002). Furthermore, thermal stimulation induces enhanced secretion of saliva, which is spread on the fur to promote heat loss as a means of heat defense in rats (Kanosue et al. 1991; Damas, 1994). Inhibition of NO production reduces saliva production during body warming (Damas, 1994). Additionally, we have shown that following heat exposure, approximately 40% of nitrergic neurones in the paraventricular nucleus of the hypothalamus were activated (Cham et al. 2006). However, it is unknown whether the nitrergic neurones present in the lower brainstem are also activated in response to hyperthermia. Therefore, the first aim of this study was to determine whether neurones in the lower brainstem that are activated by acute hyperthermia resulting from exposure to a hot environment are also capable of producing NO.
The distribution of the nitrergic neurones, and the neurones that are activated following hyperthermia, which have been reported in the lower brainstem, appears to coincide with the ‘autonomic areas’ that contain spinally projecting neurones. Neurones that project to the intermediolateral cell column (IML) of the spinal cord, where the sympathetic preganglionic motor neurones are located, can directly influence sympathetic nerve activity and could play important roles in the redistribution of blood flow, sweating and salivation in response to body temperature changes. Indeed, we have recently found that 22% of spinally projecting neurones in the hypothalamic paraventricular nucleus were activated by hyperthermia (Cham et al. 2006), a proportion that is greater than for many stimuli examined to date, including haemorrhage and elevated osmolality (Badoer et al. 1993; Kantzides & Badoer, 2003). However, whether spinally projecting neurones in the lower brainstem are activated by hyperthermia is unknown. Thus, the second aim of the present study was to determine whether exposure to a hot environment activated lower brainstem neurones that project to the spinal cord.
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Methods |
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All experimental protocols used in this study were performed in accordance with the Prevention of Gruelty 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 (Australia) and were approved by the RMIT University Animal Ethics committee. Every attempt was made to reduce animal suffering, 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. The antibiotic oxytetracycline (200 mg kg–1 S.C., Terramycin, Provet, Provet, 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 spinal cord
Under general anaesthesia, the neuronal retrogradely transported tracer rhodamine-tagged microspheres (1:1 dilution with 0.9% sterile saline, LumaFluor, New York, NY, USA) was microinjected into the spinal cord. Each rat was placed prone and its head was mounted in a Kopf stereotaxic frame. A mid-line incision was made in the upper back, and the spinal cord exposed between the T2 and T3 vertebrae. A fine glass micropipette (tip diameter 50–70 µm) filled with the tracer was inserted into the right side of the spinal cord and lowered approximately 0.7 mm below the surface. The tip of the micropipette was aimed at the IML of the spinal cord. Unilateral injections, each of 250 nl, were made into three separate rostrocaudal sites within the spinal segment. After each injection, the micropipette was left in place for several minutes before its removal to minimize tracer leakage along the route of the micropipette. After the injections, the muscles overlying the spinal cord were sutured and the incision closed. The precise locations of the spinal cord injections were verified histologically at the end of the experiment. Only animals in which the injected tracer covered the IML were used in this study.
Experimental day
Two weeks were allowed to elapse after the microinjection of the tracer to allow for its transportation. The rats were then placed into the experimental room 24 h before the experiment commenced. On the day of the temperature challenge, animals were randomly assigned to either a heated (n = 8) or control group (n = 7) 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 in the heated group were placed in the heating chamber (ambient temperature 38.9 ± 0.1°C) for 1 h. Control animals underwent similar procedures, with the exception 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 left 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 and spinal cords were then carefully removed and stored in the fixative solution for at least 2 h before being transferred into PB containing 20% sucrose solution overnight.
Detection of Fos by immunohistochemistry
Serial sections of the medulla and pons (40 µm) were cut on a cryostat and one in three sections was collected. To identify activated neurones, immunohistochemistry to detect Fos was done. The sections were incubated at room temperature and processed using standard immunohistochemical procedures as previously described (Kantzides & Badoer, 2003). 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 the 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, Australia). After 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 further 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, Nth Laverton, Victoria, 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 ß-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. The intensity of staining was examined before its termination with Tris buffer washes.
Sections were mounted onto gelatine-subbed slides and allowed to dry before another brief wash in water, and redrying. The slides were then dipped in Xylene (Analar, Merck Pty Ltd, 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 microscope fitted with a Rhodamine filter. Double-labelled neurones containing retrogradely transported tracer and either a Fos-positive nucleus or NADPH-d-positive cytoplasm 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. Triple-labelled neurones were identified by rapid switching between the bright-field and fluorescent light sources.
Labelled neurones were counted unilaterally on the side of the lower brainstem ipsilateral to the injection site (using x200 magnification). These sections were grouped to represent five different rostrocaudal levels covering a total distance of approximately 2.4 mm. Each level consisted of four sections, three of which were used for quantification. The levels of the lower brainstem examined represented approximately 1.2–3.6 mm caudal to the interaural line (Paxinos & Watson, 1986). Each level represented a rostrocaudal distance of between 0.5 and 0.6 mm. Levels 1 and 2 contained the mid- to rostral parts of the medulla, whilst levels 3, 4 and 5 encompassed the pontine raphe. For quantification purposes, the lower brainstem regions were subdivided into three areas that encompassed the mid-line, ventromedial and ventrolateral regions of the lower brainstem as shown in Fig. 1. In each region, the number of Fos-positive cell nuclei, NADPH-d-positive neurones and retrogradely labelled neurones was counted in each brain section in each animal. The number of multiple-labelled neurones was also counted. In each animal, the number of labelled neurones in each level of the lower brainstem and the total number of labelled neurones in each region of the lower brainstem (i.e. overall number) were calculated and averaged for the heated and the control group of animals.
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The overall mean values in the heated and control groups of rats were compared using Student's unpaired t test. If there was a significant difference overall between the heated and control groups, then comparisons between the groups at each of the five different levels of the lower brainstem were made 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 lower brainstem, maps were drawn from a representative section 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 version 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 5.5, Adobe Systems Inc., USA), and only the contrast and brightness were modified for presentation purposes.
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Results |
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Distribution of Fos-positive neurones. Following heat exposure, Fos-positive neurones were observed consistently forming a compact group located throughout the rostrocaudal extent of the raphe pallidus (RPa). Fos-positive neurones were also found scattered in the ventral and dorsal parts of the raphe obscurus (ROb). The total number of Fos-positive nuclei counted within the mid-line of the lower brainstem in the heated group of animals (138 ± 4) was elevated by fivefold compared with the control group of animals (25 ± 1; P < 0.01). This increase in the production of Fos occurred throughout the rostrocaudal levels of the lower brainstem examined and was significantly elevated in levels 2–5 (Fig. 2), i.e. the rostral medullary and pontine mid-line raphe. The maximum number of Fos-positive neurones was located predominantly in the pontine levels (Figs 2 and 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 rostrocaudal extent of the mid-line areas (Fig. 2).
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Distribution of spinally projecting neurones that contained Fos and NADPH-d. In both the heated and control groups of animals, there were no triple-labelled cells observed at any level of the mid-line lower brainstem examined.
Ventromedial lower brainstem
Effect of heating on Fos expression. Fos expression was markedly increased in the ventromedial lower brainstem of animals exposed to a hot environment. In the heated group of animals, Fos-positive neurones were located in the ventral gigantocellular reticular nucleus (GiV) and the ventromedial raphe magnus (RMg; Fig. 5). Overall, there was a significant eightfold increase in the total number of Fos-positive neurones in the heated group (267 ± 16) compared with the control group (34 ± 1; P < 0.0001; unilateral counts). This increase in the production of Fos was observed throughout the rostrocaudal extent of the ventromedial lower brainstem, and was significantly elevated in each of the rostrocaudal levels examined (Fig. 2). The maximum number of Fos-positive neurones was found predominantly in the rostral levels of the ventromedial lower brainstem (Figs 2 and 3). In the control group of animals, there were few Fos-positive neurones and these were observed scattered throughout the rostrocaudal extent of the ventromedial lower brainstem (Fig. 2).
Distribution of NADPH-d-positive neurones.
NADPH-d-positive neurones were observed throughout the rostrocaudal extent of the ventromedial lower brainstem (Figs 3 and 4). The NADPH-d-positive neurones were observed in a loose cluster in the GiV and became more scattered in the rostral levels of the ventromedial lower brainstem where the gigantocellular reticular nucleus 
(GiA) and the RMg are located. The distribution profiles of NADPH-d-positive neurones in both the control and the heated group of animals were similar, with the maximum number occurring in the middle to rostral levels of the ventromedial lower brainstem examined (Fig. 4). On average, a total of 271 ± 8 NADPH-d-positive neurones were counted unilaterally in the heated group of animals, which was not significantly different from that of the control group (246 ± 7).
Distribution of neurones containing NADPH-d and Fos. The average number of neurones containing both NADPH-d and Fos within the ventromedial lower brainstem were significantly different between the heated (12 ± 2) and the control groups (1 ± 0.3; P < 0.001; unilateral counts; Fig. 6). The maximum number of these double-labelled neurones was located in the middle rostrocaudal level of the ventromedial lower brainstem. In the heated group, these double-labelled neurones were predominantly located in the GiA and represented approximately 5% of the nitrergic neurones counted in this lower brainstem region.
Distribution of spinally projecting neurones. Spinally projecting neurones were scattered throughout the rostrocaudal levels of the ventromedial lower brainstem (Figs 3 and 7). The average number of neurones in the ventral lower brainstem that projected to the spinal cord was 145 ± 5 in the heated group, which was similar to the control group (149 ± 2; unilateral counts; Fig. 7). The maximum number of retrogradely labelled cells occurred in the rostral levels of the ventromedial lower brainstem (Fig. 7).
Distribution of spinally projecting neurones that also contained Fos. After exposure of the animals to the hot environment, there was a small but statistically significant increase in the number of spinally projecting neurones that contained a Fos-positive nucleus (4 ± 2; P < 0.001 compared with the control group; Fig. 6). These double-labelled neurones were found scattered throughout the rostrocaudal extent of the ventromedial lower brainstem and represented less than 3% of all the spinally projecting neurones in this region examined. In the control group, there were scarcely any double-labelled neurones present (average = 0.1 ± 0.1).
Distribution of spinally projecting neurones that contained Fos and NADPH-d. There were no triple-labelled neurones present in the ventromedial lower brainstem in either the heated or control animals.
Ventrolateral lower brainstem
Distribution of Fos-positive neurones after heat exposure. Following heat exposure, the total number of Fos-positive nuclei within the ventrolateral lower brainstem in the heated group of animals (146 ± 5) was markedly elevated by ninefold compared with the control group of animals (17 ± 1; P < 0.001). This increase in the production of Fos occurred throughout the rostrocaudal levels of the ventrolateral lower brainstem examined (Fig. 2). The maximum number of Fos-positive neurones was located predominantly in the caudal levels of the ventrolateral lower brainstem (Fig. 2). 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 different rostrocaudal levels (Fig. 2).
Within the ventrolateral lower brainstem of the heated group of animals, the Fos-positive neurones were distributed in the lateral paragigantocellular nucleus (LPGi) and included the pressor region of the rostral ventrolateral medulla (RVLM; Figs 3 and 5) found immediately caudal to the facial nucleus (Badoer et al. 1994).
Distribution of NADPH-d-positive neurones. Neurones positive for NADPH-d were observed throughout the rostrocaudal extent of the ventrolateral lower brainstem (Figs 3 and 4) and were found clustered predominantly in the LPGi and RVLM (Figs 3 and 5). The distribution profiles of NADPH-d-positive neurones in both the control and the heated group of animals were similar (Fig. 4). The total number of NADPH-d-positive neurones in the heated group averaged 81 ± 3, which was not significantly different from that of the control group (73 ± 3; unilateral counts).
Distribution of neurones containing NADPH-d and Fos. The average number of neurones containing both NADPH-d and Fos within the ventrolateral lower brainstem was 3 ± 1 (unilateral counts) in the heated group (Fig. 6). In contrast, the control group did not contain any neurones positive for both NADPH-d and Fos (Fig. 6).
In the heated group, double-labelled neurones were observed in the LPGi and RVLM of the lower brainstem. These double-labelled neurones represented less than 4% of all the nitrergic neurones in the ventrolateral lower brainstem.
Distribution of spinally projecting neurones. Spinally projecting neurones were distributed in all the rostrocaudal levels of the ventrolateral lower brainstem (Figs 3 and 7). The maximum numbers of retrogradely labelled cells were located in the middle rostrocaudal levels of the ventrolateral lower brainstem (Figs 3 and 7). The average number of neurones in the ventrolateral lower brainstem that projected to the spinal cord was 83 ± 1 in the heated group and 83 ± 2 in the control group (unilateral counts).
Distribution of spinally projecting neurones that also contained Fos. After exposure of the animals to the hot environment, there was a small but statistically significant increase in the number of spinally projecting neurones that contained a Fos-positive nucleus (2 ± 3; P < 0.001 compared with the control group 0.3 ± 0.3; Fig. 6). These double-labelled neurones represented approximately 2% of all the spinally projecting neurones in the ventrolateral lower brainstem.
Distribution of spinally projecting neurones that contained Fos and NADPH-d. In both the heated and the control group of animals, there were no triple-labelled cells observed in the ventrolateral lower brainstem.
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Discussion |
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The significant increase in Fos production observed in the mid-line regions corresponded to increases in the raphe pallidus and raphe obscurus. This is in agreement with earlier studies using different heating regimens (Kiyohara et al. 1995; Bratincsak & Palkovits, 2004). Thus, the present findings suggest that there are neurones in the mid-line raphe that are activated by elevations in body temperature, and thus may contribute to the central pathways involved in the responses elicited by a hot environment. Mid-line raphe neurones are also activated by exposure to a cold environment. Indeed, their role in cutaneous vasoconstriction has been well studied (Blessing, 2003; Ootsuka et al. 2004). Thus, these neurones may contribute to the central pathways mediating responses initiated by a cold environment. Together, these studies suggest that there are distinct populations of neurones in the lower brainstem that contribute to the central pathways involved in temperature regulation, since neurones activated by heating (i.e. in the present work) would not be expected to be activated during cooling.
Fos production was significantly elevated at all rostrocaudal levels of the ventromedial lower brainstem, which encapsulated the rostral ventromedial medulla and the raphe magnus. Our results suggest that the activated neurones were evenly spread throughout the rostrocaudal extent of this region. This contrasted with the mid-line, where Fos production was maximal at the more rostral levels examined. Our results suggest that neurones in the raphe magnus and rostral ventromedial medulla contribute to the pathways activated by elevations in body temperature. Since cooling also activates neurones in these areas, this suggests that these regions are involved in responses initiated by either decreases or increases in body temperature (Kiyohara et al. 1995; Bratincsak & Palkovits, 2004; Nakamura et al. 2004). In the ventrolateral lower brainstem, Fos-positive cells were predominantly located in the caudal levels examined, which corresponded to the rostral ventrolateral medulla.
The function of the activated neurones in the ventral lower brainstem examined in the present study cannot be determined by the present work. However, it is possible that these activated neurones may contribute to the pathways mediating vasoconstriction of the blood vessels supplying the internal organs, such as the gut and kidney, which redirects blood flow to the skin to enable heat dissipation (McAllen et al. 1995, 1997; Tanaka et al. 2002). The Fos-positive neurones may also include neurones that contribute to sweating, and the profuse salivation that occurs in the rat (McAllen et al. 1995). It is also possible that the activated neurones may represent interneurones that ultimately contribute to the vasodilatation of the skin vasculature (by inhibiting the sympathetic vasoconstrictor activity) that occurs in response to heating (Rathner & McAllen, 1999; Blessing & Nalivaiko, 2001).
Neurones positive for NADPH-diaphorase were found throughout the ventral lower brainstem examined. The neurones were found in regions that encompassed the rostral ventromedial medulla, raphe obscurus, raphe magnus and RVLM, as previously described (Vincent et al. 1992). The nitrergic neurones were found in areas in which activated neurones were also located. Following exposure to the hot environment, we found a small but statistically significant increase in the number of activated nitrergic neurones in the mid-line and ventromedial lower brainstem. In the mid-line, the activated nitrergic neurones represented less than 2% of the nitrergic neurones. In the ventromedial lower brainstem, the nitrergic neurones activated by heating represented almost 5% of the nitrergic neurones counted in this region of the lower brainstem. Nitric oxide within the central nervous system appears to play a key role in heat dissipation (Gourine, 1995; Eriksson et al. 1997; Schmid et al. 1998; Simon, 1998; Gerstberger, 1999), and these activated nitrergic neurones in the lower brainstem may contribute to this. However, based on the quantitative analysis presented in the present study, the contribution is likely to be small. This is in stark contrast to the findings in the hypothalamus, in particular in the hypothalamic paraventricular nucleus, in which we previously found almost 40% of nitrergic neurones activated by exposure to a similarly hot environment (Cham et al. 2006).
Spinally projecting neurones were found in each region of the lower brainstem examined and these included those found in the mid-line raphe, the rostral medial raphe magnus and the RVLM. Exposure to a hot environment increased the number of spinally projecting neurones activated. These activated neurones represented 2–3% of the spinally projecting neurones counted in the lower brainstem. Of course, the spinally projecting neurones mediating cutaneous vasoconstriction would be inhibited by heating and thereby contribute to the low proportion of spinally projecting neurones activated. The regions of the lower brainstem examined included the RVLM, which is known to contain neurones that can increase the activity of sympathetic nerves innervating the vasculature of internal organs, such as the kidney and mesenteric beds. Thus, it is possible that, although small, the spinally projecting neurones in the lower brainstem activated by heating may represent an important population of neurones involved in the cardiovascular responses designed to dissipate heat when body temperature rises.
The activated spinally projecting neurones may also represent neurones that mediate the increased sympathetic nerve activity that contributes to sweating and, more importantly in rats, salivation in response to heating. Spreading saliva over the body surface is an important mechanism for dissipating heat in rats, and the rats used in the present study exhibited this response.
It is interesting that the majority of spinally projecting neurones in the ventral medulla were not activated following the hot environment stimulus. Perhaps the spinal injections did not adequately label the spinally projecting neurones that play a role in the vasoconstriction of the internal vasculature that contributes to the redistribution of blood flow. Alternatively, the finding could suggest that neurones in the supramedullary/pontine regions may be more important in the heat-induced blood flow redistribution than previously realized. It is noteworthy that we have recently found that 22% of the spinally projecting neurones in the paraventricular nucleus of the hypothalamus were activated by elevations in core body temperature (Cham et al. 2006), more than with other strong stimuli, such as severe haemorrhage (Badoer et al. 1993). Functional studies, however, have suggested that suprapontine regions may not be required for the full expression of the increases in the renal, splanchnic, lumbar and splenic sympathetic nerve activities elicited by elevations in body temperature (Kenney et al. 2000). By contrast, in senescent rats, suprapontine regions appear to be critical contributors to the splanchnic sympathetic nerve responses elicited by heating (Kenney & Fels, 2002). Thus, the role of suprapontine regions in the cardiovascular responses initiated by elevations in body temperature requires further clarification.
In general, spinally projecting neurones were not nitrergic, since there were only a few that contained NADPH-diaphorase. This is consistent with the finding that the spinally projecting adrenergic C1 neurones located in the RVLM are not nitrergic (Iadecola et al. 1993). Furthermore, none of the nitrergic spinally projecting neurones in the ventral lower brainstem was activated by heating. Thus, the increase in activated nitrergic neurones observed in response to the elevated body temperature occurred in neurones that projected to regions other than the spinal cord.
Summary and conclusions
In conclusion, exposure to a hot external environment for 1 h activates neurones in the medulla and pons in regions that contain nitrergic neurones or spinally projecting neurones. Some nitrergic neurones were activated, particularly in the ventromedial lower brainstem. Spinally projecting neurones in the lower brainstem were also activated. However, no nitrergic spinally projecting neurones were activated. We hypothesize that nitrergic neurones, as well as spinally projecting (non-nitrergic) neurones, in the lower brainstem may contribute to the cardiovascular responses elicited by an acute exposure to a hot environment, but their contribution is likely to be less than that of similar neurones found in the hypothalamus.
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