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Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville 3052, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
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This study was undertaken to determine if neurons in the lamina terminalis, previously identified as projecting to the kidney (35), were responsive to alterations in stimuli associated with fluid balance homeostasis. Neurons in the lamina terminalis projecting to the kidney were identified by the retrograde transynaptic transport of Bartha's strain of pseudorabies virus in anesthetized rats. Rats were also exposed to 24-h water deprivation, intravenous hypertonic saline, or intracerebroventricular ANG II. To determine if "kidney-directed" neurons were activated following each stimulus, brain sections that included the lamina terminalis were examined immunohistochemically for viral antigen and Fos protein. With the exception of ANG II in the subfornical organ, all regions of the lamina terminalis contained neurons that were significantly activated by water deprivation, hypertonic saline, and ANG II. These results provide evidence for a neural substrate, which may underpin some of the effects of hypertonic saline and ANG II on renal function thought to be mediated through the lamina terminalis.
pseudorabies; c-Fos; angiotensin; dehydration; hypertonic saline
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE MAMMALIAN KIDNEY is under both humoral and neural control. Although most renal function studies have focused on the humoral control of the kidney, its sympathetic innervation has an extensive role in renal function (8). Recently, several groups have used retrograde viral tracing techniques to detail the extensive projections to these nerves throughout the entire neuroaxis (9, 14, 34, 35). These have been shown to extend through four orders of synaptically linked neurons from the preoptic region. Despite the extensive descriptions of the central anatomy of "kidney-directed" neurons, the physiological conditions that activate them are largely unknown. A likely function of these preoptic neurons is the coordination of renal responses to changes in fluid balance, because ablation of this midline region, which includes the lamina terminalis, severely disrupts the influence of centrally administered ANG II or hypertonic saline on renal sympathetic nerve activity (20).
The use of the protooncogene c-fos to identify activated neurons is well established (33; see also Ref. 13 for review). This technique has been used to identify the neurons in the lamina terminalis responsive to alterations in body fluid homeostasis. In response to intravenous hypertonic saline (28, 29), 24-h water deprivation (24), or centrally administered ANG II (12), neurons were activated in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, and all three regions of the lamina terminalis: the subfornical organ, median preoptic nucleus, and the organum vasculosm of the lamina terminalis (OVLT).
The distribution of neurons in the central nervous system (CNS) activated by these stimuli corresponds closely to many of the areas labeled in our retrograde tracing studies, where pseudorabies virus was injected into the kidney and its passage was traced through the nervous system (35). The aim of the present study was to combine in the same animal pseudorabies virus neural tracing from the kidney to the forebrain, with detection of c-fos expression following intravenous hypertonic saline, water deprivation, or intracerebroventricular ANG II to determine whether populations of identified kidney-directed neurons in the CNS are responsive to these perturbations. This will allow for the first time the visualization and mapping of physiologically identified neurons in the forebrain projecting multisynaptically to the kidney.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Surgical Preparation
Fifteen male Sprague-Dawley rats (250-350 g) were anesthetized (0.6 ml/kg ip pentobarbital sodium) and had intracerebroventricular metal cannulas (blunt 23-gauge needle) inserted into the right lateral cerebral ventricle using stereotaxic coordinates. The cannulas were held in place using dental acrylic and metal screws anchored to the skull. A further 13 rats anesthetized with equithesin (3 ml/kg ip) had a polyethylene cannula with a lumen diameter of 0.5 mm and external diameter of 1.5 mm inserted into the left femoral vein ~0-0.2 cm below the iliac bifurcation of the inferior vena cava. The cannulas were filled with heparinized saline and exteriorized via a small incision in the skin on the dorsal surface of the animal's neck. Animals from all groups were allowed to recover for at least 4 days before further experiments commenced. All procedures have been approved by the designated animal experimentation ethics committees at the Howard Florey Institute and the Australian Animal Health Laboratory.Pseudorabies Inoculation
Details of the preparation, characterization, and immunocytochemical detection of pseudorabies virus have been published elsewhere (6, 7), and information regarding the preparation and use of pseudorabies virus by our group has been published previously (16, 35). Rats were anesthetized with pentobarbital sodium (0.6 ml/kg), and their left kidneys were exteriorized via a flank incision. Six injections of a 1.5-µl suspension of pseudorabies Bartha (2.8 × 10
6 plaque
forming U/ml) were made into the left kidney using a 10-µl syringe
(SGE Instruments, Ringwood, Australia) with a 30-gauge needle attached.
The injections were made 1-2 mm into the cortex and spaced over
the entire surface of the kidney. The needle was held in place for 1 min following each injection, and any efflux from the puncture was
adsorbed using pledgets of cotton wool. After the injections, the
kidney was returned to the abdominal cavity, the wound was sutured, and
the skin was closed with Michelle clips. All procedures using live
virus were performed within secure facilities at the Australian Animal
Health Laboratory.
Administration of Osmotic Challenges and Intracerebroventricular ANG II
In all rats, pseudorabies infections lasted 4 days (96 h). At an appropriate time after inoculation, all rats received one of the treatments described below.Water deprivation. Twenty-four hours before being killed, 32 rats were water deprived by removing their drinking bottles for this entire period. A further 22 rats designated as controls were not exposed to the stimuli above and were allowed free access to water.
During the course of these experiments, we suspected that administration of the virus itself might lead in some ways to a state of dehydration, particularly in those rats with more advanced infections (see RESULTS and DISCUSSION). To assess the extent of dehydration in these rats, body weight was also measured over the course of the infection, and tail bleeds were taken at the time of death to determine plasma osmolality. Tail bleeds involved a small cut at the tail tip minutes before death, and blood was collected into heparinized tubes. Plasma osmolality was measured using a digimatic osmometer (Advanced Instruments).Intravenous hypertonic saline. Ninety minutes before being killed, 10 rats were injected with 1.5 ml of 1.5 M hypertonic saline (0.15 ml/min for 10 min) into the left femoral vein through preimplanted polyethylene tubing. Rats were free to move around their cages while the solution was injected. A further 12 rats with preimplanted femoral vein cannulas received control injections of 1.5 ml of 0.15 M isotonic saline under conditions identical to those above. After the injections, both groups had their water bottles removed until the rats were perfused.
Intracerebroventricular ANG II. Ninety minutes before being killed, 16 rats with preimplanted intracerebroventricular cannulas had 100 ng of ANG II in 10 µl artificial cerebrospinal fluid (CSF) injected into the lateral cerebral ventricle via a 10-µl syringe (SGE Instruments) attached to a polyethylene cannula fixed to the implanted cannulas. A further seven rats with preimplanted intracerebroventricular cannulas received a bolus injection of 10 µl artificial CSF only, under conditions identical to those above. After the injections in both groups, rats were returned to their cages, and their water bottles were removed until they were perfused.
Postmortem Analysis of Brain Tissue
After the treatments outlined above, all rats were reanesthetized with pentobarbital sodium (1 ml/kg) and were perfused transcardially with 50-100 ml of 0.15 M NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.2). Brains and spinal cords were removed and placed in a 4% paraformaldehyde solution for a further 2 h, after which they were transferred to a 20% sucrose solution in phosphate buffer overnight at 4°C. Coronal sections were cut at 40 µm throughout the forebrain on a freezing microtome.Immunohistochemistry
Brain sections free floating in phosphate buffer were transferred to 10% normal horse serum and agitated for 1 h at room temperature. Sections were placed in a solution of 2% normal horse serum with 0.3% Triton X-100 containing primary antibodies directed against acetone-inactivated pseudorabies virus raised in goat (1:5,000) and against a synthetic amino acid sequence corresponding to the residues 4-17 of human Fos raised in rabbit (1:2,000; Santacruz Biotechnology). The primary antibody for pseudorabies virus has been shown previously to recognize viral capsids and all of the major viral envelope glycoproteins (6, 7). After incubating with the primary antibodies for 2 days at room temperature, sections were transferred, respectively, to fluorescein isothiocyanate (-labeled dextran) (FITC; Rockland, PA) and Texas Red (Rockland, PA) conjugated secondary antibodies directed against the species in which the primary antibodies were raised. There was no evidence of any cross-reactivity between the two secondary antibodies. Sections were mounted on gelatin chrome alum subbed slides in a buffered glycerol solution (pH = 8.6).Counting Methods
Sections were examined using a Leica DMRBE microscope fitted with a 50-W mercury fluorescent lamp and filter blocks to detect Texas Red and FITC fluorophores. Sections were first examined for the degree of viral transport. During the course of these experiments, it was noted that with advanced infection of neuronal populations in the lamina terminalis, there was a greatly increased occurrence of dehydration responses (as evidenced by Fos distribution) albeit in control rats (i.e., water replete, intravenous hypotonic saline, and intracerebroventricular artificial CSF). However, in control groups with less extensive infections in the lamina terminalis, such dehydration responses did not occur. To exclude rats that exhibited a dehydration Fos response that was associated with a nonspecific response to advanced viral infection, we established a threshold of infection below which rats showed no elevation of Fos and were considered normally hydrated. As a guide, examination of OVLT sections from all control rats used in this study revealed that the least extensive infection that resulted in a dehydration Fos response occurred in a rat that had a total of 270 infected neurons in the OVLT. Therefore, rats in both experimental and control groups with greater than 250 infected neurons in the OVLT were designated as having "heavy" infections and were excluded from this study. Conversely, rats with less than 250 infected neurons in total in the OVLT were designated as having "light" infections. Only rats with light infections were used in the comparisons of the number of double-labeled neurons between control and experimental groups. Of all rats studied, a total of 24 rats was excluded from these comparisons on the basis of having heavy infections. Additionally, only rats with a minimum of 10 neurons in each area of the lamina terminalis were included in double-labeling counts to avoid biasing the results with calculations from small numbers. A total of 21 rats was excluded from the comparisons on this basis. The final number of rats used in each group for double-labeling comparisons following exclusion of the abovementioned data is indicated in RESULTS.Brain regions were identified in accordance with those defined by Swanson (36), and neurons were counted from these regions with the use of an eyepiece graticule using a ×40 magnification objective and ×10 magnification eyepiece. In each area, all neurons infected with the virus were counted with an FITC filter in place, and each of these neurons was then examined using a Texas Red filter to determine if it was double-labeled with a corresponding Fos-positive nucleus. Comparisons of the numbers of double-labeled (Fos and virus) neurons between groups were made using a one-way ANOVA with a significance level set at 0.05. For each animal included in the analyses, counts were made on every section taken. The numbers of sections from which counts were made were similar in each animal and for each brain region and were not significantly different when compared using a one-way ANOVA. The average number of sections taken from hypertonic and isotonic saline-treated rats ranged from 6.3 to 7.0 through the OVLT, from 7.7 to 8.3 through the median preoptic nucleus, and from 4.8 to 5.3 through the subfornical organ. In all other treatment groups, the average number of sections ranged between 3.1 and 4.5 sections in the OVLT, between 4.6 and 5.9 in the median preoptic nucleus, and between 2.8 and 3.9 in the subfornical organ. Photomicrographs were taken using a SPOT-RT digital camera (Diagnostic Instruments). These images were adjusted in Photoshop version 5.5 (Adobe) to attain consistent levels of brightness and contrast. Each image was also adjusted using an unsharp mask filter set at 100% with a diameter setting of 2.0 and a threshold setting of zero, otherwise no further postcapture processing was made.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Brain Areas Labeled Following Retrograde Transport of Pseudorabies Virus from the Kidney
After 96 h, viral antigen was detected in both third- and fourth-order neurons, where the term order refers to the number of synaptic relays traversed by the virus. The order of neurons in each area was based on the graded appearance of infected neurons at timed intervals following inoculation (35). Importantly, the pattern of brain regions labeled and the spatial distribution of pseudorabies virus within each of these regions were the same for all rats in all groups. In third-order neurons, the virus was detected in rostral ventromedial medulla, rostral ventrolateral medulla, A5 pontine area, and paraventricular nucleus as well as the nucleus of the solitary tract, locus ceruleus, and subceruleus nuclei. At 96 h, the level of infection was moderate to extensive within these areas. At the same survival time, fourth-order neurons in all three regions of the lamina terminalis were labeled. In addition, fourth-order infected neurons were present within the bed nucleus of the stria terminalis, retrochiasmatic area, medial preoptic area, periaqueductal gray, primary motor area of the cortex, nucleus of the solitary tract, area postrema, anterior hypothalamic area, suprachiasmatic nucleus, lateral hypothalamic area, anteroventral periventricular nucleus of the hypothalamus, lateral preoptic area, and visceral area of the insular cortex. In the OVLT, viral antigen was distributed equally among the lateral boundaries, dorsal cap, and ventromedial components, although the virus was absent from the central vascularized region (Fig. 1A). In the median preoptic nucleus, the virus was evenly dispersed in the dorsal and ventral components (Fig. 1B). In the subfornical organ, viral labeling was mainly concentrated around its periphery so that in that section, labeled neurons formed an annular arrangement (Fig. 1C).
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Fos Immunoreactivity Following Water Deprivation, Intravenous Hypertonic Saline, or Intracerebroventricular ANG II in Lightly Infected Rats
The following observations are restricted to the preoptic region. Sections taken from rats following 24-h water deprivation, intravenous hypertonic saline, or intracerebroventricular ANG II combined with light viral labeling (see METHODS for definition) showed Fos-immunoreactive (Fos-IR) nuclei in a range of CNS sites. After all stimuli, Fos-IR nuclei were found in the supraoptic nucleus, paraventricular nucleus, OVLT, median preoptic nucleus, and subfornical organ. For each stimulus, the distribution of Fos immunoreactivity within each of these areas did not appear to be altered following pseudorabies infection, as the distributions were identical in rats with infections not reaching the lamina terminalis. Within the component structures of the lamina terminalis, there was a number of differences in the distribution of Fos immunoreactivity following the three stimuli. In the OVLT, water depletion elicited the most extensive Fos response, with labeling confined to its lateral boundaries and dorsal cap, forming an arched band around the perimeter of this structure (Fig. 2A). In contrast, hypertonic saline activated neurons in the dorsal cap of the OVLT more than those in its lateral boundaries (Fig. 2B). ANG II, on the other hand, generated an inverse response to hypertonic saline, with more Fos-IR nuclei in the lateral boundaries than the dorsal cap (Fig. 2C). Very few cells were labeled in the central vascularized region of the OVLT with any of the three stimuli used. In the median preoptic nucleus, all stimuli resulted in a Fos response in neurons situated in both the ventral and dorsal aspects (Fig. 2, D-F). In this nucleus, water deprivation (Fig. 2D) elicited the most extensive and dense Fos response, with neurons activated by hypertonic saline present in reduced numbers that were restricted to the midline, particularly in its ventral aspect (Fig. 2E). The response in the median preoptic nucleus following ANG II (Fig. 2F) was reduced in density and extent and was restricted to the midline of the dorsal and ventral aspects. In the subfornical organ, water deprivation elicited the most extensive response, with Fos-IR nuclei present throughout the structure (Fig. 2G) and with a concentration in the central regions and only a small number of cells labeled elsewhere. Hypertonic saline-activated neurons were restricted more to the annular regions of the subfornical organ (Fig. 2H), and ANG II resulted in more Fos-IR nuclei within the core and particularly the anterior and periventricular regions (Fig. 2I). Other areas of the hypothalamus and regions more caudal were not examined in this study.
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Kidney-Directed Neurons in the Lamina Terminalis Double-Labeled with Fos Following Water Deprivation, Intravenous Hypertonic Saline, and Intracerebroventricular ANG II
Only rats that were lightly infected were used in counts of virus and Fos double-labeled neurons. Colocalized neurons were found in all regions of the lamina terminalis, including the dorsal cap and lateral boundaries of the OVLT in response to 24-h water deprivation (Fig. 3, A-C), the median preoptic nucleus in response to ANG II (Fig. 3, D-F), and the subfornical organ in response to hypertonic saline (Fig. 3, G-I). In each of the control groups (artificial CSF, intravenous isotonic saline, and free access to water), there were varying numbers of double-labeled cells within the component regions of the lamina terminalis (range 3.5-21.7%). This may, to some extent, represent the confounding expression of Fos that occurs with advancing infection of neurons (see below and DISCUSSION). However, for all of these comparisons, the numbers of double-labeled neurons in the treated groups were more than double those of rats in their respective control groups (Table 1). In all comparisons between treatment and control groups, there were no significant differences in the number of virally infected neurons in each area of the lamina terminalis. In the animals receiving artificial CSF infusions, the numbers of infected neurons were lower in general than those in rats with injections of intracerebroventricular ANG II. Although there is no obvious reason for this, and although the difference between the groups was insignificant, smaller numbers of newly infected neurons are always associated with a reduced expression of Fos. In this case, an additional comparison has been made with infected rats that had free access to water. For this comparison, there were significantly greater numbers of virally infected neurons that were activated by ANG II in the OVLT and median preoptic nucleus, but not in the subfornical organ. For the regions with significant activation, between 27 and 53% of all neurons projecting to the kidney from each area of the lamina terminalis were double-labeled for Fos. Comparisons across treatments showed that there were greater numbers of double-labeled neurons in the subfornical organ following hypertonic saline infusion than water deprivation. There were also greater numbers of double-labeled neurons in the OVLT following both hypertonic saline and ANG II compared with water deprivation. By comparison, there were fewer double-labeled neurons in the median preoptic nucleus following ANG II compared with water deprivation and hypertonic saline (Table 1).
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Fos Immunoreactivity in Heavily Infected Rats
During the course of these experiments, it was apparent that there was a positive correlation between the extent of viral infection and a "dehydration-like" distribution of Fos-IR neurons in the lamina terminalis. This was evident in heavily infected rats treated with intravenous isotonic saline, intracerebroventricular artificial CSF, and other control rats, all of which were allowed unrestricted access to water. In many of these control rats with heavy infections in the lamina terminalis, this distribution of Fos-IR neurons was identical to the pattern arising from dehydration as described above and in previous studies (24). This pattern included large numbers of Fos-IR neurons in the lamina terminalis and regions of the hypothalamus, namely the supraoptic and paraventricular nuclei. By contrast, in control rats with only light viral infections, occasional Fos-IR nuclei were observed in the lamina terminalis and supraoptic nucleus, and these numbers decreased with reduced infection. The few scattered Fos-IR neurons that were present in the lamina terminalis of lightly infected rats were not necessarily confined to the distribution seen after dehydration but, in some cases, they were associated with virus-infected neurons. In all likelihood, the pattern of Fos labeling present in heavily infected rats with access to water was due to the reduced fluid intake, which may arise as a consequence of viral infection (see DISCUSSION).As the Fos immunoreactivity in the lamina terminalis in heavily infected rats appeared "dehydration-like," we also measured the body weights and plasma osmolalities in rats with free access to water (n = 5) and in water-deprived (n = 3) rats with similarly heavy viral infections. All heavily infected rats with access to water had a "dehydration-like" distribution of Fos-IR neurons. Water-deprived rats decreased their body weight by an average of 37.8 ± 3.8 g over the entire course of viral infection, and those rats with free access to water experienced a reduction of 25.5 ± 4.2 g. Similarly, water-deprived rats had extremely high plasma osmolalities (366.0 ± 20.1 mosmol/kgH2O) and those with free access to water were also elevated (323.3 ± 8.4 mosmol/kgH2O), with both of these being considerably higher than normal values.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The brain areas labeled in this study following retrograde transynaptic transport of pseudorabies virus from the kidney are consistent with our previous study (35) and those of other groups (9, 14, 34). In the present study, we have focused on fourth-order labeling in the preoptic region, particularly, the lamina terminalis, because these neurons are known to be critically involved in the control of fluid and electrolyte balance (see Ref. 23 for a review). As the virus appears coincidentally in all three structures of the lamina terminalis, it is likely that these neurons all belong to the same synaptic order. However, as extensive reciprocal connections are known to exist between the component regions of the lamina terminalis (4, 19, 25, 31), it is possible that with advancing infection, these connections are also recruited and labeled. Therefore, the neurons labeled in the lamina terminalis may represent both interconnections between its component regions and neurons projecting to the third-order structures labeled in this study.
We have illustrated that many of the kidney-directed neurons in the lamina terminalis have a distribution overlapping that of neurons that are activated in response to intracerebroventricular ANG II, water deprivation, or intravenous infusion of hypertonic saline. The degree of overlap varies depending on the stimulus used, as each evokes a different distribution of Fos-IR nuclei. Previous studies have shown similar distributions of Fos immunoreactivity in response to intravenous hypertonic saline (28, 29), 24-h water deprivation (24), or centrally administered ANG II (12). Kidney-directed neurons were activated significantly following all stimuli in all three areas of the lamina terminalis, with the exception of ANG II in the subfornical organ, compared with control rats with access to water. The latter result is not surprising given that viral labeling in the subfornical organ is largely confined to an annular distribution, and ANG II-activated neurons are found mostly in the core of the organ. In contrast to the subfornical organ, both the OVLT and the median preoptic nucleus contained a large proportion of kidney-directed neurons responsive to ANG II. In the OVLT, these were concentrated in its lateral margins and extended into the adjacent preoptic region. The lateral aspects of the OVLT have recently been shown to project to the parvocellular paraventricular nucleus among other sites (unpublished data from our group). Kidney-directed neurons in the median preoptic nucleus with elevated levels of Fos protein have probably been activated directly by centrally administered ANG II. ANG II receptors are particularly abundant in the median preoptic nucleus (1) as are ANG II-containing terminals (30). Many of these terminals arise from neurons with cell bodies in the subfornical organ (19). It is likely that the endogenous ligand for the AT1 receptors in this nucleus is derived from local nerve terminals (37). Finally, AT1A mRNA has recently been shown to be associated with kidney-directed neurons in the in the median preoptic nucleus (11).
The neurons double-labeled in the OVLT and subfornical organ following intravenous hypertonic saline are likely to be directly responsive to elevated plasma osmolality as previous studies suggest that these regions are the site of cerebral osmoreceptors (3, 22, 29, 39). The present results are consistent with these neurons having direct projections to autonomic premotor regions en route to the kidney, although they may also project through an interposed synapse in the median preoptic nucleus (31), as kidney-directed neurons showed Fos activation in this structure.
In response to 24-h water deprivation, activated kidney-directed neurons were found in all regions of the lamina terminalis, although the number of double-labeled cells was reduced compared with that observed with intravenous hypertonic saline. This reduction may be due, in part, to the relatively weaker hypertonic challenge provided by water deprivation for 24 h. The increase in plasma tonicity following 24-h water deprivation is ~4-5% (24), whereas there is a 9% increase following 1.5 M hypertonic saline (29) when administered identically to that in the present experiments. Consistent with this suggestion, neurons in the dorsal cap of the OVLT are activated intensely by hypertonic saline but to a lesser extent by water deprivation (Fig. 2, A and B). Water deprivation causes loss of fluid from both intracellular and extracellular compartments. Extracellular fluid loss stimulates the kidney to release renin with subsequent generation of ANG II that is likely to activate neurons in the OVLT and subfornical organ. Therefore, Fos immunoreactivity in the lateral boundaries of the OVLT and core of the subfornical organ following water deprivation is likely to represent that due to the effects of circulating ANG II. In the present study, the number of activated kidney-directed neurons in the lateral boundaries of the OVLT was greater following intracerebroventricular infusion of ANG II compared with 24-h water depletion. This finding is in accordance with a previous study where the levels of circulating ANG II following 24-h water deprivation were measured and found to be considerably lower than estimates of the levels of ambient ANG II acting in the brain intracerebroventricularly, albeit when using a concentration of intracerebroventricular ANG II lower than that used here (21).
The present finding that neurons in the preoptic region that are sensitive to fluid balance perturbations have a neural link to the kidney is consistent with a number of electrophysiological and pharmacological findings. Centrally and peripherally administered hypertonic saline has, in the majority of studies, been shown to decrease renal sympathetic nerve activity (2, 18, 27, 41). In one of these studies, central administration of hypertonic saline and ANG II in conscious sheep resulted in a decrease in renal sympathetic nerve activity, and these effects were abolished by lesions of the lamina terminalis (20).
The present results reflect patterns of distribution of neurons responsive to a range of perturbations rather than absolute numbers. In this regard, numbers of double-labeled neurons in each case are likely to be underestimates for the following reasons. Some of the Fos-responsive neurons in the lamina terminalis may send projections to sites other than the kidney, such as those mediating thirst and vasopressin release. In addition, greater numbers of neurons may be labeled with virus from the kidney if rats are allowed to survive for longer times. Conversely, virus-infected neurons not expressing Fos following any of the stimuli used in the present experiment may be responsive to other substances known to act through the preoptic region to alter renal sympathetic nerve activity such as prostaglandin E2 (15) or endothelin-1 (26).
The reliability of the viral tract tracing technique used in the present experiments depends on the specificity with which the virus traverses only synaptically connected pathways. Many previous studies have shown pseudorabies to be a highly specific transneuronal tracer (see Ref. 5 for a review). Furthermore, the technique has previously been used to track polysynaptic pathways to the kidney from the lamina terminalis (35). Labeling in the lamina terminalis has also been reported following pseudorabies tracing from the heart (38), salivary glands (16), and sympathetic ganglia (42). However, the extent to which neurons may form mixed populations or represent, at least in some part, single neurons that project to multiple end organs remains to be determined (see Ref. 17).
There is a number of technical issues that need to be considered in relation to the combination of viral labeling with c-fos methodology. Several studies have examined the relationship of elevated levels of c-fos mRNA or Fos protein in neurons following infection with neurotropic viruses. Weiss and Chowdhury (40) have found elevated levels of Fos protein before the appearance of immunohistochemically detectable pseudorabies in neurons of the spinal cord and medulla; however, no attempt was made to quantify the degree of coexpression. Herpes simplex virus-1 (HSV-1) induces Fos protein in some neurons of the rat brain stem following infection of the cervical vagus (10). From the evidence presented, only ~22% of HSV-1 neurons appeared colocalized with Fos in the rostral ventrolateral medulla. It has also been concluded that the attenuated HSV-1 and highly neuroinvasive HSV-2 strain both induce Fos expression in spinal cord neurons after injection of the virus into the mouse hind-paw planter skin (32). In that study, the attenuated HSV-1 strain did not induce Fos expression in spinal cord neurons when the virus was present in the neurons 3 days after inoculation, but only at later survival times. Considering all of the data together, there is general consensus that levels of Fos protein are elevated subsequent to advanced viral infections.
Our findings are in agreement with this correlation between levels of viral infection of neurons and the expression of Fos protein. In this respect, it is critical to point out that only time periods and infection levels, which resulted in newly infected neurons in the lamina terminalis, were considered in the present experiments. This focus on the "first wave" of viral infection in a particular region offers a window of opportunity where viral tracking and c-fos methodology can be combined with confidence.
The use of appropriate control groups in conjunction with the strategy outlined above and statistical analysis of counts largely circumvents the confounding influence of viral-induced Fos expression.
In the present experiments, advanced infections were implicated in the generation of Fos responses in other ways. Heavily infected rats with access to water had reduced body weights and elevated plasma osmolalities, indicating the animals were dehydrated. Some of this dehydration is likely to be the result of reduced water intake as we have also noted that heavily infected rats allowed free access to water had reduced water and food intakes over the last day of infection compared with the 2 preceding days. The reduced water intakes may be due to either general malaise following viral infection, a specific immune response, or they may be related to infection and impairment of the function of neurons within the lamina terminalis. Those rats (control and experimental) with heavy infections (see METHODS) were excluded from the data set.
Perspectives
Recent studies have shown that the lamina terminalis influences body fluid regulation through activation of the renal sympathetic nerves. This effect is in addition to the known influence of the lamina terminalis on the regulation of thirst and vasopressin secretion. These findings extend this notion, having identified specific populations of kidney-directed neurons within each region of the lamina terminalis that are responsive to intravenous hypertonic saline, intracerebroventricular ANG II, and water depletion.| |
ACKNOWLEDGEMENTS |
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This study was supported by the National Health and Medical Research Council of Australia Block Grant 983001.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Oldfield, Howard Florey Institute, Univ. of Melbourne, Parkville 3052, Australia (E-mail: b.oldfield{at}hfi.unimelb.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2001; accepted in final form 20 July 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Allen, AM,
Oldfield BJ,
Giles ME,
Paxinos G,
McKinley MJ,
and
Mendelsohn FAO
Localization of angiotensin receptors in the nervous system.
In: Handbook of Chemical Neuroanatomy, edited by Quirion R,
Bjorklund A,
and Hokfelt T.. Amsterdam: Elsevier, 2000, p. 79-124.
2.
Bealer, SL,
Delle M,
Skarphedinsson JO,
Carlsson S,
and
Thoren P.
Differential responses in adrenal and renal nerves to CNS osmotic stimulation.
Brain Res Bull
39:
205-209,
1996[ISI][Medline].
3.
Bisley, JW,
Rees SM,
McKinley MJ,
Hards DK,
and
Oldfield BJ.
Identification of osmoresponsive neurons in the forebrain of the rat: a Fos study at the ultrastructural level.
Brain Res
13:
25-34,
1996.
4.
Camacho, A,
and
Phillips MI.
Horseradish peroxidase study in rat of the neural connections of the organum vasculosum of the lamina terminalis.
Neurosci Lett
25:
201-204,
1981[ISI][Medline].
5.
Card, JP.
Practical considerations for the use of pseudorabies virus in transneuronal studies of neural circuitry.
Neurosci Biobehav Rev
22:
685-694,
1998[ISI][Medline].
6.
Card, JP,
and
Enquist LW.
Use of pseudorabies virus for definition of synaptically linked populations of neurons.
In: Methods in Molecular Genetics. New York: Academic, 1994.
7.
Card, JP,
Rinaman L,
Lynn RB,
Lee BH,
Meade RP,
Miselis RR,
and
Enquist LW.
Pseudorabies virus infection of the rat central nervous system: ultrastructural characterization of viral replication, transport, and pathogenesis.
J Neurosci
13:
2515-2539,
1993[Abstract].
8.
DiBona, GF,
and
Kopp UC.
Neural control of renal function.
Physiol Rev
77:
75-197,
1997
9.
Ding, ZQ,
Li YW,
Wesselingh SL,
and
Blessing WW.
Transneuronal labelling of neurons in rabbit brain after injection of herpes simplex virus type 1 into the renal nerve.
J Auton Nerv Syst
42:
23-31,
1993[ISI][Medline].
10.
Gieroba, ZJ,
Zhu BS,
Blessing WW,
and
Wesselingh SL.
Herpes simplex virus induces Fos expression in rat brainstem neurons.
Brain Res
675:
329-332,
1995[ISI][Medline].
11.
Giles, ME,
Sly DJ,
McKinley MJ,
and
Oldfield BJ.
Neurons in the lamina terminalis which project polysynaptically to the kidney express angiotensin AT(1A) receptor.
Brain Res
898:
9-12,
2001[ISI][Medline].
12.
Herbert, J,
Forsling ML,
Howes SR,
Stacey PM,
and
Shiers HM.
Regional expression of c-fos antigen in the basal forebrain following intraventricular infusions of angiotensin and its modulation by drinking either water or saline.
Neuroscience
51:
867-882,
1992[ISI][Medline].
13.
Herrera, DG,
and
Robertson HA.
Activation of c-fos in the brain.
Prog Neurobiol
50:
83-107,
1996[ISI][Medline].
14.
Huang, J,
and
Weiss ML.
Characterization of the central cell groups regulating the kidney in the rat.
Brain Res
845:
77-91,
1999[ISI][Medline].
15.
Huang, XC.
Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit.
Int J Biometeorol
37:
222-228,
1993[ISI][Medline].
16.
Hubschle, T,
McKinley MJ,
and
Oldfield BJ.
Efferent connections of the lamina terminalis, the preoptic area and the insular cortex to submandibular and sublingual gland of the rat traced with pseudorabies virus.
Brain Res
806:
219-231,
1998[ISI][Medline].
17.
Jansen, AS,
Nguyen XV,
Karpitskiy V,
Mettenleiter TC,
and
Loewy AD.
Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response.
Science
270:
644-646,
1995
18.
Kawano, Y,
and
Ferrario CM.
Neurohormonal characteristics of cardiovascular response due to intraventricular hypertonic NaCl.
Am J Physiol Heart Circ Physiol
247:
H422-H428,
1984.
19.
Lind, RW,
Swanson LW,
and
Ganten D.
Angiotensin II immunoreactivity in the neural afferents and efferents of the subfornical organ of the rat.
Brain Res
321:
209-215,
1984[ISI][Medline].
20.
May, CN,
McAllen RM,
and
McKinley MJ.
Renal nerve inhibition by central NaCl and ANG II is abolished by lesions of the lamina terminalis.
Am J Physiol Regulatory Integrative Comp Physiol
279:
R1827-R1833,
2000
21.
McKinley, MJ,
Badoer E,
Vivas L,
and
Oldfield BJ.
Comparison of c-fos expression in the lamina terminalis of conscious rats after intravenous or intracerebroventricular angiotensin.
Brain Res Bull
37:
131-137,
1995[ISI][Medline].
22.
McKinley, MJ,
Denton DA,
and
Weisinger RS.
Sensors for antidiuresis and thirst
osmoreceptors or CSF sodium detectors?
Brain Res
141:
89-103,
1978[ISI][Medline].
23.
McKinley, MJ,
Gerstberger R,
Mathai ML,
Oldfield BJ,
and
Schmid H.
The lamina terminalis and its role in fluid and electrolyte homeostasis.
J Clin Neurosci
6:
289-301,
1999[ISI][Medline].
24.
McKinley, MJ,
Hards DK,
and
Oldfield BJ.
Identification of neural pathways activated in dehydrated rats by means of Fos-immunohistochemistry and neural tracing.
Brain Res
653:
305-314,
1994[ISI][Medline].
25.
Miselis, RR,
Shapiro RE,
and
Hand PJ.
Subfornical organ efferents to neural systems for control of body water.
Science
205:
1022-1025,
1979
26.
Mosqueda-Garcia, R,
Fernandez-Violante R,
Hamakubo T,
and
Stainback R.
Vasopressin mediates the pressor effects of endothelin in the subfornical organ of the rat.
J Pharmacol Exp Ther
277:
1034-1042,
1996
27.
Nishida, Y,
Sugimoto I,
Morita H,
Murakami H,
Hosomi H,
and
Bishop VS.
Suppression of renal sympathetic nerve activity during portal vein infusion of hypertonic saline.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R97-R103,
1998
28.
Oldfield, BJ,
Badoer E,
Hards DK,
and
McKinley MJ.
Fos production in retrogradely labelled neurons of the lamina terminalis following intravenous infusion of either hypertonic saline or angiotensin II.
Neuroscience
60:
255-262,
1994[ISI][Medline].
29.
Oldfield, BJ,
Bicknell RJ,
McAllen RM,
Weisinger RS,
and
McKinley MJ.
Intravenous hypertonic saline induces Fos immunoreactivity in neurons throughout the lamina terminalis.
Brain Res
561:
151-156,
1991[ISI][Medline].
30.
Oldfield, BJ,
Ganten D,
and
McKinley MJ.
An ultrastructural analysis of the distribution of angiotensin II in the rat brain.
J Neuroendocrinol
1:
121-128,
1989.
31.
Oldfield, BJ,
Hards DK,
and
McKinley MJ.
Neurons in the median preoptic nucleus of the rat with collateral branches to the subfornical organ and supraoptic nucleus.
Brain Res
586:
86-90,
1992[ISI][Medline].
32.
Ozaki, N,
Sugiura Y,
Yamamoto M,
and
Nishiyama Y.
Induction of Fos protein expression in spinal cord neurons by herpes simplex virus infections in the mouse.
Neurosci Lett
216:
61-64,
1996[ISI][Medline].
33.
Sagar, SM,
Sharp FR,
and
Curran T.
Expression of c-fos protein in brain: metabolic mapping at the cellular level.
Science
240:
1328-1331,
1988
34.
Schramm, LP,
Strack AM,
Platt KB,
and
Loewy AD.
Peripheral and central pathways regulating the kidney: a study using pseudorabies virus.
Brain Res
616:
251-262,
1993[ISI][Medline].
35.
Sly, DJ,
Colvill L,
McKinley MJ,
and
Oldfield BJ.
Identification of neural projections from the forebrain to the kidney, using the virus pseudorabies.
J Auton Nerv Syst
77:
73-82,
1999.
36.
Swanson, LW.
Brain Maps (2nd ed.). Amsterdam: Elsevier, 1999.
37.
Tanaka, J,
Saito H,
and
Kaba H.
Subfornical organ and hypothalamic paraventricular nucleus connections with median preoptic nucleus neurons: an electrophysiological study in the rat.
Exp Brain Res
68:
579-585,
1987[ISI][Medline].
38.
Ter Horst, GJ,
Hautvast RW,
De Jongste MJ,
and
Korf J.
Neuroanatomy of cardiac activity-regulating circuitry: a transneuronal retrograde viral labelling study in the rat.
Eur J Neurosci
8:
2029-2041,
1996[ISI][Medline].
39.
Thrasher, TN.
Osmoreceptor mediation of thirst and vasopressin secretion in the dog.
Fed Proc
41:
2528-2532,
1982[ISI][Medline].
40.
Weiss, ML,
and
Chowdhury SI.
The renal afferent pathways in the rat: a pseudorabies virus study.
Brain Res
812:
227-241,
1998[ISI][Medline].
41.
Weiss, ML,
Claassen DE,
Hirai T,
and
Kenney MJ.
Nonuniform sympathetic nerve responses to intravenous hypertonic saline infusion.
J Auton Nerv Syst
57:
109-115,
1996[ISI][Medline].
42.
Westerhaus, MJ,
and
Loewy AD.
Sympathetic-related neurons in the preoptic region of the rat identified by viral transneuronal labeling.
J Comp Neurol
414:
361-378,
1999[ISI][Medline].
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