Patterns of Neuronal Activation During Development of Sodium Sensitive Hypertension in SHR
Abstract The effects of regular (RNa) or high (HNa) sodium diet for 3, 7, and 14 days on Fra-like immunoreactivity (Fra-LI) in the brains of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were examined using an antibody that recognizes all known members of the Fos family (Fos, Fos-B, Fra-1, and Fra-2). Two weeks of HNa significantly exacerbated hypertension in SHR but had no effects in WKY. On RNa, compared with WKY, SHR showed higher Fra-LI in the median preoptic nucleus, supraoptic nucleus, both parts of the paraventricular nucleus, nucleus of the solitary tract, and central gray. Fra-LI in the subfornical organ did not differ between the two strains. On RNa, Fra-LI in the anterior hypothalamic area could be detected only in WKY. In osmoregulatory areas, HNa diet increased Fra-LI in both SHR and WKY to comparable extents, but in the median preoptic nucleus, Fra-LI was increased to a greater extent in SHR. HNa produced smaller increases in the subfornical organ of SHR compared with WKY. In both the parvocellular and magnocellular paraventricular nuclei, increases in Fra-LI by HNa were more pronounced in SHR than in WKY. In the anterior hypothalamic area, Fra-LI could no longer be detected in WKY on HNa, whereas it appeared in SHR. HNa increased Fra-LI in the NTS and central gray to similar levels in WKY and SHR. These results indicate that WKY and SHR differ in the pattern of neuronal activation accompanying maturation on RNa. HNa activates neurons in a number of brain areas, and the pattern of these changes also differs between WKY and SHR.
In sodium-sensitive rats, HNa increases sympathetic outflow and exacerbates the hypertension within 4 to 5 days of ingesting an HNa diet.1 2 3 Our previous study indicated that the primary event in this process may be increased release of brain “ouabain,” which subsequently activates the brain RAS.4 Blockade of either brain “ouabain” or the brain RAS prevents both the decreased sympathoinhibition and enhanced sympathoexcitation and the hypertension.4
High sodium intake may intermittently or chronically increase sodium concentration in the plasma and cerebrospinal fluid, resulting in activation of not only osmoreceptors but also sodium receptors centrally or peripherally.5 So far, there is no clear evidence documenting which populations of neurons sense changes in osmolality and/or sodium concentrations during this process, as well as which central nervous system pathways are activated leading to the increase in sympathetic outflow. Recent advances in the understanding of the regulation of gene expression associated with activation of neurons may provide insight into this process. It is now well accepted that immediate-early genes such as c-fos and NGFI-A encode transcriptional factors that are considered to be markers for neuronal activation.6 In particular, immunohistochemical detection of Fos has been used to map activation of neurons involved in cardiovascular regulation.7 8 9 Because Fos down-regulates very quickly during chronic exposure to a variety of treatments, it is not possible to use it as a marker of chronic neuronal activation.10 11 In contrast, other Fos-related proteins show prolonged induction, and the expression persists during chronic exposure.10 12 This group of proteins includes products of fos-related genes such as Fos-B, Fra-1, and Fra-2. Immunohistochemical detection of these proteins has been found to be a very useful tool with which to identify chronically activated neuronal populations in a wide variety of central nervous system regions.13 14 15 16 Recently, it was reported that Fra-like proteins are expressed in the PVN, SON, and lamina terminalis nuclei for up to 7 days after a single intraperitoneal injection of 1.35% saline.17
In the present study, we used immunohistochemical detection of Fos family proteins (Fra-LI) to study the effects of HNa intake on the activity of various neuronal populations involved in the regulation of cardiovascular and osmotic homeostasis. In particular, we compared changes in Fra-LI in these neuronal populations during development of sodium-sensitive hypertension in SHR with changes in sodium-resistant WKY fed an HNa.
All animals were treated in strict accordance with the procedures outlined in the “Guide for the Care and Use of Experimental Animals” endorsed by the Medical Research Council of Canada. Young, male WKY (n=24) and SHR (n=24) (3.5 weeks; Taconic Farms) were housed two or three per cage in a temperature-controlled environment with a 12-hour light/dark cycle and free access to an RNa (101 μmol Na/g) and water for 3 to 4 days before continuing on RNa or starting HNa (1370 μmol Na/g; Harlan Sprague-Dawley). After 3, 7, or 14 days of diet, between 9 and 11 am, rats were deeply anaesthetized with sodium pentobarbital (100 mg/kg IP) and perfused transcardially with 200 mL of 0.9% saline followed by 150 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). The brains were removed and postfixed in 4% paraformaldehyde for 24 to 48 hours at 4°C. Coronal sections (30 μm thick) were cut using a vibratome from the forebrain starting at the vertical limb of the diagonal band of Broca and ending at the arcuate nucleus and from the medulla starting at the level of the obex to the parabrachial nucleus.
Fra-like immunoreactivity was detected using an affinity-purified rabbit polyclonal antibody (c-fos K-25; Santa Cruz Biotechnology) that recognizes Fos, Fos-B, Fra-1, and Fra-2 proteins.18 Immunohistochemistry was performed as described previously.13 Briefly, after blockade of endogenous peroxidase activity (0.3% H2O2 in 0.01 mol/L PBS for 10 minutes) and three washes in PBS, sections were incubated in PBS containing 0.3% Triton X-100, 0.02% sodium azide, and primary antibody 1:2000 for 48 hours. Next, the sections were washed three times with PBS and incubated with biotin-labeled donkey anti-rabbit secondary antisera (1:200; Jackson Laboratories) for 16 hours. The sections were washed three times with PBS and incubated for 3 hours with PBS containing 0.3% Triton X-100 and streptavidin-horseradish peroxidase (1:100; Amersham Canada). After three washes in PBS, the sections were rinsed in 0.1 mol/L acetate buffer, pH 6.0. The reaction was visualized using a glucose oxidase-DAB-nickel method. After termination of DAB reaction by washing in acetate buffer, the sections were mounted on chromium-alum–coated slides, dried, dehydrated through a graded series of alcohols and two changes of xylene, and cover-slipped for microscopic observation.
Fra-LI was quantified in the different brain areas using an image analysis system equipped with Image 1.47 software (Wayne Rasband, NIMH).13 Digitization of sampled areas (660×800 μm) was performed at 100× magnification using a CCD camera linked to a microscope. Then, the area of interest was outlined, and thresholding was performed on the digitized image to allow for exclusion of small positive profiles such as fragments of nuclei (<8 μm), glial cells, and weakly stained nuclei from the final analysis. To ensure consistency between measurements, the threshold value was chosen on the basis of the background staining. In every rat, for each area a total of two measurements of Fra-LI positive nuclei were done, each performed on a separate section. Averages from these two measurements were used for statistical analysis.
Blood Pressure Measurements
Because surgical stress may increase Fra-LI in general, and possibly to a varying extent between strains and/or diets, blood pressure measurements were performed in separate groups of young WKY (n=16) and SHR (n=16) kept on either RNa or HNa from 4 to 6 weeks of age. At the end of this period, under halothane anesthesia, polyethylene catheters were implanted into the carotid artery. After ≈24 hours of recovery baseline blood pressure was recorded using a Data Science International system (St Paul, MN).
Data are presented as mean±SEM. Factorial ANOVA was performed on the cell count for each region with strain, diet, and time as factors followed by the Newman-Keuls test were applicable.19 Individual differences between groups of rats were separated using modified t test.19
Distribution of Fra-LI Neurons
The whole brain was initially scanned for the presence of immunoreactive neurons after feeding WKY and SHR a RNa or HNa for 3, 7, or 14 days. Immunoreactive neurons were detected in the osmosensitive nuclei of the lamina terminalis including the MnPO (ventral and dorsal parts) and SFO. Immunoreactive neurons were also observed in the SON and in areas participating in cardiovascular regulation including the PVN, AHA, NTS, and CG. No Fra-LI–positive neurons were detected in the VLM, locus ceruleus, and parabrachial nucleus.
Fra-LI in Neurons of the Lamina Terminalis and Other Osmosensitive Nuclei
On RNa diet, WKY and SHR displayed few Fra-LI–positive neurons in the SFO (Fig 1⇓). HNa diet significantly increased the number of Fra-LI–positive neurons in the SFO of both strains. These increases were significantly higher in WKY than in SHR after 7 and 14 days on HNa diet. In both strains, these neurons were located exclusively at the SFO border with the third ventricle.
On RNa diet, a few scattered Fra-LI–positive neurons were present in the MnPO in both strains; the number of these neurons was higher in SHR than in WKY after 7 and 14 days (Fig 1⇑). Fra-LI expression in the MnPO of SHR was significantly higher 7 and 14 days on RNa diet than that after 3 days. In WKY, 7 and 14 days of HNa diet resulted in significant increases in Fra-LI in the MnPO over the level observed on control diet. Fra-LI in SHR was increased after 3 and 14 but not after 7 days on HNa diet, and these increases were larger than in WKY on high sodium (Fig 1⇑).
On RNa, the number of Fra-LI–positive neurons in the SON of both SHR and WKY increased with age but was significantly higher in SHR than WKY at all time points. HNa diet resulted in a significant increase in Fra-LI in both strains. At all time points, increases in Fra-LI by high sodium were more pronounced in WKY, resulting in comparable levels of Fra-LI on high sodium in the two strains (Fig 1⇑).
Fra-LI in Neurons of the PVN, AHA, NTS, and CG
On RNa, low levels of Fra-LI were observed in both parts of the PVN throughout its rostrocaudal extent. Few immunoreactive neurons were noted in both divisions of the PVN in WKY on RNa diet (Figs 2⇓ and 3⇓). In SHR on RNa, the number of Fra-LI–positive neurons in both divisions of the nucleus was significantly higher than in WKY. HNa diet increased Fra-LI in the parvocellular part of the PVN in WKY markedly after 7 days and to a minor extent after 14 days but at all time points in SHR. The number of Fra-LI neurons was significantly higher in SHR than in WKY after 3 and 14 days on HNa diet. On HNa diet in the magnocellular part of the PVN, Fra-LI initially showed a decrease in SHR, but after 7 and 14 days in both strains, Fra-LI increased compared with RNa intake (Fig 2⇓). By 14 days on HNa, almost all magnocellular PVN neurons were Fra-LI positive (Fig 3⇓).
On RNa diet in the AHA, a small number of Fra-LI–positive neurons were detected in WKY (7 and 14 days) but not in SHR (Fig 1⇑). HNa diet resulted in the disappearance of Fra-LI in WKY, whereas in SHR Fra-LI–positive neurons were now detected at all time points.
In both strains, Fra-LI was detected in a few neurons in the rostral but not in other parts of the NTS. On RNa diet, Fra-LI was significantly higher in SHR than in WKY, particularly after 7 and 14 days. On HNa intake, Fra-LI increased in both strains but more in WKY, resulting in similar expression in both strains except for 14 days on HNa when it remained higher in SHR (Fig 2⇑).
In the central gray, Fra-LI was detected in all its parts and throughout its rostro-caudal extend. On RNa intake, very few neurons were Fra-LI positive in both strains except after two weeks when clear expression was detected in SHR. On RNa, SHR showed higher levels of Fra-LI than WKY, increasing with age (Fig 2⇑). In both strains of rats HNa diet resulted in increases in Fra-LI. The extent of these increases by HNa was more marked in WKY than in SHR, resulting in similar expression in the strains on HNa.
Blood Pressure and Body Weight
At 6 weeks of age, mean blood pressure in SHR was significantly higher than in WKY. Two weeks of HNa diet significantly exacerbated hypertension in SHR but did not change blood pressure in WKY (Table⇓). WKY were consistently heavier that SHR. In both strains, consistent with previous studies,1 2 3 rats on HNa gained less weight than rats on RNa intake (Table⇓).
The present study demonstrates significant differences between normotensive WKY and hypertensive SHR in the pattern of neuronal activation accompanying both maturation and responses to increased dietary sodium intake.
The K-25 antibody recognizes all proteins of the Fos family.18 As a consequence, the observed immunoreactivity may not only represent an increase in Fra proteins but also increases in Fos or Fos-B. On the other hand, the time elapsing from the introduction of the stimulus (HNa) to the detection of immunoreactivity is most likely long enough to allow for a down-regulation of Fos because Fos typically peaks at ≈30 minutes to 1 hour, starts decreasing at 2 hours, and is essentially absent at 4 hours after introduction of the stimulus.10 With continuous stimulation, the elevated Fos levels decrease and stay at baseline values.10 11 20 Between 1 to 3 hours after the stimulus, Fra appears and can persist for up to 7 days.10 17 This evidence strongly suggest that the immunoreactivity detected in the present study should represent, mainly Fra proteins.
Changes in Fra-LI During Maturation on Regular Sodium Intake
In the SFO, Fra-LI did not differ between WKY and SHR. In contrast, in both parts of the PVN, MnPO, SON, NTS, and central gray, Fra-LI expression was significantly higher in SHR than in WKY, whereas in the AHA, immunoreactive neurons were only detected in WKY. Fra-LI increased during maturation in SHR but not in WKY in the magnocellular PVN, MnPO, NTS, and central gray. In the SON, Fra-LI increased with age in both strains but at higher activity in SHR throughout the observation period. In contrast, the parvocellular PVN and SFO showed no maturation related increases in either strains.
Inasmuch as immediate-early genes activation may be indicative of increased neuronal activity, the observation of increased Fra-LI in MnPO neurons of SHR on RNa diet is in agreement with a study by Tanaka et al,21 demonstrating higher spontaneous discharge rate of MnPO neurons projecting to the PVN in this strain in comparison with WKY.
The presence of the AP-1 binding site on the vasopressin gene establishes a functional link between an increase in Fos family proteins and increase in gene expression because the AP-1 site is the receptor binding Fos-Jun complex.22 23 Therefore, higher Fra-LI expression in the PVN and SON of SHR seems to be consistent with previous observations indicating increased metabolic activity of these neurons in SHR and other studies showing elevated plasma AVP and significant depressor responses to peripheral administration of a V1 receptor antagonist in this strain.24 25 26 27
Interestingly, in AHA neurons on RNa Fra-LI expression was observed in WKY but not in SHR. Other studies examining the release of norepinephrine and the role of the AHA in the regulation of sympathetic activity suggest that the activity of sympathoinhibitory neurons is higher in SHR than in WKY.28 The reason for this discrepancy is not clear. In the present study, no morphological markers such as α2 receptors were identified, and it is therefore possible that Fra-LI was detected in a different population of neurons.
In both SHR and WKY, Fra-LI was detected only in the rostral parts of the NTS. In SHR versus WKY, Fra-LI levels were significantly higher and increased with age. The significance of these differences is unclear. Increasing blood pressure in SHR is unlikely to play a role because the rostral NTS is not innervated by cardiovascular afferents but receives gustatory afferents (fifth and seventh cranial nerves).29
Responses to an Increase in Dietary Sodium Consumption
Areas Involved in Central Osmoregulation
The response to HNa intake differed between various neuronal populations and between strains. SFO neurons in SHR responded with significantly smaller activation than in WKY. In contrast, in the MnPO, increases in Fra-LI were higher in SHR than in WKY. In the SON, on HNa the number of Fra-LI neurons did not differ between the strains. The pattern of activation in the PVN appears to be more complex. In WKY on HNa diet, activation of the parvocellular part of the PVN clearly preceded activation of its magnocellular division. This appears not be the case in SHR, in which both divisions of the PVN were activated simultaneously 7 days after introduction of HNa chow, but subsequently only the magnocellular division remained markedly activated.
Previous studies have demonstrated that peripheral administration of hyperosmotic solutions increases Fos expression in the SFO, organum vasculosum laminae terminalis, MnPO, PVN, SON, and medullary catecholaminergic neurons.17 30 31 32 The present study indicates that a similar pattern of neuronal activation is induced in normotensive WKY by HNa intake. The biological significance of the differences in the extent and pattern of neuronal activation observed in WKY versus SHR is not clear. Higher activation of the MnPO in SHR may be consistent with our recent observation indicating that release of brain “ouabain” in this structure mediates sodium-sensitive hypertension in SHR.33 In the SFO, Fra-LI-positive neurons were exclusively located at the periphery of the SFO adjoining the third ventricle, which was also observed after peripheral administration of hypertonic saline and intracerebroventricular angiotensin II but not after hypovolemia, hemorrhage, or intravenous angiotensin II.7 34 35
In WKY, significant activation of the SFO accompanied by an intense activation of the parvocellular PVN after 7 days and subsequent activation of the magnocellular PVN after 14 days of HNa diet may represent a pattern of neuronal activation underlying sodium resistance. It has been demonstrated that central release of AVP in normotensive WKY but not in SHR exerts hypotensive action.36 Interestingly, in contrast to the magnocellular PVN, the intense activation by HNa of the parvocellular division of this nucleus did not persist beyond 7 days, and after 14 days of HNa diet, Fra-LI was almost similar to that observed after 3 days and only moderately above levels on RNa. It may suggest that the parvocellular PVN contains a pool of neurons in which the initial activation is followed by an adaptation to the stimulus.
Other Brain Areas Involved in Regulation of Cardiovascular Homeostasis
Introduction of HNa diet increased Fra-LI in the NTS of WKY. In SHR, these increases were only minimal, and on HNa, the absolute number of Fra-LI–positive neurons in the two strains showed only minor differences. Fra-LI neurons were located in the rostral part of the NTS, indicating perhaps an increased activity of the gustatory afferents. If so, in WKY, activity of these afferents was higher than in SHR. This observation may be consistent with findings indicating impairment of early events of salt taste transduction in SHR.37
In the AHA, changes in Fra-LI in response to HNa diet in WKY and SHR were reciprocal. In WKY, Fra-LI disappeared, whereas it appeared in SHR. SHR, in contrast to WKY, respond to HNa diet with a decrease in the release of norepinephrine from nerve terminals in the AHA, resulting in a decrease in the activity of sympathoinhibitory neurons in this area.3 Our study shows at first glance opposite changes. However, it is conceivable that neurons, in which changes in Fra-LI were detected, represent a different neuronal population (see also discussion above).
In the central gray, the number of Fra-LI neurons increased >25-fold in normotensive WKY on HNa diet. In SHR, these increases were comparable. Considering the uniform distribution of Fra-LI–positive neurons and complexity of cardiovascular responses evoked by central gray stimulation,38 the relevance of these changes remains unclear.
Sodium-sensitive hypertension in SHR is accompanied by an increase in sympathetic outflow,1 3 and therefore one might expect a concordant presence of Fra-LI-positive neurons in the VLM, which is a key brain structure generating tonic sympathetic activity.38 Moreover, existence of basal Fos expression in the VLM has been documented in adult normotensive WKY and in SHR.39 In the present study, Fra-LI was not detected in the VLM on RNa and HNa diets in either strain. The antibody used in the present study recognizes Fos, and therefore the reason for this discrepancy is not clear. It appears that the lack of Fra-LI in the VLM did not result from the conditions under which sections were processed because in sections in which Fra-LI was detected in the NTS, no immunoreactivity was observed in VLM. It is possible that basal expression becomes apparent with further maturation.
In the absence of assessments of, for example, function, it is not known which differences in Fra-LI between strains and diets indeed relate to the development of hypertension in SHR versus WKY or the progression of the hypertension in SHR by high sodium intake. Further studies involving, for example, lesions of specific nuclei or blockade of brain “ouabain” and angiotensin II4 may identify which nuclei are indeed involved in the pathways leading to sympathetic hyperactivity and worsening of hypertension by high sodium intake.
In conclusion, the present study demonstrates that on RNa, the development of hypertension in SHR is paralleled by larger increases in Fra-LI in the magnocellular PVN and MnPO compared with WKY. On the other hand, early intense activation of the parvocellular PVN and more intense activation of the MnPO appear to be associated with the exacerbation of hypertension in this strain by high sodium intake. In WKY, early and intense activation of the SFO and parvocellular PVN may represent a mechanism related to the prevention of an increase in blood pressure on HNa.
Selected Abbreviations and Acronyms
|AHA||=||anterior hypothalamic area|
|HNa||=||high sodium diet|
|MnPo||=||median preoptic nucleus|
|NTS||=||nucleus of the solitary tract|
|RNa||=||regular sodium diet|
|SHR||=||spontaneously hypertensive rats|
This study was supported by an operating grant for the Medical Research Council of Canada and an unrestricted grant form Apotex Inc. Dr Budzikowski is a postdoctoral fellow from the Department of Clinical and Applied Physiology, Warsaw School of Medicine, and is supported by a Research Fellowship from the Heart and Stroke Foundation of Ontario. Dr Leenen is a Career Investigator of the Heart and Stroke Foundation of Ontario. The authors would like to thank Dr Leo P. Renaud (Neurosciences, Loeb Research Institute, Ottawa Civic Hospital) for the use of facilities and constructive comments regarding the manuscript.
Reprint requests to Frans H.H. Leenen, MD, PhD, FRCP(C), Hypertension Unit, University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, Ontario, Canada K1Y 4E9.
- Received March 16, 1997.
- Revision received April 28, 1997.
- Accepted July 18, 1997.
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