Patterns of Neuronal Activation During Development of Sodium Sensitive Hypertension in SHR

Immunohistochemistry

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.

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Figure 1.

Number of Fra-LI–positive neurons per section in the SFO (−1.4 mm from the bregma), MnPO (−0.4 mm from the bregma), SON (−1.8 mm from the bregma), and AHA (−1.8 mm from the bregma) of SHR and WKY on RNa and HNa. Data are presented as mean±SEM (n=4 per group). ^aP<.05 vs time-matched control on RNa diet within the strain. ∗*P<.05 vs WKY on similar diet at the same time point.

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⇓).

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Figure 2.

Number of Fra-LI–positive neurons per section in the PVN (−2.12 mm from the bregma), rostral part of the NTS (−11.6 mm from the bregma), and central gray (−7.64 mm from the bregma; note different scale) of SHR and WKY on RNa and HNa. Data are presented as mean±SEM (n=4 per group). ^aP<.05 vs time-matched control on RNa diet within the strain. *P<.05 vs WKY on similar diet at the same time point.

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Figure 3.

Typical distribution of Fra-LI immunoreactive neurons in the PVN (−2.12 mm from the bregma) of WKY and SHR on RNa or HNa for 14 days. Scale bar, 100 μm.

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⇓).

Methodological Considerations

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

Study Limitations

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.