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The development of two subnuclei of the nucleus tractus solitarius in spontaneously hypertensive rats
Developmental Brain Research, 12 (1984) 39--44
39
Elsevier
The Development of Two Subnuclei of the Nucleus Tractus Solitarius in Spontaneously Hypertensive Rats T. L. KRUKOFF* and T. M. SCOTT
Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfld., AI B 3V6 (Canada) (Accepted August 23rd, 1983)
Key words: spontaneously hypertensive rat - - nucleus tractus solitarius - - nucleus commissuralis nucleus medialis - - metabolism - - neuronal densities
Changes in relative metabolic requirements and neuronal densities in the nucleus commissuralis (NC) and nucleus medialis (NM) of the nucleus tractus solitarius were studied in spontaneously hypertensive rats (SHR) during development. The changes in relative metabolic requirements in the two subnuclei of SHR between 2 and 12 weeks of age were similar to those previously reported for normotensive WKY at the same ages. However, the relative metabolic activity in the NC of 2- and 4-week SHR was significantly higher than in normotensive rats. The differences in metabolic requirements of the NC could not be explained by differences in the neuronal densities of this subnucleus in young SHR and may reflect abnormalities in developmental or functional activities in the pre-hypertensive rat. Neuronal densities in the NC of 8- and 12-week SHR and in the NM of 4-, 8- and 12-week SHR were significantly higher than in WKY controls. Differences in the neuronal densities in the NC and NM of SHR may be explained by a smaller brain size characteristic of this strain, but differences in the NC of SHR suggest that the alterations may underlie or result from the cardiovascular abnormalities associated with the spontaneous hypertension of this strain. INTRODUCTION It is generally accepted that the nucleus tractus solitarius (NTS) is involved in cardiovascular function (see ref. 27 for review) and that it is the site of the first synapse of the b a r o r e c e p t o r reflexl,18,29, 32. It has been suggested that two subnuclei of the NTS, the nucleus commissuralis (NC) and nucleus medialis (NM), are of particular i m p o r t a n c e in blood pressure regulationS,6,9-11,15,17. The relative metabolic requirements of the NC and N M have recently been studied at various stages in the d e v e l o p m e n t of the rat20 Using [3H]2-deoxy-D-glucose ([3H]2-DG) aut o r a d i o g r a p h y it was shown that relative metabolic requirements in the NC and N M are not constant between 2 and 12 weeks of age and that these changes in metabolism are not related to changes in blood pressure of the animals or to changes in neuronal densities within the N C and NM. The spontaneously hypertensive rat ( S H R ) has
,been used extensively as a m o d e l of h u m a n essential hypertension. H o w e v e r , very little attention has been paid to the d e v e l o p m e n t of the CNS structures which are considered to be i m p o r t a n t in the develo p m e n t and/or maintenance of hypertension in the SHR. F o r this reason, we studied the relative metabolic requirements and neuronal densities in the N C and N M of S H R at the various stages in the develo p m e n t of hypertension and c o m p a r e d these to previous findings m a d e in normotensive W K Y rats20 in o r d e r that we might d e t e r m i n e w h e t h e r the develo p m e n t of these subnuclei is altered in the S H R during the d e v e l o p m e n t of hypertension. MATERIALS AND METHODS SH rats, bred from rats originally purchased from the Charles River Lakeview L a b o r a t o r i e s (New York), were m a i n t a i n e d by b r o t h e r - s i s t e r matings and weaned at 4 weeks of age. A n i m a l s were housed,
Correspondence at present address: T. L. Krukoff, Department of Physiology, University of Western Ontario, London, Ontario, Can-
ada N6A 5C1.
40 3 to a cage, in a 12 h light/dark schedule. They consumed water and Purina rat chow ad libitum. Blood pressure profiles of the rat colony were made by means of direct femoral artery measurements in male rats aged 4, 8 and 12 weeks which were anesthetized with 3 5 ~ 0 mg/kg body weight sodium pentobarbital. The procedure used to quantitate relative metabolic requirements in the NC and NM has been described previously x0. Briefly, male SHR at ages 2, 4, 8 and 12 weeks, 4 animals per group, were given i.p. injections of [3H]2-DG (New England Nuclear, Boston, MA) at a dosage of 200 ktCi/100 g body weight. After 45 rain the rats were anesthetized with ether and perfused through the left ventricle of the heart with 100 ml/kg of 0.1 M phosphate buffer followed by 200 ml/kg of 2% glutaraldehyde in 0.05 M phosphate buffer. The brainstems were removed and prepared for sectioning with a cryostat. Frozen sections of 6 #m were cut in the dark and prepared for autoradiography. After exposure in the dark ( l - 2 months), slides were processed according to standard autoradiographic procedures. The brainstem was studied between levels 1.0 mm caudal to the obex and 1.5 mm rostral to the obex. Borders of the NC and NM were the same as those described 20. [3H]2-DG uptake was quantified from photographs of autoradiograms or directly from autoradiograms as previously described20. Representative counts of silver grains for each NC were made from a photographic montage of one-half of that NC. Grain counts of the NM were made directly from the autoradiograms. Counts for both subnuclei were standardized against counts made from the hypoglossal nucleus (XII) of each section. The mean of the values for the uptake of [3H]2-DG into the NC or NM in one animal was calculated and used to represent the relative metabolic activity for that subnucleus in that animal. The method for calculating the neuronal densities in the NC and NM of experimental rats is identical to the methods described previously 2°. Data were expressed as 'n' neurons/3.6 x 103/lm 2. Counts were made in rats at 2, 4, 8 and 12 weeks of age (4 animals per group). In all aspects of this study, data obtained for SHR in each age group were compared to data for WKY at
corresponding ages (obtained previously 2.)) with ~t Student's t-test. Data among groups were analyzed with the Duncan Multiple Range Test. RESULTS
The body weights and mean arterial pressures (MAP) of the WKY and SHR colonies are plotted and compared in Fig. 1. There were no significant differences between the weights of the two strains at any of the ages studied. There was no significant difference between the MAP of WKY and SHR at 4 weeks. At 8 and 12 weeks, the MAP of SHR was significantly higher than those of the WKY. At 8 weeks the MAPs were 9 2 _ + 6 m m H g a n d l l l + 7mmHg for WKY and SHR, respectively. At 12 weeks, WKY A
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41 in the S H R was not significantly different from that in the WKY. The uptake of [3H]2-DG into the NM of the S H R was not significantly different from that into the NM of the W K Y at any of the ages studied. The trends in metabolic requirements that were observed at different ages in the NC were also observed in the NM.
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Fig. 2. Relative metabolic activity in the nucleus commissuralis (NC) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; ** = significant difference, P < 0.05). had a M A P of 98 + 7 m m Hg and S H R had a M A P of 124 + 6 mm Hg.
Quantification of relative metabolism [3H]2-DG uptake was quantified in the NC and NM of S H R at 2, 4, 8 and 12 weeks of age. The data are plotted in Figs. 2 and 3 along with data obtained from W K Y at the corresponding ages. Uptake of [3H]2-DG into the NC of S H R generally followed the same trends as did the [3H]2-DG uptake into the NC of WKY. That is, a significant decrease from 2 to 4 weeks was followed by a significant increase at 8 weeks and a significant decrease at 12 weeks. At 2 and 4 weeks, however, the [3H]2-DG uptake into the NC was significantly higher in the S H R compared with the WKY. At 8 and 12 weeks, uptake
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Neuronal densities were measured in the NC and NM of S H R at 2, 4, 8 and 12 weeks of age. The data are plotted in Figs. 4 and 5 along with the data obtained from W K Y at the corresponding ages. In the NC of WKY, the neuronal density decreased linearly as the animals matured so that the density of neurons at 12 weeks was significantly lower than at 2 weeks. The density of neurons in the NC of 2- and 4-week S H R was the same as that of W K Y at the corresponding ages. However, at 8 and 12 weeks of age the neuronal densities in S H R were significantly higher than in WKY. The density of neurons in the NC of S H R did not change significantly after 4 weeks of age. In WKY, as was the case for the NC, the neuronal densities in the NM decreaed as the animals matured so that neuronal density at 12 weeks was significantly lower than at 2 weeks. In 2-week S H R the density of neurons in the N M was not significantly higher than that of 2-week WKY. At 4, 8 and 12 weeks of age, however, the neuronal densities in S H R were significantly higher than in W K Y at the corresponding ages. As in W K Y the density of neurons in S H R decreased significantly as the animals matured.
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Fig. 3. Relative metabolic activity in the nucleus medialis (NM) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group).
8
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Fig. 4. Density of neurons in the nucleus commissuralis (NC) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; *** = significant difference, P < 0.005).
42 higher than those in WKY. The neuronal density in the NM of SHR decreased with development in the same way as in WKY. However, at 4, 8 and 12 weeks, the densities of neurons in SHR were significantly higher than those in WKY. Recently it was re-
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Fig. 5. Density of neurons in the nucleus medialis (NM) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; ** = significant difference, P < 0.025). DISCUSSION
Changes in metabolic activity This investigation has demonstrated that the changes in relative metabolic activity which occur in the NC and NM are similar in SHR and WKY rats. It has been suggested that the two phases of metabolic activity in the NC and NM of SHR and WKY (2 and 4 weeks versus 8 and 12 weeks) may reflect different energy requirements in the brainstem which are manifested at different developmental stages 20. The reason for the increase in energy requirements which occurred between 4 and 8 weeks of age in both strains is not clear but may be a reflection of an altered state of metabolic activity in the internal standard, XII tg. It is possible that the weaning process which occurred just after 4 weeks of age, for example, had an effect on the metabolic activity of XII between 4 and 8 weeks of age. Such a change could then be partly responsible for the apparent increase in metabolic activity within the NC and NM between 4 and 8 weeks. Neuronal densities in the N C and N M In SHR, the neuronal densities in both the NC and NM differed developmentally from those in WKY. In the case of the NC, the neuronal densities were not significantly different from those in the NC of WKY at the corresponding ages. Whereas the density of neurons in WKY continued to decrease with age, the density in the NC of SHR remained unchanged from 4 to 12 weeks of age. Thus, the neuronal densities in the NC of SHR at 8 and 12 weeks were significantly
ported that there is no significant difference in the neuronal density of the NTS between adult SHR and WKY 26. Since the latter study did not differentiate between specific subnuclei of the NTS, as was done in the present study, the data from the two studies cannot be directly compared. It is suggested that, when the neuronal densities of the entire NTS were calculated in the former study 26, the differences in neuronal densities of the NC and NM between SHR and WKY which are reported in the present study could not be observed. It has recently been shown that the brains of WKY are larger than the brains of SHR at 12 weeks 25 and at 8 months of age 2t. In the former study it was found that the SHR brain volume and brain weight were 11.8% and 10.6% below that of the WKY brain. A general decrease in the size of the SHR brain may explain the differences seen in the neuronal densities within the NM of WKY and SHR. That is, if the absolute number of neurons in this area is not different in WKY and SHR, the relative decrease in volume available for the same number of neurons in the SHR would cause the neuronal densities to be higher in these animals. The lack of a significant difference in the neuronal density of the very young animals (2 weeks of age) may imply that differences in brain size between WKY and SHR do not begin to appear until a later point in development (4 weeks of age or older). The same explanation may apply in part to the NC of WKY and SHR. The differences in brain size of the SHR may again explain the significant differences in the neuronal densities between the NC of WKY and SHR at 8 and 12 weeks of age. However, since the density of neurons in the NC of SHR did not decrease with development, as would be expected as brain size increased, other factors may be involved. There are numerous examples in the literature which demonstrate a correlation between morphometric changes in the CNS and functional activity of specific brain areas 2,3,13,2~26,28, These changes include densities of cells, somal cross-sectional areas, nucleoli numbers, and dendritic orientation. There-
43 fore, because of the importance of this area in cardiovascular regulation it is possible that the observed differences in the neuronal densities of the NCs between S H R and W K Y at 8 and 12 weeks of age may underlie or result from the cardiovascular abnormalities associated with spontaneous hypertension. General considerations In the NC, but not in the NM, the relative metabolic activity of 2- and 4-week S H R was found to be significantly higher than in the normotensive WKY. In the older animals (8 and 12 weeks) no difference was found between the relative metabolic activity in the NC (or NM) of W K Y and SHR. It is possible that the differences which were found between the N C of young S H R and W K Y were also present in the NM but were not distinguishable using the method of [3H]2-DG quantification which was used for that subnucleus. In contrast to the NC, where a larger area was studied and representative counts were made, all of the silver grains within a much smaller area of the NM were counted. Because of the smaller area of the NM and the resulting smaller counts, final results were more greatly affected by variations in counts. Thus the standard deviations in the NM counts were greater than in the NC counts and real differences in the metabolic activity of the NM between young W K Y and S H R may have been hidden. To date, only one other study has addressed the question of differences in glucose utilization in S H R brains compared to W K Y controls TM. As in the present study, Hiyashi and N a k a m u r a 14 found that 2-DG
uptake into the NC of 4-week S H R was significantly higher than in W K Y ; in the more rostral NTS, no difference was found. The same findings were made in 20-week rats. The latter finding suggests a possible discrepancy with the results obtained for the older animals in the present study. Because the cell densities in the NC of 2- and 4week W K Y and S H R were found to be the same, the different energy requirements in the NC of the two strains cannot be explained by greater energy demands of a higher density of cells. The differences in relative metabolic activity in the NC between S H R and W K Y at 2 and 4 weeks of age may reflect increased developmental metabolic requirements or increased rates of functional activities (e.g. transmission, maintenance, etc.) in young SHR. Since the relative metabolic activity in the NC of S H R is not elevated compared to W K Y at 8 and 12 weeks of age, it is possible that this increased metabolic activity in the young S H R is associated with an altered central regulation of the cardiovascular system which has been hypothesized to occur during the development but not the maintenance of hypertension 12. It cannot be concluded from the present study that the trigger for the alteration in central regulation of the cardiovascular system in S H R is located in the NC. However, because of the extensive connection between the NC and other CNS structures which are involved in the regulation of blood pressure4, 7, 8,16,22,27,29--31,33, it can be concluded that, at the very least, the altered activity caused by this so-called trigger is expressed within the NC.
REFERENCES 1 Beckstead, R. M. and Norgren, R., An autoradiographic examination of the central distribution of the trigeminal, glossopharyngeal, and vagal nerves in the monkey, J. comp. Neurol., 184 (1979) 455-472. 2 Bereiter, D. A. and Jeanrenaud, B., Altered neuroanatomical organization in the central nervous system of the genetically obese (ob/ob) mouse, Brain Res., 165 (1979) 249-260. 3 Bereiter, D. A. and Jeanrenaud, B., Altered dendritic orientation of bypothalamic neurons from genetically obese (ob/ob) mice, Brain Res., 202 (1980) 201-206. 4 Chalmers, J. P., Blessing, W. W., West, M. J., Howe, P. R. C., Costa, M. and Furness, J. B., Importance of new catecholamine pathways in control of blood pressure, Clin. Exp. Hypertension, 3 (1981) 393-416. 5 Chiba, T. and Doba, N., The synaptic structure of catecholaminergic varicosities in the dorsomedial portion of the
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nucleus tractus solitarius of the cat: possible roles in the regulation of cardiovascular reflexes, Brain Res., 84 (1975) 31--46. Chiba, T. and Doba, N., Catecholaminergic axo-axonic synapses in the nucleus tractus solitarius (pars commissuralis) of the cat: possible relation to presynaptic regulation of baroreceptor reflexes, Brain Res., 102 (1976) 255-265. Ciriello, J. and Calaresu, F. R., Autoradiographic study of ascending projections from cardiovascular sites in the nucleus tractus solitarii in the cat, Brain Res., 186 (1980) 488-453. Ciriello, J. and Calaresu, F. R., Monosynaptic pathway from cardiovascular neurons in the nucleus tractus solitarii to the paraventricular nucleus in the cat, Brain Res., 193 (1980) 529--533. Czachurski, J., Lackner, K. J., Ockert, D. and Seller, H., Localization of neurons with baroreceptor input in the medial solitary nucleus by means of intracellular application of HRP in the cat, Neurosci. Len., 28 (1982) 133-137.
44 10 De Jong, W., Zandberg, P., Palkovits, M. and Bohus, B., Acute and chronic hypertension after lesions and transections in the rat brain stem. In W. De Jong, A. P. Provoost and A. P. Shapiro (Eds.), Hypertension and Brain MechanLs'ms, Progr. Brain Res., Vol. 47, Elsevier, Amsterdam, 1977, pp. 189-197. 11 Doba, N. and Reis, D. J., Acute fulminating neurogenic hypertension produced by brainstem lesions in the cat, Circular. Res., 32 (1976) 584-593. 12 Folkow, B., Physiological aspects of primary hypertension, Physiol. Rev., 62 (t982) 347-504. 13 Hatton, G. I. and Waiters, J. K., Induced multiple nucleoli, nucleolar margination, and cell size changes in supraoptic neurons during dehydration and rehydration in the rat, Brain Res., 59 (1973) 137-154. 14 Hayashi, T. and Nakamura, K., Cerebral neuronal activity in spontaneously hypertensive rats as demonstrated by the 14C-deoxyglucose method, Naunyn-Schmiedeberg's Arch. Pharmacol., 316 (1981) 331-339. 15 Healy, D. P., Jew, J. Y., Black, A. D. and Williams, T., Bradycardia following injection of 6-hydroxydopamine into the intermediate portion of nucleus tractus solitarius medialis, Brain Res., 206 (1981) 415-420. 16 Hopkins, D. A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 17 Kalia, M. and Mesulam, M.-M., Brainstem projections of sensory and motor components of the vagus complex in the cat. I. The cervical vagus and nodose ganglion, J. comp. Neurol., 193 (1980) 435-465. 18 Kerr, F. W. L., Facial, vagal, and glossopharyngeal nerves in the cat. Afferent connections, Arch. Neurol., 6 (1962) 164-281. 19 Krukoff, T. L., A Study of the Metabolic Development in the Nucleus Tractus Solitarius in Normotensive and Hypertensive Rats, Ph.D. Thesis, Memorial Univ. of Newfoundland, St. John's, Canada, 1982. 20 Krukoff, T. L. and Scott, T. M., The postnatal metabolic development of the nucleus commissuralis and nucleus medialis of the nucleus tractus solitarius, Develop. Brain Res., in press. 21 Lehr, R. P., Browning, R. A. and Hurley Myers, J., Gross morphological brain differences between Wistar-Kyoto and spontaneously hypertensive rats, Clin. Exp. Hypertension. 2 (1980) 123-127. 22 Miura, M. and Reis, D. J., Termination and secondary pro-
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26 27
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jections of carotid sinus nerve in the cat brain stem, J. Physiol. (Lond.), 217 (1969) 142-153. Mohishita, H., Kawamoto. M., Masuda, Y.. Higuchi, R.. Nagamachi, N., Mitani, H., Ozasa, T. and Adachi, H., Quantitative histological changes in the hypothalamic nuclei in the prepuberal, puberal and postpuberal female rat, Brain Res.. 76 (1974) 41-47. Morris, J. R. and Dyball. R. E. J., A quantitative study of the ultrastructural changes in the hypothalamo-neurohypophyseal system during and after experimentally induced hypersecretion, Cell Tiss. Res., 36 (1974) 415-416. Nelson, D. O. and Boulant, J. A., Altered CNS neuroanatomical organization of spontaneously hypertensive (SHR) rats, Brain Res., 226 (1981) 119-130. Nelson, D. O. and Boulant, J. A.. Altered brain stem structure of SHRs, Brain Res., 261 (1983) 145-150. Palkovits, M. and Zaborsky, L., Neuroanatomy of central cardiovascular control. Nucleus tractus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc. In W. De Jong, A. P. Provoost and A. P. Shapiro (Eds.), Hypertension and Brain Mechanisms, Progr. Brain Res., Vol. 47, Elsevier, Amsterdam. 1977, pp. 9-34. Pfaff, D. W., Morphological changes in the brains of adult male rats after neonatal castration, J. Endocrin., 36 (1966) 415-416. Rhoton, A. L., O'Leary, J. L. and Ferfuson, J. P., The trigeminal, facial, vagal, and glossopharyngeal nerves in the monkey, Arch. Neurol., 14 (1966) 530-540. Ricardo, J. A. and Koh, E. T., Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Brain Res., 153 (1978) 1-26. Ross, C. A., Ruggiero, D. A. and Reis, D. J., Afferent projections to the cardiovascular portion of the nucleus tractus solitarius in the rat, Brain Res., 223 (1981) 402-408. Saper, C. B., Loewy, A. D., Swanson, L. W. and Cowan, W. M., Direct hypothalamo-autonomic connections, Brain Res., 117 (1976) 305-312. Torvik, A., Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures. An experimental study in the cat, J. comp. Neurol., 106 (1956) 51-141. Warren Cottle. M. K. and Calaresu, F. R., Projections from the nucleus and tractus solitarius m the cat, J. comp. Neurol., 161 (1975) 143,-158.
39
Elsevier
The Development of Two Subnuclei of the Nucleus Tractus Solitarius in Spontaneously Hypertensive Rats T. L. KRUKOFF* and T. M. SCOTT
Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfld., AI B 3V6 (Canada) (Accepted August 23rd, 1983)
Key words: spontaneously hypertensive rat - - nucleus tractus solitarius - - nucleus commissuralis nucleus medialis - - metabolism - - neuronal densities
Changes in relative metabolic requirements and neuronal densities in the nucleus commissuralis (NC) and nucleus medialis (NM) of the nucleus tractus solitarius were studied in spontaneously hypertensive rats (SHR) during development. The changes in relative metabolic requirements in the two subnuclei of SHR between 2 and 12 weeks of age were similar to those previously reported for normotensive WKY at the same ages. However, the relative metabolic activity in the NC of 2- and 4-week SHR was significantly higher than in normotensive rats. The differences in metabolic requirements of the NC could not be explained by differences in the neuronal densities of this subnucleus in young SHR and may reflect abnormalities in developmental or functional activities in the pre-hypertensive rat. Neuronal densities in the NC of 8- and 12-week SHR and in the NM of 4-, 8- and 12-week SHR were significantly higher than in WKY controls. Differences in the neuronal densities in the NC and NM of SHR may be explained by a smaller brain size characteristic of this strain, but differences in the NC of SHR suggest that the alterations may underlie or result from the cardiovascular abnormalities associated with the spontaneous hypertension of this strain. INTRODUCTION It is generally accepted that the nucleus tractus solitarius (NTS) is involved in cardiovascular function (see ref. 27 for review) and that it is the site of the first synapse of the b a r o r e c e p t o r reflexl,18,29, 32. It has been suggested that two subnuclei of the NTS, the nucleus commissuralis (NC) and nucleus medialis (NM), are of particular i m p o r t a n c e in blood pressure regulationS,6,9-11,15,17. The relative metabolic requirements of the NC and N M have recently been studied at various stages in the d e v e l o p m e n t of the rat20 Using [3H]2-deoxy-D-glucose ([3H]2-DG) aut o r a d i o g r a p h y it was shown that relative metabolic requirements in the NC and N M are not constant between 2 and 12 weeks of age and that these changes in metabolism are not related to changes in blood pressure of the animals or to changes in neuronal densities within the N C and NM. The spontaneously hypertensive rat ( S H R ) has
,been used extensively as a m o d e l of h u m a n essential hypertension. H o w e v e r , very little attention has been paid to the d e v e l o p m e n t of the CNS structures which are considered to be i m p o r t a n t in the develo p m e n t and/or maintenance of hypertension in the SHR. F o r this reason, we studied the relative metabolic requirements and neuronal densities in the N C and N M of S H R at the various stages in the develo p m e n t of hypertension and c o m p a r e d these to previous findings m a d e in normotensive W K Y rats20 in o r d e r that we might d e t e r m i n e w h e t h e r the develo p m e n t of these subnuclei is altered in the S H R during the d e v e l o p m e n t of hypertension. MATERIALS AND METHODS SH rats, bred from rats originally purchased from the Charles River Lakeview L a b o r a t o r i e s (New York), were m a i n t a i n e d by b r o t h e r - s i s t e r matings and weaned at 4 weeks of age. A n i m a l s were housed,
Correspondence at present address: T. L. Krukoff, Department of Physiology, University of Western Ontario, London, Ontario, Can-
ada N6A 5C1.
40 3 to a cage, in a 12 h light/dark schedule. They consumed water and Purina rat chow ad libitum. Blood pressure profiles of the rat colony were made by means of direct femoral artery measurements in male rats aged 4, 8 and 12 weeks which were anesthetized with 3 5 ~ 0 mg/kg body weight sodium pentobarbital. The procedure used to quantitate relative metabolic requirements in the NC and NM has been described previously x0. Briefly, male SHR at ages 2, 4, 8 and 12 weeks, 4 animals per group, were given i.p. injections of [3H]2-DG (New England Nuclear, Boston, MA) at a dosage of 200 ktCi/100 g body weight. After 45 rain the rats were anesthetized with ether and perfused through the left ventricle of the heart with 100 ml/kg of 0.1 M phosphate buffer followed by 200 ml/kg of 2% glutaraldehyde in 0.05 M phosphate buffer. The brainstems were removed and prepared for sectioning with a cryostat. Frozen sections of 6 #m were cut in the dark and prepared for autoradiography. After exposure in the dark ( l - 2 months), slides were processed according to standard autoradiographic procedures. The brainstem was studied between levels 1.0 mm caudal to the obex and 1.5 mm rostral to the obex. Borders of the NC and NM were the same as those described 20. [3H]2-DG uptake was quantified from photographs of autoradiograms or directly from autoradiograms as previously described20. Representative counts of silver grains for each NC were made from a photographic montage of one-half of that NC. Grain counts of the NM were made directly from the autoradiograms. Counts for both subnuclei were standardized against counts made from the hypoglossal nucleus (XII) of each section. The mean of the values for the uptake of [3H]2-DG into the NC or NM in one animal was calculated and used to represent the relative metabolic activity for that subnucleus in that animal. The method for calculating the neuronal densities in the NC and NM of experimental rats is identical to the methods described previously 2°. Data were expressed as 'n' neurons/3.6 x 103/lm 2. Counts were made in rats at 2, 4, 8 and 12 weeks of age (4 animals per group). In all aspects of this study, data obtained for SHR in each age group were compared to data for WKY at
corresponding ages (obtained previously 2.)) with ~t Student's t-test. Data among groups were analyzed with the Duncan Multiple Range Test. RESULTS
The body weights and mean arterial pressures (MAP) of the WKY and SHR colonies are plotted and compared in Fig. 1. There were no significant differences between the weights of the two strains at any of the ages studied. There was no significant difference between the MAP of WKY and SHR at 4 weeks. At 8 and 12 weeks, the MAP of SHR was significantly higher than those of the WKY. At 8 weeks the MAPs were 9 2 _ + 6 m m H g a n d l l l + 7mmHg for WKY and SHR, respectively. At 12 weeks, WKY A
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AGE (wk) Fig. 1. A'. mean arterial pressures (MAP) (mm Hg) in WistarKyoto rats (white bars) and spontaneously hypertensive rats (black bars) at 4, 8 and 12 weeks of age (n = 8 in each group; ** = significant difference, P < 0.05). B: mean weights (gm) of Wistar-Kyoto rats (white bars) and spontaneously hypertensive rats (black bars) at 2, 4, 8 and 12 weeks of age (n = 12 in each group).
41 in the S H R was not significantly different from that in the WKY. The uptake of [3H]2-DG into the NM of the S H R was not significantly different from that into the NM of the W K Y at any of the ages studied. The trends in metabolic requirements that were observed at different ages in the NC were also observed in the NM.
U
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Fig. 2. Relative metabolic activity in the nucleus commissuralis (NC) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; ** = significant difference, P < 0.05). had a M A P of 98 + 7 m m Hg and S H R had a M A P of 124 + 6 mm Hg.
Quantification of relative metabolism [3H]2-DG uptake was quantified in the NC and NM of S H R at 2, 4, 8 and 12 weeks of age. The data are plotted in Figs. 2 and 3 along with data obtained from W K Y at the corresponding ages. Uptake of [3H]2-DG into the NC of S H R generally followed the same trends as did the [3H]2-DG uptake into the NC of WKY. That is, a significant decrease from 2 to 4 weeks was followed by a significant increase at 8 weeks and a significant decrease at 12 weeks. At 2 and 4 weeks, however, the [3H]2-DG uptake into the NC was significantly higher in the S H R compared with the WKY. At 8 and 12 weeks, uptake
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Neuronal densities were measured in the NC and NM of S H R at 2, 4, 8 and 12 weeks of age. The data are plotted in Figs. 4 and 5 along with the data obtained from W K Y at the corresponding ages. In the NC of WKY, the neuronal density decreased linearly as the animals matured so that the density of neurons at 12 weeks was significantly lower than at 2 weeks. The density of neurons in the NC of 2- and 4-week S H R was the same as that of W K Y at the corresponding ages. However, at 8 and 12 weeks of age the neuronal densities in S H R were significantly higher than in WKY. The density of neurons in the NC of S H R did not change significantly after 4 weeks of age. In WKY, as was the case for the NC, the neuronal densities in the NM decreaed as the animals matured so that neuronal density at 12 weeks was significantly lower than at 2 weeks. In 2-week S H R the density of neurons in the N M was not significantly higher than that of 2-week WKY. At 4, 8 and 12 weeks of age, however, the neuronal densities in S H R were significantly higher than in W K Y at the corresponding ages. As in W K Y the density of neurons in S H R decreased significantly as the animals matured.
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Neuronal densities
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12
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Fig. 3. Relative metabolic activity in the nucleus medialis (NM) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group).
8
12
AGE (wk)
Fig. 4. Density of neurons in the nucleus commissuralis (NC) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; *** = significant difference, P < 0.005).
42 higher than those in WKY. The neuronal density in the NM of SHR decreased with development in the same way as in WKY. However, at 4, 8 and 12 weeks, the densities of neurons in SHR were significantly higher than those in WKY. Recently it was re-
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,R
4
12
AGE (wk)
Fig. 5. Density of neurons in the nucleus medialis (NM) of WKY (white bars) and SHR (black bars) at 2, 4, 8 and 12 weeks of age (n = 4 in each group; ** = significant difference, P < 0.025). DISCUSSION
Changes in metabolic activity This investigation has demonstrated that the changes in relative metabolic activity which occur in the NC and NM are similar in SHR and WKY rats. It has been suggested that the two phases of metabolic activity in the NC and NM of SHR and WKY (2 and 4 weeks versus 8 and 12 weeks) may reflect different energy requirements in the brainstem which are manifested at different developmental stages 20. The reason for the increase in energy requirements which occurred between 4 and 8 weeks of age in both strains is not clear but may be a reflection of an altered state of metabolic activity in the internal standard, XII tg. It is possible that the weaning process which occurred just after 4 weeks of age, for example, had an effect on the metabolic activity of XII between 4 and 8 weeks of age. Such a change could then be partly responsible for the apparent increase in metabolic activity within the NC and NM between 4 and 8 weeks. Neuronal densities in the N C and N M In SHR, the neuronal densities in both the NC and NM differed developmentally from those in WKY. In the case of the NC, the neuronal densities were not significantly different from those in the NC of WKY at the corresponding ages. Whereas the density of neurons in WKY continued to decrease with age, the density in the NC of SHR remained unchanged from 4 to 12 weeks of age. Thus, the neuronal densities in the NC of SHR at 8 and 12 weeks were significantly
ported that there is no significant difference in the neuronal density of the NTS between adult SHR and WKY 26. Since the latter study did not differentiate between specific subnuclei of the NTS, as was done in the present study, the data from the two studies cannot be directly compared. It is suggested that, when the neuronal densities of the entire NTS were calculated in the former study 26, the differences in neuronal densities of the NC and NM between SHR and WKY which are reported in the present study could not be observed. It has recently been shown that the brains of WKY are larger than the brains of SHR at 12 weeks 25 and at 8 months of age 2t. In the former study it was found that the SHR brain volume and brain weight were 11.8% and 10.6% below that of the WKY brain. A general decrease in the size of the SHR brain may explain the differences seen in the neuronal densities within the NM of WKY and SHR. That is, if the absolute number of neurons in this area is not different in WKY and SHR, the relative decrease in volume available for the same number of neurons in the SHR would cause the neuronal densities to be higher in these animals. The lack of a significant difference in the neuronal density of the very young animals (2 weeks of age) may imply that differences in brain size between WKY and SHR do not begin to appear until a later point in development (4 weeks of age or older). The same explanation may apply in part to the NC of WKY and SHR. The differences in brain size of the SHR may again explain the significant differences in the neuronal densities between the NC of WKY and SHR at 8 and 12 weeks of age. However, since the density of neurons in the NC of SHR did not decrease with development, as would be expected as brain size increased, other factors may be involved. There are numerous examples in the literature which demonstrate a correlation between morphometric changes in the CNS and functional activity of specific brain areas 2,3,13,2~26,28, These changes include densities of cells, somal cross-sectional areas, nucleoli numbers, and dendritic orientation. There-
43 fore, because of the importance of this area in cardiovascular regulation it is possible that the observed differences in the neuronal densities of the NCs between S H R and W K Y at 8 and 12 weeks of age may underlie or result from the cardiovascular abnormalities associated with spontaneous hypertension. General considerations In the NC, but not in the NM, the relative metabolic activity of 2- and 4-week S H R was found to be significantly higher than in the normotensive WKY. In the older animals (8 and 12 weeks) no difference was found between the relative metabolic activity in the NC (or NM) of W K Y and SHR. It is possible that the differences which were found between the N C of young S H R and W K Y were also present in the NM but were not distinguishable using the method of [3H]2-DG quantification which was used for that subnucleus. In contrast to the NC, where a larger area was studied and representative counts were made, all of the silver grains within a much smaller area of the NM were counted. Because of the smaller area of the NM and the resulting smaller counts, final results were more greatly affected by variations in counts. Thus the standard deviations in the NM counts were greater than in the NC counts and real differences in the metabolic activity of the NM between young W K Y and S H R may have been hidden. To date, only one other study has addressed the question of differences in glucose utilization in S H R brains compared to W K Y controls TM. As in the present study, Hiyashi and N a k a m u r a 14 found that 2-DG
uptake into the NC of 4-week S H R was significantly higher than in W K Y ; in the more rostral NTS, no difference was found. The same findings were made in 20-week rats. The latter finding suggests a possible discrepancy with the results obtained for the older animals in the present study. Because the cell densities in the NC of 2- and 4week W K Y and S H R were found to be the same, the different energy requirements in the NC of the two strains cannot be explained by greater energy demands of a higher density of cells. The differences in relative metabolic activity in the NC between S H R and W K Y at 2 and 4 weeks of age may reflect increased developmental metabolic requirements or increased rates of functional activities (e.g. transmission, maintenance, etc.) in young SHR. Since the relative metabolic activity in the NC of S H R is not elevated compared to W K Y at 8 and 12 weeks of age, it is possible that this increased metabolic activity in the young S H R is associated with an altered central regulation of the cardiovascular system which has been hypothesized to occur during the development but not the maintenance of hypertension 12. It cannot be concluded from the present study that the trigger for the alteration in central regulation of the cardiovascular system in S H R is located in the NC. However, because of the extensive connection between the NC and other CNS structures which are involved in the regulation of blood pressure4, 7, 8,16,22,27,29--31,33, it can be concluded that, at the very least, the altered activity caused by this so-called trigger is expressed within the NC.
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