The human hypothalamo-neurohypophyseal system in relation to development, aging and Alzheimer's disease

D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 16

The human hypothalamo-neurohypophysealsystem in relation to development, aging and Alzheimer’s disease Elmer Goudsmit, Angela Neijmeijer-Leloux and Dick F. Swaab Netherlands Institute for Brain Research, 1105 AZ Amsterdam, The Netherlands

Introduction The hypothalamo-neurohypophyseal system (HNS) is one of the best studied neuroendocrine systems both in experimental animals and in humans. The system consists of hypothalamic magnocellular neurosecretory neurons located in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN) (Fig. 1). These neurons project to the posterior lobe of the pituitary. In addition to the SON and PVN, clusters of magnocellular neurons belonging to the HNS are found in the hypothalamic gray in between these two nuclei. These clusters are referred to as accessory neurosecretory nuclei (Dierickx and Vandesande, 1977). Immunocytochemical studies have shown.that the peptides vasopressin and oxytocin are present in mutually exclusive sets of neurons (Vandesande and Dierickx, 1975). Transport of neurosecretory material from magnocellular neurons to the neural lobe of the pituitary and the release of this material into the blood stream had already been demonstrated by Bargmann in 1949 (Bargmann, 1949). The magnocellularvasopressin and oxytocin neurons in the SON are considered to project exclusively to the neural lobe of the pituitary (Sofroniew, 1985), although central projections originating in this nucleus were also reported in the rat (Alonso et al., 1986). In contrast to the SON, the PVN not only contains magnocellular vasopressin and oxytocin cells, but also parvocellular neurons which are im-

munoreactive for these peptides. In the rat these parvocellular neurons are situated in distinct parvocellular subdivisions of the nucleus which have been shown to project to central brain regions where the peptides probably act as neurotransmitters or neuromodulators (Buijs, 1983; Sofroniew, 1985). An additional group of parvocellular vasopressin neurons was shown to project to the median eminence in the rat (see Sofroniew, 1985). In these cells vasopressin is colocalized with corticotropinreleasing hormone (CRH) (Whitnall et al., 1985). The human PVN cannot readily be divided into parvocellular and magnocellular subdivisions as in the rat. The nucleus rather consists of a mixture of cells of different sizes (Fig. 2). The vasopressin and oxytocin cell populations in this nucleus are more or less overlapping (Dierickx and Vandesande, 1977). No data are available on the relative distribution of centrally and peripherally projecting vasopressin and oxytocin cells within this nucleus in the human brain. Colocalization of vasopressin with CRH was recently observed in small neurons in the human PVN (Raadsheer et al., 1991) and might be used as a marker for neurons which project to the median eminence. In the peripheral circulation vasopressin and oxytocin act as hormones on several target organs. Vasopressin plays an important role in the regulation of blood pressure and plasma osmolality; it induces water retention and natriuresis in the kidneys and is a potent vasoconstrictor. In addition, effects

The neuronal circuitry involved in the regulation of vasopressin and oxytocin release has not yet been completely disclosed. Projections from a number of brain-stem nuclei, notably the nucleus of the solitary tract, appear to play an important role (Cunningham and Sawchenko, 1988). In addition, evidence has been presented for hormone-, volumeand osmo-receptive elements in periventricular organs like the subfornical organ which project either directly or indirectly to the SON and PVN (Swanson, 1987). Magnocellular neurons themselves also appear to be sensitive to changes in osmolality (Mason, 1980; Oliet and Bourque, 1991). Changes in HNS function during aging: early studies In the older literature, a functional impairment of the rodent HNS was proposed to occur in senescence. This view was based on evidence that vasopressin levels in plasma and urine were reduced in aged animals in combination with a diminished antidiuretic response following osmotic stimulation (Friedman et al., 1956; Turkington and Everitt, 1976).

Fig. 1. Human hypothalamus, frontal section stained immunocytochemicallyfor vasopressin. Vasopressin cells are stained in the supraoptic, paraventricular and suprachiasmatic nuclei. Abbreviations: OC, optic chiasm; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; 111, third ventricle. Bar, 1 mm.

on carbohydrate metabolism in the liver, on platelet aggregation and on blood coagulation have been described (for a recent review, see Cunningham and Sawchenko, 1991). Oxytocin is well known for its role in labor and lactation (Swaab and Boer, 1979). In males oxytocin peaks during ejaculation, acting on the smooth musculature of the vas deferens. Recent work inexperimental animals has shown the involvement of oxytocin in fat and carbohydrate metabolism (for a review, see Cunningham and Sawchenko, 1991).

In the sixties, an impairment in retention in a passive avoidance paradigm was observed in rats following hypophysectomy (De Wied, 1965). This impairment could be restored by both peripheral and central administration of vasopressin and vasopressin analogues which lacked antidiuretic and vasopressor activity (De Wied, 1983). These results were interpreted as an enhancement of memory processes by vasopressin which was proposed to act as a hormone on the brain (for a review, see De Wied, 1983). This hypothesis was of course of great interest in view of the decline in posterior pituitary function which had been observed in aged rats. A similar decrease in HNS function appeared to occur in human aging. Legros (1975) reported a decrease in plasma neurophysin levels in men after the age of 50. These and other studies gave rise to the hypothesis that an age-related decrease in vasopressin secretion might, at least in part, account for cognitive impairments in the elderly (for a review, see De

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Fig. 2. Frontal section through the human paraventricular nucleus stained immunocytochemicallyfor vasopressin. Cells of various sizes are scattered throughout the nucleus. P V N , Paraventricular nucleus; 111, third ventricle. Bar, 250 pm.

Wied and Van Ree, 1982). Subsequently, intranasally administered vasopressin was reported to enhance memory in middle-aged men (Legros et al., 1978) and numerous trials with vasopressin administration in elderly and demented subjects were started with the aim of improving cognitive impairments in these conditions.

Evidence of increased HNS activity during aging

The putative relationship between age-related memory decline and impaired HNS function, which became topical in the early 1980s, also gave rise to the question whether degenerative alterations might take place in the magnocellular neurons in the SON

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and PVN during aging. This question prompted several groups to study the aging HNS from a morphological point of view. In the rat, no changes in cell numbers were observed in the SON and PVN during aging (Hsu and Peng, 1978; Peng and Hsu, 1982; Flood and Coleman, 1983). Absence of cell loss during aging was also observed in the human SON and PVN (Hofman et al., 1988, 1990; Goudsmit et al., 1990a). When the morphology of the vasopressin and oxytocin cells in human postmortem tissue was studied in more detail using immunocytochemical and morphometric techniques, some unexpected results were obtained. Fliers et al. (1985) observed an increase in the size of the vasopressin cells in the human SON and PVN in subjects older than 80 years. In contrast, the oxytocin cells in these nuclei showed no such change. In order to find out whether this increased vasopressin cell size was due to hypertrophy or to degenerative changes (e.g., accumulation of age pigments), Hoogendijk et al. (1985) determined the size of the nucleoli in the vasopressin and oxytocin cells in the same material. An increase in nucleolar size was observed in the vasopressin cells in the SON and PVN in senescent subjects, but not in the oxytocin cells, suggesting an increase in peptide synthesis in vasopressin cells, but not in oxytocin cells in senescence. Studies in Wistar rats also showed morphological signs of increased neurosecretory activity in the SON and PVN with aging (Fliers and Swaab, 1983). The results of these morphological studies clearly disagreed with the earlier data on decreased plasma levels of vasopressin and neurophysin in aged humans and rodents (see above). However, when Legros extended his studies on plasma neurophysin levels in aging humans to men aged 60 - 100 years, he observed a secondary rise in plasma neurophysin levels after 70 years of age (Legros et al., 1980). Subsequently, other investigators also presented evidence for a gradual increase in plasma vasopressin levels in aging humans and rodents (Frolkis et al., 1982; Kirkland et al., 1984; 0 s et al., 1985). In addition, an increase in vasopressin secretion upon osmotic stimulation was observed in elderly subjects

as compared to young controls (Helderman et al., 1978; Robertson and Rowe, 1980; Philips et al., 1984). In the early studies, which showed declining vasopressin levels in aging rodents, the peptide had been extracted with phenol and measured in a bioassay in which the antidiuretic effect of the extracts was determined in young male animals. This might explain part of the discrepancies with more recent studies which make use of radioimmunoassays. However, the literature on age-related changes in rodent HNS function has remained controversial. The reported data change from marked decreases in vasopressin synthesis, secretion and excretion in senescence to marked increases. Strain differences between the animals used in the various studies might offer an explanation for these discrepancies. In addition, the data are confounded by the use of animals which fail to meet the 50% survival criterion for senescence (cf., Zurcher et al., 1982). When studies with animals which were not old enough according to this criterion are disregarded, decreases in HNS activity and responsiveness were only found in 30 months-old Fisher 344 and Sprague-Dawley rats (Sladek et al., 1981; Zbuzek and Wu, 1982; Zbuzek et al., 1983,1987). Increases in HNS activity were reported in 33 - 34-months-old Brown-Norway rats (Goudsmit et al., 1988), in 32-months-old Wistar rats (Fliers and Swaab, 1983) and in 20 - 23months-old Long-Evans rats (Miller, 1985, 1987). These data suggest the presence of strain differences in HNS aging between Fisher 344 and SpragueDawley rats on the one hand and Brown-Norway, Wistar and Long-Evans rats on the other. All five strains were found to develop an increase in urine production and water intake during aging (Bengele et al., 1981; Beck and Yu, 1982; Miller, 1985; Corman and Michel, 1987; Goudsmit et al., 1988; Phelps et al., 1989). In Fisher 344 and SpragueDawley rats this decline in renal concentrating ability goes together with pronounced histopathological changes in the kidneys (Coleman et al., 1977; Gray et al., 1982). In contrast, histopathological changes in the kidneys of senescent Brown-Norway and Wistar rats are only moderate (Burek, 1978; Gray et

24 1

al., 1982; Ravid et al., 1987). Yet, these two strains show a marked age-related loss in the number and affinity of renal binding sites for vasopressin (Swaab et al., 1986; Ravid et al., 1987; Herzberg et al., 1989). In view of the absence of major renal histopathology these changes might be considered to be due to normal aging rather than to pathology. A loss in renal vasopressin receptors might be expected to result in a decreased renal sensitivity to vasopressin. This appears indeed to be the case since cyclic-AMP production in response to vasopressin was found to be diminished in renal medullary cells of senescent mice as compared to tissue of younger animals (Goddard et al., 1984). Hence, the decreased urinary concentrating ability of aged rodents might be due to a decrease in renal sensitivity to vasopressin. The observation that aged rats drink more than young animals and produce more vasopressin suggests a feedback action mediated by osmo- and/or volume-receptors in order to compensate for the polyuria which occurs in these animals (Fig. 3). Impaired urinary concentrating ability and decreased renal responsiveness to vasopressin also occur in human aging (Miller and Shock, 1953; Rowe et al., 1976). Therefore, an increase in vasopressin production in the human SON and PVN in senes24h Urine volume

Urine osmolality

5

5

1000

$

24h WateP intake

800

3

4

600

0"

400

z *

200

1

4

n

g -- 3

E

3

8.' E

2 1

young old

Fig. 3. Urine osmolality, urine volume and water intake in young (4 months) and old (34 months) Brown-Norway rats. Urine osmolality was significantly reduced in aged animals, while urine volume and water intake were significantly increased. Since vasopressin excretion was also increased in the aged animals, these results indicate the development of a moderate renal diabetes insipidus with aging and a subsequent stimulation of drinking behavior and vasopressin release. Bars represent means k S . E . M . ; * , P < O.O5;**,P < 0.01.(FromGoudsmitetal., 1988, with permission from the publisher.)

"- 30 40

I Ctr c60

Ctr >60

Alz >60

Fig. 4. Vasopressin (light bars) and oxytocin (dark bars) cell numbers in the human PVN in young (< 60 years) and old (> 60 years) controls (Ctr) and in Alzheimer patients (Alz). Vasopressin cell numbers increase with age in controls. Alzheimer patients fail to show a similar increase. Oxytocin cell numbers remain unaltered during aging and in Alzheimer's disease. *, Different from young controls and from Alzheimer patients ( P < 0.05). Redrawn using data from Wierda et al. (1991) and Van der Woude et al. (1992).

cence might also be due to renal changes. Recently, we observed a gradual increase in the number of neurons which were immunoreactive for vasopressin in the human PVN with aging (Fig. 4)(Van der Woude et al., 1992). This finding is in line with the morphological and physiological evidence of an increase in vasopressin synthesis in this nucleus during aging (see above). The number of oxytocin-immunoreactive neurons in the PVN was found to remain constant during aging (Fig. 4) (Wierda et al., 1991) which is in line with the absence of morphological signs of activation in these cells in senescence (Fliers et al., 1985; Hoogendijk et al., 1985). The evidence for an activation of the vasopressin cells in the human HNS in senescence might be corroborated by the application of mRNA in situ hybridization to human hypothalamic tissue obtained post-mortem (see Mengod et al., this volume). Increased levels of vasopressin mRNA were demonstrated in patients with clinical evidence of antemortem dehydration (Rivkees et al., 1989). In this small study, the highest levels of vasopressin mRNA were found in the oldest subjects, suggesting that vasopressin synthesis is indeed increased in senescence.

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Alzheimer’s disease

Alzheimer’s disease is the most common form of dementia in the elderly. The pre-senile form of the disease is much less common but tends to be more severe (Hansen et al., 1988). The disease is characterized by a progressive impairment of cognitive function and the presence of characteristic neuropathological changes observed at autopsy (McKhann et al., 1984). Neurochemical research into the nature of this disease has revealed disturbances in numerous transmitter systems, including cholinergic, monoaminergic, peptidergic and amino acid transmitter systems which exceed the changes seen during normal aging (for a review, see Goudsmit et al., 1990b). Decreased levels of vasopressin have been reported in plasma, cerebrospinal fluid and brain tissue in Alzheimer’s disease, suggesting an involvement of both centrally and peripherally projecting vasopressin cells in this disease (Sarensen et al., 1983, 1985; Sundquist et al., 1983; Mazurek et al., 1986a,b; Raskindet al., 1986). Ofcourse, thesefindings were of interest in view of the presumed role of declining vasopressin levels in age-related memory decline (see above). In a recent study (Van der Woude et al., 1992), we found that the number of vasopressin-immunoreactive cells in the PVN of Alzheimer patients was 37% lower than in agematched controls, although values did not drop below values of young controls (Fig. 4). This finding suggests a failure of the vasopressin cells in Alzheimer’s disease to respond to the increased demand for vasopressin in senescence (see above) and appears to support the data on reduced vasopressin levels in Alzheimer patients. In the same study, the nuclei of the vasopressin cells in the PVN of Alzheimer patients were found to be enlarged, suggesting hypertrophy of the unaffected cells, or, alternatively, a selective loss of staining in small vasopressin neurons. Whether a disturbance of vasopressin synthesis in Alzheimer’s disease has any clinical relevance seems doubtful. The results of trials with vasopressin administration to elderly and demented subjects have been rather disappointing (e.g., Wolters et al., 1990)

and at present there remains little evidence that vasopressin administration is an effective treatment for age-related memory disorders, although the peptide apparently enhances certain aspects of cognitive function in healthy volunteers (for a review, see Jolles, 1986). Suprachiasmatic nucleus

In close vicinity to the SON and PVN lies another hypothalamic nucleus which contains vasopressinimmunoreactive cells, viz. the suprachiasmatic nucleus (SCN) (Dierickx and Vandesande, 1977) (Fig. 1). Neurons in the rat SCN have been shown to project exclusively to central brain regions (Watts and Swanson, 1987; Watts et al., 1987). Animal experiments have shown that the SCN is the principal circadian pacemaker of the mammalian brain (Rusak and Zucker, 1979). The rodent SCN receives information about the environmental light-dark cycle from the retina via the retinohypothalamic tract (Moore, 1973) and indirectly via projections of neuropeptide Y-containing neurons in the intergeniculate leaflet (Card and Moore, 1989). Data on the organization and connections of the primate SCN give roughly the same picture, although the presence of a geniculate-hypothalamic projection in the human brain is uncertain (for a review see Moore, this volume). During aging, the amplitude of circadian rhythmicity declines in both rodents and humans (for reviews, see Van Goo1 and Mirmiran, 1986, and Mirmiran et al., this volume). This impairment is even more pronounced in Alzheimer patients (Witting et al., 1990). Cell counts in the SCN of both rats and humans revealed a decrease in the number of vasopressin-immunoreactive neurons in this nucleus during aging (Fig. 5 ) (Swaab et al., 1985, 1987; Roozendaal et al., 1987). In Alzheimer patients this cell loss was even more pronounced, amounting to a 75% reduction in the number of vasopressin cells in this nucleus (Swaab et al., 1985, 1987). Since the integrity of the SCN has been shown to be directly reIated to the expression of its pacemaker qualities (Pickard and Turek, 1983), these degenerative

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input via osmo- and/or volume-receptors (see above). In view of the different aging patterns of the SCN on the one hand and the SON and PVN on the other, the stability of these latter nuclei might be related to the fact that they contain neurons which remain highly active throughout the aging process (Swaab, 1991). Evidence that neuronal activation might also prevent or postpone cell death in other neuronal systems (paraphrased as ”use it or lose it”) was presented in a recent review (Swaab, 1991).

SUPRACHIASMATIC NUCLEUS

Fetal development 20

60 Age (years )

40

00

100

Fig. 5 . Linear regression between vasopressin cell numbers in the human SCN and age. A statistically significant decrease was observed in controls after 60 years of age (P < 0.05). Triangles represent males and circles represent females. Values of Alzheimer patients (closed symbols) are delineated by a minimum convex polygon and were reduced as compared to agematched controls (P< 0.01). Redrawn using data from Swaab et al. (1985, 1987).

changes might form the anatomical basis for disturbances in circadian rhythmicity in Alzheimer’s disease. Degenerative changes have also been reported in the retina and optic tract in Alzheimer’s disease (Hinton et al., 1986; Katz et al., 1989). In addition, Alzheimer patients are generaly exposed to lower light intensities than controls (Campbell et al., 1988). Since the input to the SCN via the retinohypothalamic tract is essential for the entrainment of circadian rhythmicity (Johnson et al., 1988), these changes might cause a decrease in the input which is required for proper functioning of the SCN in Alzheimer’s disease. Hence, the degenerative changes which occur in the SCN in this disease might be secondary to changes in regulatory input to this nucleus. The changes in the SCN during aging and in Alzheimer’s disease are quite distinct from the aging pattern seen in the SON and PVN. The SON and PVN show a striking absence of cell loss during normal aging. The vasopressin cells in these nuclei are probably activated due to an increased stimulatory

The stability of the SON and PVN is not limited to the aging process, but appears to encompass the entire life span, including the fetal period. Cell counts in premature and mature fetuses revealed that adult numbers of vasopressin and oxytocin cells are already present from 26 weeks of gestation onwards (Fig. 6) (Neijmeijer-Leloux et al., in preparation). However, the maturation of the nuclei appeared to continue well beyond this timepoint, since the volume of the vasopressin and oxytocin cell populations was found to increase rapidly from 26 weeks gestation towards term. At term, mean volumes were still only about half of the values observed in the adult (e.g., 1.79 f 0.14 mm3 for the volume of the oxytocin cell population in the PVN in mature infants as compared to 4.04 f 0.21 mm3 in adults),

I

5 t 40

7

30

1

Premature

Mature

Adults

Fig. 6 . Vasopressin (light bars) and oxytocin (dark bars) cell numbers in the PVN of premature (26 - 37 weeks) and mature (37-42 weeks) fetuses and in adults. Adult numbers were already present around 26 weeks gestation (preliminary results from Neijmeijer-Leloux et al., in preparation; for details see text).

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suggesting maturation of this cell group does not stop at birth, but continues post-natally. Data on vasopressin and oxytocin concentration and secretion during the fetal period are scant, but tend to support a gradual maturation of the HNS. Vasopressin and oxytocin can be detected in the human hypothalamus from 11 weeks gestation onwards and concentrations were shown to increase gradually between 11 and 28 weeks gestational age (Burford and Robinson, 1982). Vasopressin levels in the umbilical blood were shown to correlate positively with gestation length (Oosterbaan and Swaab, 1989). From these data it might be inferred that vasopressin and oxytocin neurons are already present and functioning during early fetal life, but continuetomaturethroughoutthefetalperiodandprobably also post-natally. Determination of the nuclear diameter of the vasopressin and oxytocin cells in the SON and PVN during fetal development revealed a rapid increase in nuclear diameter of oxytocin cells in the PVN between 26 and 42 weeks gestation (Neijmeijer-Leloux et al., in preparation), suggesting an increase in neurosecretory activity during this period (cf., Palkovits and Fischer, 1968). The oxytocin cells in the SON showed a similar trend. The vasopressin cells in these nuclei failed to show consistent changes in nuclear diameter during this period. An activation of oxytocin synthesis and release towards term would be in line with a role of oxytocin produced by the fetus in the onset and progression of labor (Swaab and Boer, 1979). Summary and conclusions

The research reviewed in the present paper indicates that vasopressin and oxytocin cells in the human HNS constitute an extremely stable population of neurons throughout the human life span. Increases in the activity of these cells, which are probably related to maturation of the system were observed during fetal development and probably extend well beyond term. During senescence an increase in the activity of the vasopressin cells in the human HNS was observed which is probably a compensation for age-related changes in kidney function. These data

do not support a role of declining vasopressin secretion in age-related memory decline. Although there is some evidence for an impairment of vasopressin synthesis and release in Alzheimer patients, vasopressin cell numbers in Alzheimer’s disease do not fall below values observed in young controls. Furthermore, peripheral administration of vasopressin or vasopressin analogues to AD patients have not yielded consistent results.

Acknowledgements

The secretarial help of Ms. T. Eikelboom in preparing the manuscript is gratefully acknowledged. Expert technical assistance was provided by Mr. B. Fisser. We thank Mr. H. Stoffels and Mr. G. Van der Meulen for preparing the illustrations. Human brain tissue was obtained from the Netherlands Brainbank (Coordinater: Dr. R. Ravid). This work was supported by the Sandoz Foundation for Gerontological Research, The Innovation Fund of the Royal Netherlands Academy of Sciences and the fund ‘‘Beoefening Wetenschap” from the Institute for Obstetrics and Gynecology, St. Radboud ziekenhuis, Nijmegen. References Alonso, G . , Szafarczyk, A. and Assenmacher, I. (1986) Radioautographic evidence that axons from the area of supraoptic nuclei in the rat project to extrahypothalamic brain regions. Neurosci. Lett., 66: 251 - 256. Bargmann, W. (1949) Ueber die neurosekretorische Verknupfung von Hypothalamus und Neurohypophyse. Z . Zellforsch. Mikrosk. Anat., 34: 610-634. Beck, N. and Yu, B.P. (1982) Effect of aging on urinary concentrating mechanism and vasopressin-dependent CAMPin rats. Am. 1.Physiol., 243: F121 -F125. Bengele, H.H., Mathias, R.S., Perkins, J.H. and Alexander, E.A. (1981) Urinary concentrating defect in the aged rat. Am. J. Physiol., 240: F147-Fl50. Buijs, R.M. (1983) Vasopressin and oxytocin - their role in neurotransmission. Pharmacol. Ther., 22: 127 - 141. Burek, J.D. (1978) Pathology of Aging Rats, CRC Press, Boca Raton, FL. Burford, G.D. and Robinson, I.C.A.F. (1982) Oxytocin, vasopressin and neurophysins in the hypothalamo-neurohypophyseal system of the human fetus. J. Endocrinol., 95: 403 - 408.

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Discussion M. Mirmiran: You have made an important point by saying that the differences in stability between certain hypothalamic nuclei (such as the supraoptic and paraventricular vs. the suprachiasmatic nucleus) are based on differential effects of the input (no change for the SON/PVN and a decrease for the SCN). Do you think that the changes found in the development of the SON/PVN in premature infants can be the result of dehydration or changes in the osmolality in these infants? E. Goudsmit: Our data show a gradual increase in the volumes

248 of the vasopressin and oxytocin cell populations in the SON and PVN during fetal development. At term the volumes are still only half of the volumes observed in adults. Therefore, it is likely that these volume changes are related to a gradual process of maturation which extends well into post-natal life. As far as I know, there is no evidence for sustained hyperosmolahty during fetal and early post-natal life. The differences in nuclear diameters between premature and mature infants are probably not due to changes in plasma osmolality since these changes occurred only in oxytocin cells and not in vasopressin cells. W.A. Scherbaum: Osmotic stimulation of vasopressin secretion is regulated minute by minute. Most patients during the last hours before death will have a certain degree of dehydration so that I would expect that vasopressin and its mRNA are increased in all your hypothalami. Therefore, I would suggest that you collect data on plasma and urine osmolality in these individuals

before death in order to correlate your mRNA values with the hydration state. E. Goudsmit: I wonder whether dehydration really is a general phenomenon indying subjects. Fluid and electrolyte equilibrium are usually carefully controlled in critically ill patients. Moreover, vasopressin mRNA levels appear to rise more in terms of days than in terms of hours following dehydration (Zingg el al., 1986). Therefore, I expect that elevated levels will only be observed in cases of chronic osmotic stimulation.

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