Alterations of local cerebral glucose utilization during chronic dehydration in rats

Brain Research, 330 (1985) 329-336 Elsevier

329

BRE 10629

Alterations of Local Cerebral Glucose Utilization During Chronic Dehydration in Rats PAUL M. GROSS 1, MASSAKO KADEKARO 1, LOUIS SOKOLOFF1, HENRY H. HOLCOMB 2 and JUAN M. SAAVEDRA3

1Laboratory of Cerebral Metabolism*, 2Biological Psychiatry Branch and 3Laboratory of Clinical Science, National Institute of Mental Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, MD 20205 (U.S.A.) (Accepted July 5th, 1984)

Key words: conscious rats - - water deprivation - - saline ingestion - - thirst - - 2-[14C]deoxyglucose- subfornical organ - - pituitary neural lobe

The quantitative autoradiographic deoxyglucose method was used to study changes in local cerebral glucose utilization in conscious dehydrated rats. Animals were either given saline to drink or were deprived of water for 5 days. Saline ingestion did not alter the rates of glucose metabolism in any brain region when compared to the rates of glucose metabolism in animals which had free access to water. Glucose utilization was increased by 140%, however, in the pituitary neural lobe. Water deprivation produced both increases and decreases in glucose metabolism, depending on the particular structure. In 20 of 44 brain structures analyzed, there were significant decreases from -18 to -34% in glucose utilization. Four forebrain structures, the subfornical organ, septal triangular nucleus, and hypothalamic paraventricular and supraoptic nuclei, had increases in glucose utilization of 30-73%. The rate of glucose utilization in the pituitary neural lobe was increased by 367% in water-deprived rats. The results demonstrate that metabolic activity is stimulated in some, but not all, of the structures participating in fluid regulation during an intense thirst challenge. Many brain regions have depressed metabolism in chronic severe dehydration. INTRODUCTION The sensation of thirst and the corresponding behavioral and h o r m o n a l responses to sustain fluid balance are k n o w n to be mediated through a complex, integrated circuit of cerebral structures8,28. The stimuli that trigger activation of this circuit include hyperosmolarityZ.24, baroreceptor input28,39 and the hormone, angiotensin II, which is formed from either renal or brain renin during volume contraction8,25,28, 35. The actions of these factors are presumably initiated by stimulation of receptive zones primarily in the forebrain, including such structures as the preoptic area, the septum, specific nuclei in the hypothalamus, and the subfornical organ 4,16,21,33,34. Substantive evidence has highlighted the importance and anatomical connections of two circumventricular organs, the subfornical organ and the o r g a n u m vasculosum of the lamina terminalis, in mediating responses to thirst stimuli17,18,20,25,26. F u r t h e r m o r e , the princi-

pal hormones responding to dehydration, vasopressin and angiotensin II, have cells of origin and fiber tracts in the brain5,9A2,14,27,38. These findings provide an anatomical and functional basis for the present studies of local cerebral glucose utilization in thirsty rats. The quantitative autoradiographic deoxyglucose technique has been used extensively to map functional alterations in neural activity in n u m e r o u s physiological models 37. Its application in the present experiments allowed simultaneous determinations of metabolic activity in regions of the brain that have been implicated in the regulation of thirst, drinking behavior, and fluid balance. MATERIALS AND METHODS Adult male S p r a g u e - D a w l e y rats weighing 260-400 g were used. Catheters were inserted into a femoral vein and artery during light halothane anes-

* Address for reprints: Laboratory of Cerebral Metabolism, Building 36, Room 1A-05 NIMH, Bethesda, MD 20205, U.S.A. Correspondence: P. M. Gross. Present address: Department of Neurological Surgery, Health Sciences Center 12T-080, State University of New York at Stony Brook, Stony Brook, NY 11794-8122, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

330 thesia. The animals were immobilized by means of a loose-fitting plaster cast covering the abdomen and hindquarters. The experiments were begun at least 2 h after the termination of surgery and anesthesia. The condition of each animal was assessed before and during the experimental procedure by measurements of arterial blood pressure, hematocrit, blood gases and pH, and plasma osmolality and glucose concentration. Rectal temperature was measured continuously. Three groups of rats were studied: (1) 8 rats had free access to food and water until the time of surgery; (2) 7 rats had free access to food and were offered 2% saline for 5 days; and (3) 8 rats had free access to food but were deprived of fluids for 5 days. The theory and application of the deoxyglucose method for measuring local cerebral glucose utilization have been described previously in detaiP 6. Briefly, the period of measurement was initiated by the intravenous injection of a pulse of 2-deoxy-D-[1Aac]glucose (125 pCi/kg) in a volume of 0.5 ml saline. Several samples of arterial blood, approximately 70 pl each, were obtained during the first minute after the intravenous injection. Additional samples were obtained at prescribed times over the following 44 min to define the entire history of arterial plasma [14C]deoxyglucose and glucose concentration in each animal. The brain was removed and frozen in 2-methylbutane cooled to -55 °C. The pituitary gland was mounted in frozen embedding matrix in a small foil container. Coronal sections of brain and horizontal sections of pituitary gland were cut in a cryostat at -20 °C. Each section was 20 p m in thickness. The sections were thaw-mounted on coverslips at 70 °C and were placed in cassettes for autoradiography. Calibrated [14C]methylmethacrylate standards in a range of 48-1804 nCi/g were autoradiographed with the tissue sections. A computer-based image-processing system was used to analyze the amount of radioactivity represented in the autoradiographs10. Together with the values for arterial radioactivity and glucose concentration for each rat, and the rate and lumped constants used for conscious rats 36, the computations permitted determination of cerebral glucose utilization from the operational equation of the method 36. Because the rate and lumped constants have not

been directly determined for the subfornical organ and pituitary neural lobe, those used for cerebral gray matter were applied. The anatomical definition of structures analyzed in this report was confirmed by referring to anatomical atlases~5, 23 and to thionin stains of sections that were adjacent to, or the same as, those used to generate the autoradiographs. For bilateral structures, at least 10 measurements of optical density were made in one animal; an average value was then calculated to represent the individual region. A minimum of 6 readings was made for midline structures. Differences among the three treatments were evaluated for significance at the 0.05 level by a oneway analysis of variance in a randomized design. The Bonferroni t-test was used to discern differences between treatments. RESULTS

Physiological status The dehydration procedures affected several of the physiological measurements in the rats. Treatment for 5 days with saline drinking was associated with a 5% loss in body weight whereas the rats deprived of water for 5 days lost 22% of their body weight. The body weights on the day of the experiments were 365 + 10,310 + 13, and 262 + 8 g (mean + S.E.M.), respectively, for the control, saline, and water-deprived groups. Arterial blood pressure and pH were significantly lower, and arterial hematocrit, osmolality and plasma glucose were higher, in water-deprived rats compared to either the saline or control group (Table I). Local cerebral glucose utilization Saline ingestion produced no significant effect on glucose metabolism in any brain region (Table II). An increase of 140% in the rate of glucose utilization was found, however, in the pituitary neural lobe of rats that had drunk saline (Table III; Fig. 1). In rats that had been deprived of water for 5 days, there were both decreases and increases in local glucose metabolism. Generally, the tendency was for metabolism to be depressed as there were decreases in glucose utilization of -18 to - 3 4 % in several brain regions (Table II). Four brain structures demonstrated increases in the rate of glucose metabolism.

331 The subfornical organ, septal triangular nucleus, and hypothalamic paraventricular and supraoptic nuclei had elevated rates of metabolism (+30 to +73%, Table III, Figs. 2, 3). Additionally, glucose utilization in the pituitary neural lobe was increased by 367% (Table III, Fig. 1). DISCUSSION

The principal finding of this study was that the two conditions of dehydration, water deprivation and salt loading, affected local glucose utilization in the brain differently. Chronic salt ingestion, a model for intracellular dehydration, did not change glucose utilization in any of the brain structures examined. Chronic water deprivation, which constitutes a model of compounded thirst stimuli resulting from intracellular and extracellular dehydration, produced both increases and decreases in local cerebral glucose utilization. Increases in metabolic activity were found in 4 circumscribed forebrain structures: the subfornical organ, septal triangular nucleus, and the hypothalamic paraventricular and supraoptic nuclei. All these regions have anatomical connections with each other and with other structures known to participate in body fluid regulation18.20,38,41. In contrast, a depression of glucose metabolism occurred in many other structures throughout the brain. Depletion of body fluids produces peripheral stimuli that act in concert as feedback signals at target areas in the brain7,8,28,39. Hypertonicity of plasma and cerebrospinal fluid, low- and high-pressure baro-

receptors, and the peripheral and central renin-angiotensin systems are known to participate in stimulation of cardiovascular, hormonal, and behavioral responses from brain receptor sites. The preoptic area, septum and circumventricular organs mediate responses both to osmotic stimuli and to angiotensin I14,8,21,24,25,35. The subfornical organ specifically has high rates of glucose utilization in rat models with increased circulating levels of angiotensin 1111,13. Furthermore, baroreceptor input influences hypothalamic magnocellular neurons and, consequently, the release of vasopressin from the pituitary neural 1obe28,39, 41. T h u s , multiple forms of input probably converge at the paraventricular and supraoptic nuclei during dehydration. These two regions receive afferent projections from several forebrain and brainstem structures that would be under neural and/ or humoral stimulation in dehydrated animals, such as the subfornical organlS, 20, organum vasculosum6, septum 41, and medullary centers participating in cardiovascular regulation 41. Arterial hypotension produces stimuli that provoke increases in glucose utilization in the paraventricular and supraoptic nuclei29,30. Thus, inputs to these two nuclear regions from baroreceptors and from angiotensin and osmotic receptors during dehydration are probably important determinants of the increases in glucose metabolism that we observed. In water-deprived rats in the present study, we found decreases in glucose metabolism in the medial preoptic area and lateral hypothalamus, both of which putatively contain cells that participate in the

TABLE I

Physiological status of rats immediately prior to commencement of deoxyglucose experiments Values are m e a n + S.E.M.

Arterial blood pressure ( m m Hg) Rectal temperature ( ° C ) Arterial blood gases and p H PaO2 ( m m Hg) paCO2 ( m m Hg) pH Hematocrit (%) Plasma osmolality ( m O s m / k g ) Plasma glucose (mg/ml)

Ad libitum water

5 Days 2 % saline

5 Days water deprivation

(n = 8)

(n = 7)

(n = 8)

132 + 3 37.0 + 0.3

131 + 3 37.0 + 0.2

119 + 5* 36.4 + 0.3

89 35 7.45 49 298 1.77

81+3 37+1 7.45 + 0.01 52 + 1 314 __+6 1.43 + 0.06*

90+2 34+1 7.40 + 0.01",** 63 + 2",** 364 + 10",** 2.66 + 0.27*,**

+ + + + + +

3 2 0.01 1 4 0.14

* Different from ad libitum water group; P < 0.05. ** Different from 2% saline group; P < 0.05.

332 T A B L E II

Structures displaying decreases or no change in glucose utilization (l~rnol/lO0 g/min) during chronic dehydration Values are mean + S.E.M.

Structures Hindbrain Nucleus of solitary tract Dorsal motor nucleus of vagus Nucleus cuneatus Nucleus ambiguus Reticular formation Medial raph6 Locus coeruleus Substantia grisea (lat.) Cerebellum Cortex Nuclei Midbrain Substantia nigra (pars reticulata) Forebrain Cortex visual auditory sensorimotor anterior cingulate olfactory Hippocampus CA 1 molecular layer dentate gyrus Caudate nucleus Globus pallidus Amygdala Lateral habenula Thalamic nuclei parafascicular ventrolateral anterior Hypothalamic areas and nuclei suprachiasmatic retrochiasmatic periventricular lateral ventromedial arcuate mamillary Medial forebrain bundle Medial preoptic area Nucleus medianus Bed nucleus of stria terminalis Septum lateral nuclei medial nucleus Genu of corpus callosum

A d libitum water (n = 8) 72 69 67 65 66 90 82 77

+ 6 + 7 + 7 _+ 5 + 5 _+ 5 + 7 _+ 5

5 Days 2% saline (n = 7) 65 67 66 68 65 92 79 77

+ 4 + 6 + 3 _+ 6 + 4 + 7 _+ 3 + 6

5 Days water deprivation % Change from ad (n = 8) libitum water group*** 52 62 44 48 45 73 64 59

+ 3* + 2 + 3",** + 4*,** + 4*,** _+ 5 + 4* + 4*.**

59 + 4 101 _+ 9

63 + 3 98 + 2

45 + 3*,** 77 + 6**

59 _+ 3

57 + 3

51 + 2

-22 -23 -24

+ 7 + 8 + 6 _+ 5 _+ 6

119 137 105 118 101

_+ 8 _+ 12 _+ 8 _+ 7 + 10

62 77 69 103 58 50 107

+ 3 + 5 + 4 _+ 6 + 3 _+ 2 + 3

66 78 65 105 55 46 106

+ 4 _+ 5 + 4 _+ 7 + 4 + 2 + 5

52_+ 3** 61 + 2*,** 56 + 1" 75 _+ 5".** 45 + 3* 41 + 2* 86 + 4*,**

-21 -19 -27 -22 -18 -21

93 _+ 5 84 _+ 6 126 + 7

99 + 6 82 _+ 5 119 + 6

75 + 6** 64 _+ 4* 96 _+ 5*,**

-24 -24

65 54 60 67 52 48 112 58 63 80 68

66 49 61 69 50 42 117 57 54 76 63

67 43 52 52 45 49 95 65 46 80 56

66 + 4 84 __+4 26 + 1

* Different from ad libitum water group; P < 0.05. ** Different from 2% saline group; P < 0.05. *** Where statistically different.

+ 6 + 4 + 5 + 4 + 3 _+ 2 + 8 + 1 _+ 4 + 6 + 4

64 _+ 3 84 + 6 27 + 2

+ 7*,** + 7 _+ 6 + 6*,** + 5**

-34 -26 -32

115 140 105 113 87

_+ 5 + 3 _+ 4 _+ 3 _+ 2 + 4 + 6 + 2 _+ 2 + 4 + 4

89 118 87 87 69

-28

+ 4 + 2* + 3 + 4*.** + 3 _+ 4 _+ 6 _+ 5 + 3* + 7 _+ 3

53 + 4 71 +_ 6 23 _+ 3

-23

-23

-20 -22

-27

333

Fig. l. Autoradiographic deoxyglucose images of pituitary glands from rats that had free access to water (A), had ingested 2% saline for 5 days (B), and had been deprived of fluids for 5 days (C). The increased optimal density of the neural lobes (NL, arrows) in B and C reflect increased rates of glucose utilization. Water deprivation caused a 367% increase in glucose utilization in the neural lobe. regulation of water balance4A6,21,26. It is unclear what factors account for the depression of m e t a b o l i s m in these and o t h e r brain structures that had r e d u c e d rates of glucose utilization during water deprivation. One purpose of this study was to extend a previous qualitative analysis from this laboratory31 by a quantitative application of the deoxyglucose m e t h o d so that any changes in local cerebral glucose m e t a b o lism would be m o r e easily discerned. In doing so, and also by studying a m o r e complicated m o d e l of dehydration, water deprivation, we could c o m p a r e quantitatively the effects on local cerebral m e t a b o lism of two d e h y d r a t i o n conditions of different nature and magnitude. The authors of a n o t h e r deoxyglucose study f o r m u l a t e d ratios of optical density to represent metabolic activity in structures of thirst regulation in w a t e r - d e p r i v e d rats 40. Chronic salt ingestion p r o d u c e d m o d e r a t e systemic hypertonicity and no change in arterial b l o o d pres-

sure. Because extracellular volume is e x p a n d e d in this model, circulating angiotensin II would not be expected to have a role in thirst mechanisms. It is likely, however, that salt-loading causes both excitatory and inhibitory input to converge on mechanisms for vasopressin secretion. H y p e r t o n i c i t y increases the rate of firing of paraventricular and supraoptic neurons~. Osmotic stimuli p r o m o t e increases in plasma vasopressin42,45,46, whereas h y p e r v o l e m i a inhibits vasopressin release 32. Such opposing stimuli may have competing effects on cellular metabolic activity, a p h e n o m e n o n that could partly explain why there is no effect of salt-loading on local cerebral glucose utilization, even in the paraventricular and supraoptic nuclei where vasopressin is synthesized. Anatomically, the final point of brain integration to e l a b o r a t e vasopressin for p e r i p h e r a l distribution is at the level of the pituitary neural lobe, a structure rich in nerve endings and axons of magnocellular ori-

TABLE III Structures displaying increases in glucose utilization (l~mol/lO0 g/min) during chronic dehydration

Values are mean + S.E.M. Structures

A d libitum water (n = 8)

Subfornical organ Septal triangular nucleus Hypothalamus Paraventricular nuclei Supraoptic nuclei Pituitary neural lobe

47 + 5 49 + 2

53 + 5 57 + 5

81 _+4*,** 85 _+6"~**

+72 +73

60 + 2 66 + 4 42 + 7

58 + 3 67 + 5 101 + 8*

78 _+6*.** 88 _+6*.** 196 + 19",**

+30 +33 +367

* Different from ad libitum water group; P < 0.05. ** Different from 2% saline group; P < 0.05.

5 Days 2% saline (n = 7)

5 Days water deprivation (n = 8)

% Change from ad libitum water group

334

8

Fig. 2. Thionin-stained sections showing the following locations (arrows). A: subfornical organ (SFO). B: septal triangular nucleus (STN). C: hypothalamic paraventricular nuclei (PVN). D: hypothalamic supraoptic nuclei (SON).

gin22. Our findings indicate that the compounded stimuli of chronic water deprivation are synergistic at the neural lobe because the rate of its glucose utilization was considerably higher than in animals having the simpler stress of salt-loading. Two recent reports have suggested that the pituitary neural lobe does not oxidize glucose, but rather selectively metabolizes free fatty acids, to support its functional activity43.44. These findings likely pertain only to unstimulated conditions. The present results clearly demonstrate marked increases in glucose utilization in the neural lobe when its functional activity is stimulated during saline ingestion and water deprivation. We have considered whether selective increases in glucose metabolism arise from stimulation of the axonal terminals of afferent inputs or from somal activity. In the saline-loaded rats, no increases in glucose

metabolism were found in the cell bodies of vasopressin neurons which have increased rates of firing during osmotic stimulation 1. Previous reports from this laboratory19,31 proposed that it is the surface-to-volume ratio and axonal pumping of sodium ions that explain why the neural lobe has increased rates of glucose utilization during chronic salt ingestion when the paraventricular and supraoptic nuclei do not. Furthermore, synaptic activity may partly account for increases in neuronal metabolism 3. Thus, it is possible that the increased glucose metabolism of the subfornical organ, septal triangular nucleus, and paraventricular and supraoptic nuclei reflects projected stimulation from other neural substrates for thirst. The present experiments demonstrate that chronic severe dehydration depresses metabolic activity in much of the brain, but, in localized areas participat-

335

Fig. 3. Top: from left to right are enlarged thionin-stained sections (A-D) showing the subfornical organ, septal triangular nucleus, hypothalamic paraventricular nuclei, and hypothalamic supraoptic nuclei. Middle: corresponding deoxyglucose images (E-H) from water-sated control animals. Bottom: corresponding deoxyglucose images (I-L) from rats deprived of water for 5 days. The darker optical densities of these structures correspond to their increased rates of glucose utilization.

ing in fluid regulation, there are increases in glucose utilization. Important information in future studies will be to identify in more detail chemical factors and

ACKNOWLEDGEMENTS

neural inputs that determine local levels of cerebral metabolism during chronic dehydration.

D. Brown for assistance. P . M . G . was a Fellow of the C a n a d i a n Heart Foundation.

REFERENCES

7 Epstein, A. N., The physiology of thirst, Canad. J. Physiol. Pharmacol., 54 (1976) 639-649. 8 Fitzsimons, J. T., The Physiology of Thirst and Sodium Appetite, Cambridge University Press, Cambridge, 1979. 9 Ganten, D., Fuxe, K., Phillips, M. I., Mann, J. F. E. and Ganten, U., The brain isorenin-angiotensinsystem: biochemistry, localization, and possible role in drinking and blood pressure regulation. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Vol. 5, Raven, New York, 1978, pp. 61-99. 10 Goochee, C., Rasband, W. and Sokoloff, L., Computerized densitometry and color coding of [14C]deoxyglucose autoradiographs, Ann. Neurol., 7 (1980) 359-370. 11 Gross, P. M., Kadekaro, M., Andrews, D. W., Sokoloff, L. and Saavedra, J. M., Selective metabolic stimulation of the subfornical organ and pituitary neural lobe by peripheral angiotensin II, Peptides, in press. 12 Harding, J. W., Stone, L. P. and Wright, J. W., The distribution of angiotensin II binding sites in rodent brain, Brain Research, 205 (1981) 265-274. 13 Kadekaro, M., Gross, P. M., Sokoloff, L., Holcomb, H. H. and Saavedra, J. M., Elevated glucose utilization in subfornical organ and pituitary neural lobe of the Brattleboro rat, Brain Research, 275 (1983) 189-193.

1 Akaishi, T., Negoro, H. and Kobayasi, S., Responses of paraventricular and supraoptic units to angiotensin II, Sar lIlea-angiotensin II and hypertonic NaC1 administered into the cerebral ventricle, Brain Research, 188 (1980) 499-511. 2 Andersson, B., Regulation of water intake, Physiol. Rev., 58 (1978) 582-603. 3 Auker, C. R., Meszler, R. M. and Carpenter, D. O., Apparent discrepancy between single-unit activity and [14C]deoxyglucose labeling in optic tectum of the rattlesnake, J. Neurophysiol., 49 (1983) 1504-1516. 4 Buggy, J., Hoffman, W. E., Phillips, M. I., Fisher, A. E. and Johnson, A. K., Osmosensitivity of rat third ventricle and interactions with angiotensin, Amer. J. Physiol., 236 (1979) R75-R82. 5 Buijs, R. M., Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat, Cell Tiss. Res., 192 (1978) 423-435. 6 Camacho, A. and Phillips, M. I., Horseradish peroxidase study in rat of the neural connections of the organum vasculosum of the lamina terminalis, Neurosci. Lett., 25 (1981) 201-204.

We thank B. Gabriel, E. Lewis, R. Bower, and J.

336 14 Kilcoyne, M. M., Hoffman, D. L. and Zimmerman, E. A., Immunocytochemical localization of angiotensin II and vasopressin in rat hypothalamus: evidence for production in the same neuron, Clin. Sci., 59 (1980) 57s-60s. 15 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 16 Kucharczyk, J., Assaf, S. Y. and Mogenson, G. J., Differential effects of brain lesions on thirst induced by the administration of angiotensin If to the preoptic region, subfornical organ and anterior third ventricle, Brain Research, 108 (1976) 327-337. 17 Lind, R. W. and Johnson, A. K., Subfornical organ - - median preoptic connections and drinking and pressor responses to angiotensin II, J. Neurosci., 2 (1982) 1043-1051. 18 Lind, R. W., van Hoesen, G. W. and Johnson, A. K., An HRP study of the connections of the subfornical organ of the rat, J. comp. Neurol., 210 (1982) 265-277. 19 Mata, M., Fink, D. J., Gainer, H., Smith, C. B., Davidsen, L., Savaki, H., Schwartz, W. J. and Sokoloff, L., Activitydependent energy metabolism in rat posterior pituitary primarily reflects sodium pump activity, J. Neurochem., 34 (1980) 213-215. 20 Miselis, R. R., The efferent projections of the subfornical organ of the rat: a circumventricular organ within a neural network subserving water balance, Brain Research, 230 (1981) 1-23. 21 Mogenson, G. J. and Kucharczyk, J., Central neural pathways for angiotensin-induced thirst, Fed. Proc., 37 (1978) 2683-2688. 22 Nordmann, J. J., Ultrastructural morphometry of the rat neurohypophysis, J. Anat., 123 (1977) 213-218. 23 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 24 Peck, J. W. and Blass, E. M., Localization of thirst and antidiuretic osmoreceptors by intracranial injections in rats, Amer. J. Physiol., 228 (1975) 1501-1509. 25 Phillips, M. I., Angiotensin in the brain, Neuroendocrinology, 25 (1978) 354-377. 26 Phillips, M. I., Hoffman, W. E. and Bealer, S. L., Dehydration and fluid balance: central effects of angiotensin, Fed. Proc., 41 (1982) 2520-2527. 27 Rhodes, C. H., Morrell, J. I. and Pfaff, D. W., Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and number of cells containing neurophysin, oxytocin and vasopressin, J. comp, Neurol., 198 (1981) 45-64. 28 Rolls, B. J. and Rolls, E. T., Thirst, Cambridge University Press, Cambridge, 1982. 29 Savaki, H. E., McCulloch, J., Kadekaro, M. and Sokoloff, L., Influence of a-receptor blocking agents upon metabolic activity in nuclei involved in central control of blood pressure, Brain Research, 233 (1982) 347-358. 30 Savaki, H. E., Macpherson, H. and McCulloch, J., Alterations in local cerebral glucose utilization during hemorrhagic hypotension in the rat, Circulat. Res., 50 (1982) 633-644. 31 Schwartz, W. J., Smith, C. B., Davidsen, L., Savaki, H., Sokoloff, L., Mata, M., Fink, D. J. and Gainer, H., Metabolic mapping of functional activity in the hypothalamoneurohypophysial system of the rat, Science, 205 (1979) 723-725.

32 Share, L., Blood pressure, blood volume, and the release of vasopressin. In R. O. Greep and E. B. Astwood (Eds.), Handbook of Physiology, Endocrinology, Vol. IV, Am. Physiol. Soc./Williams and Wilkins, Baltimore, 1974, pp. 243-255. 33 Simonnet, G., Bioulac, B., Rodriguez, F. and Vincent, J. D., Evidence of a direct action of angiotensin II on neurones in the septum and in the medial preoptic area, Pharmacol. Biochem. Behav., 13 (1980) 359-363. 34 Simpson, J. B., Mangiapane, M. L. and Dellmann, H. D., Central receptor sites for angiotensin-induced drinking: a critical review, Fed. Proc., 37 (1978) 2676-2682. 35 Simpson, J. B., The circumventricular organs and the central actions of angiotensin, Neuroendocrinology, 32 (1981) 248-256. 36 Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. 37 Sokoloff, L., Localization of functional activity in the central nervous system by measurement of glucose utilization with radioactive deoxyglucose, J. Cereb. Blood Flow Metabol., 1 (1981) 7-36. 38 Sterba, G., Naumann, W. and Hoheisel, G., Extrahypothalamic axons of the classic neurosecretory system and their synapses, Progr. Brain Res., 53 (1980) 141-158. 39 Stricker, E. M., Thirst, sodium appetite, and complementary physiological contributions to the regulation of intravascular fluid volume. In A. N. Epstein, H. R. Kissileff and E. Stellar (Eds.), The Neuropsychology of Thirst: New Findings and Advances in Concepts, V. H. Winston and Sons, Washington, D.C., 1973, pp. 73-98. 40 Sutherland, R. C., Martin, M. J., McQueen, J. K. and Fink, G., Water deprivation results in increased 2-deoxyglucose uptake by paraventricular neurones as well as pars nervosa in Wistar and Brattleboro rats, Brain Research, 271 (1983) 101-108. 41 Swanson, L. W. and Mogenson, G. J., Neural mechanisms for the functional coupling of autonomic, endocrine and somatomotor responses in adaptive behavior, Brain Res. Rev., 3 (1981) 1-34. 42 Thrasher, T. N., Jones, R. G., Keil, L. C., Brown, C. J. and Ramsay, D. J., Drinking and vasopressin release during ventricular infusions of hypertonic solutions, Amer. J. Physiol., 238 (1980) R340-R345. 43 Vannucci, S. and Hawkins, R., Substrates of energy metabolism of the pituitary and pineal glands, J. Neurochem., 41 (1983) 1718-1725. 44 Vina, J. R., Page, R. B., Davis, D. W. and Hawkins, R. A., Aerobic glycolysis by the pituitary gland in vivo, J. Neurochem., 42 (1984) 1479-1482. 45 Wade, C. E., Bie, P., Keil, L. C. and Ramsay, D. J., Osmotic control of plasma vasopressin in the dog, Amer. J. Physiol., 243 (1982) E287-E292. 46 Wang, B. C., Share, L., Crofton, J. T. and Kimura, T., Effect of intravenous and intracerebroventricular infusion of hypertonic solutions on plasma and cerebrospinal fluid vasopressin concentrations, Neuroendocrinology, 34 (1982) 215-221.