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Cerebral sodium sensors in the sodium-deplete sheep
Brain R
352
s'~ i~h, 5()ti ~ i~'~:-;~ ~52--",:',,'-
BRES 14876
Cerebral sodium sensors in the sodium-deplete sheep Eva Tarjan, Derek A. Denton and Richard S. Weisinger Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Vic. (Australia) (Accepted 14 March 1989)
Key words. Monensin; Ouabain; Push-pull perfusion; Sheep; Sodium appetite; Thirst
The sodium intake of sodium deplete sheep was studied during local, push-pull perfusion of different solutions within the third cerebral ventricle. Sheep were made sodium deplete by continuous loss of parotid saliva, and were allowed access to 0.6 M NaHCO 3 solution for 2 h daily. Local perfusion within the third cerebral ventricle was performed before and during the access to sodium solution. Four perfusion sites were used: anterior dorsal and ventral, and posterior dorsal and ventral. Perfusion of 200 mM Na-csf caused a decrease in sodium intake at each perfusion site. Perfusion of ouabain, 10 -6 M, caused a reduction in sodium intake only during perfusions within the anterior portion of the third ventricle. The results may indicate that specific neuronal elements sensitive to changes in intracellular sodium concentration are located around the anterior portion of the third cerebral ventricle. These neurones, however, are not exclusive sites from where sodium intake of sodium deplete sheep can be influenced.
INTRODUCTION Studies on sodium deficiency d e m o n s t r a t e d that sheep m a d e sodium deplete by losing sodium-rich saliva through chronic p a r o t i d fistula drink amounts of sodium solution c o m m e n s u r a t e with their deficit s . T h e exact mechanism of accurate correction of body sodium deficit is unknown, but several contributing factors have been studied. Interference with the renin-angiotensin system by systemic administration of converting enzyme inhibitor disrupted the correction of sodium deficit in sheep 22. A d a p t a t i o n to the taste of a given sodium concentration also plays an i m p o r t a n t role in thc precise r e m e d y of sodium deficit 15. O t h e r studies d e m o n s t r a t e d that changes in the cerebrospinal fluid (csf) sodium concentration induced by infusions o f different substances into the lateral cerebral ventricle (i.c.v.) also modify the sodium i n t a k e of sodium-deficient sheep 23. In these e x p e r i m e n t s sodium concentration was increased by infusion o f artificial csf with a sodium concentration of 0.5 M (hyperosmotic Na-csf), and resulted in a decrease in sodium intake. W h e n the csf sodium concentration was decreased by i.c.v, infusion of
hyperosmotic manuitol solution it caused an increase in sodium intake 23. B a s e d on the above and later observations, utilising saccharides with different abilities to p e n e t r a t e into cells and cross the b l o o d brain barrier, it was suggested that there are specific areas in the brain, accessible by i,c.v, infusion but some distance into the neuropil which, having sensed the change in the sodium concentration of the brain extracellular fluid (e.c.f.), will act to modify sodium intake 21. The aim of the present e x p e r i m e n t s was to investigate w h e t h e r these sodium sensors can be localized to any discrete brain a r e a surrounding the third cerebral ventricle. Local push-pull perfusion was used to create a m i c r o e n v i r o n m e n t of localised change in a discrete area within one of the 4 quadrants of the third cerebral ventricle. Earlier this technique was found successful in localising a brain area, surrounding the anterior dorsal q u a d r a n t of the third cerebral ventricle, responsible for the natriuresis elicited by increased csf sodium concentration 3. The effect on sodium appetite of the local increase in csf sodium concentration caused by perfusion of 200 m M Na-csf was c o m p a r e d to that of the
Correspondence: E. Tarjan, Howard Florey Institute, University of Melbourne, Parkville, Vic. 3052, Australia. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
353 generalised increase in csf sodium concentration induced by i.c.v, infusion of 200 mM Na-csf, and local perfusion with glucose solution of equal osmolality. In addition, the effect on sodium appetite of local perfusion of ouabain, a Na+-K + ATPase inhibitor, and monensin, a Na+/H ÷ ionophore, both known to increase intracellular sodium concentration, were evaluated. MATERIALS AND METHODS Twenty-one crossbred Merino ewes, b. wt. 30-40 kg, were used. The animals were surgically prepared in two stages, both under general inhalation anaesthesia (Fluothan/oxygen mixture) after induction with i.v. sodium thiopentone. In the first stage oophorectomy was performed and a permanent unilateral parotid fistula was made. In the second stage a 16-gauge stainless steel guide cannula was implanted above the anterior portion of the third cerebral ventricle and another guide cannula was placed either above the lateral ventricle or above the posterior portion of the third cerebral ventricle. Placement of the guide cannulae was aided by intraoperative ventriculography using iopamidol (Iopamino, Schering AG.) and verified by postmortem examination. The animals were held in individual metabolism cages and were fed 0.8-0.9 kg oaten-lucerne chaff (sodium content 50-150 mmol/kg, potassium content 200-300 mmol/kg) at 16.30 h daily. The chaff was supplemented with 10 g KH2PO 4 daily to replace salivary phosphate loss. Sheep were trained to press one pedal in order to obtain delivery of sodium solution into a drinking cup (15 ml of 0.6 M N a H C O 3 = 9 mmoi/delivery). Pressing a second pedal caused delivery of 50 ml of water into a second drinking cup. The animals had 2 h access daily (12.00-14.00 h) to sodium after the sounding of a tone and learned rapidly to replace daily salivary sodium loss during 2 h TM. They had continuous access to water. All deliveries were consumed. The number of deliveries was counted and recorded continuously by computer (IBM Series I). Local push-pull perfusions were performed at 4 different sites in the third cerebral ventricle: anterior dorsal lAD) at or 1 mm above the level of the anterior commissure, anterior ventral (AV) at the
level of the optic recess, posterior dorsal (PD) at or 1 mm below the level of the pineal recess and posterior ventral (PV) 1 mm above the entrance to the aquaeduct cerebri. A sterile double-barrelled needle (push-pull cannula, Plastic Products, Roanoke, VA) of appropriate length was inserted via the guide tube into the anterior or posterior portion of the third cerebral ventricle. The inner needle tip was 1 mm longer than the outer one. The inner needle was used for infusion and the outer one for withdrawal, both at the same 1.2 ml/h rate with a Harvard infusion pump (model 55-5557) 3. The perfusion solutions used were artificial cerebrospinai fluid (composition in mM: Na 150; K 2.8; Ca 1.2; Mg 0.9; C1 135; HCO~ 22; PO 4 0.5) 13. 200 mM Na-csf, 100 mmol D-glucose (hyperosmotic glucose-csf, sodium concentration 150 raM, osmolality 400 mosmol/kg). Ouabain, 10" M, and monensin, 10 5 M, solutions were prepared immediately before perfusion in artificial csf. The pH of the perfusate was adjusted to 7.3-7.4 before the experiment. Perfusion commenced 1 h before the access to sodium solution and continued during the first hour of access period. After the termination of perfusion the sheep had one more hour access to sodium. Six sheep received i.c.v, infusion with a 2()-gauge needle of appropriate length attached to a metal luer-lok cap inserted through the guide tube into the lateral ventricle. The needle was connected via a polyethylene cannula to a 10-ml syringe held in an infusion pump (Perfusor, Braun). A 3 h infusion (1 ml/h) was begun 1 h prior to and continued until the end of the sodium access period. Urine and saliva were collected daily before the access to sodium solution. Daily sodium loss was measured two days before and one day after the experiment. The sodium concentration of saliva and urine was measured with a Technicon autoanalyser. The sodium and potassium concentration of perfusate samples were measured by Corning flame photometer (model 450). Osmolality of the perfusates was determined by freezing point depression on an Advanced Cryomatic osmometer (model 3CII). Statistical analysis. The number of deliveries of sodium solution counted at each 10-min interval during the second hour of perfusion and the following 1-h of sodium access was compared with the
354 mean of the number of deliveries during baseline days by analysis of variance (repeated measures design) with a subsequent t-test 2. The same statistical analysis was used to compare water intake at each 10-rain interval during the 2 h of perfusion and the following 1 h of sodium access between the experimental day and the baseline days. The mean of preceding baseline days was calculated as the mean of the two days preceding the perfusion of a given solution at the A D (or PD) site and the mean of the two days preceding the perfusion of the same solution at the AV (or PV) site. In no instances were there differences between the intakes during the baseline days. Daily sodium loss was calculated as the sum of salivary and urinary sodium loss. Correction of sodium deficit was calculated as the ratio of intake (number of deliveries counted multiplied by 9 mmol/delivery) to sodium loss during the 22 h preceding the access period. The sodium and potassium concentration and osmolality of the infused and withdrawn solutions were compared by paired ttests. RESULTS The time course of repletion of sodium deficit during the 2-h access to sodium solution for animals perfused at the AD3V with artificial csf, 200 mM Na-csf or ouabain is presented in Fig. 1. During the first 10 min of access, sheep drank amounts of sodium equivalent to 50-60% replacement of their deficit. During the following 10 min the intake of sodium during perfusions of 200 mM Na-csf and
LOCAL PERFUSION WITHIN THE A D 3 V
f n ~z u,t
START BAR PRESS
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oz :f m© :ici
•
~
~. =. :-:
;
•
oo,.
"
20
"=P, ,o -6'0
-30
0 r mu
30
80
9'0
120
mifll
Ik"dd
Fig. l. Cumulative number of deliveries of 0.6 M NaHCO 3 drank during local perfusion of artificial csf (n = 5), 200 mM Na-csf (n = 10) and ouabain, 10 .6 M (n = 6), within the anterior dorsal third ventricle. *P < 0.05 compared to artificial csf.
ouabain solutions were significantly lower than the intakes during perfusion of artificial csf. At 20 rain sheep perfused with artificial csf drank 34.4 ± 6.1 deliveries, those perfused with 200 mM Na-csf drank 26.6 + 2.6 deliveries (P < 0.05 compared to csf) and those perfused with ouabain consumed 23.7 _ 1.~ deliveries (P < 0.05 compared to csf). Intake remained lower throughout the first 60 min of access while perfusion of 200 mM Na-csf or ouabain was maintained. After the end of perfusion, sheep previously perfused with 200 mM Na-csf drank further amounts of sodium and at the end of the 2-h access period intake was not different from those perfused with csf. Sheep perfused with ouabain, however, did not correct their sodium deficit after the end of perfusion. Sodium intake at the end of perfusion (60 min) and at the end of sodium access period (120 min) for the different perfused solutions at the 4 perfusion sites is detailed in Table I. Perfusion of artificial csf did not influence the sodium intake of the animals compared to the intake during the preceding baseline, control days. This is in agreement with previous findings: i.c.v, infusion of artificial csf did not influence the sodium intake of sodium-deplete sheep 23. Sheep corrected their sodium deficit incurred during the 22 h before access to sodium by 96% with the A D perfusion (loss was 335 ± 48 mmol), and by 83% with the AV perfusion (loss was 422 + 47 mmol) at 60 min. Infusion of 200 mM Na-csf for 3 h into the lateral cerebral ventricle (presented among the A D perfusions in Table I) did not influence sodium intake. In contrast, local perfusion of 200 mM Na-csf decreased the sodium intake at all 4 peffusion sites during perfusion, but intake increased to baseline levels during the second hour of access with the exception of perfusion at PD. The sodium deficit ranging from 339 to 411 mmol incurred during the 22 h prior to access to sodium was corrected at 65% at AD, 75% at AV and PD, and 71% at PV perfusion sites during the first 60 min of perfusion (all significantly, P < 0.05, different from baseline). Perfusion of hyperosmotic glucose-csf did not influence the sodium intake of sodium-depleted sheep: at every time interval and each perfusion site it was not different from the intake during baseline. The same results were obtained during perfusion of
355
TABLE I Number of deliveries o f O.6 M NaHCO ~(15 ml = 9 retool~delivery) consumed after 60 and 120 rain o f access to sodium solution by 22 h sodium-deficient sheep during local perfusion of different solutions in the 4 quadrants of the third cerebral ventricle and during the preceding baseline days Values arc mean _+ S.E.M. Number of deliveries drank
Perfl~sate
Baseline" O0 rain Anterior dorsal third ventricle Artificial csf i.c.v. 200 mM Na-csf 200 mM Na-csf Hyperosmotic glucose-csf Ouabain 10 ~M Monensin 10 5 M Anterior ventral third ventricle Artificial csf 200 mM Na-csf Hyperosmotic glucose-csf Ouabain 10-~' M Monensin 10 5 M Posterior dorsal third ventricle 2(10 mM Na-csf Hyperosmotic glucose-csf Ouabain 10 o M Monensin 10 s M Posterior ventral third ventricle 200 mM Na-csf H ype rosmotic glucose -csf Ouabain 10 aM Monensin 10 s M
5 6 10 4 6 7
35.0 ± 40.0 + 38.9 ± 35.4 ± 37.0 ± 36.9±
5 10 4 6 7
35.0 38.9 35.4 37.0 36.9
1.8 2.3 1.3 2.8 1.7 1.4
+ 1.8 ± 1.3 +_ 2.8 +_ 1.7 _+ 1.4
Perfusion 120 min
60 min
120 min
37.4 ± 1.8 40.8_+ 2.4 42.5 ± 1.4 39.5 ± 2.9 39.2 +_ 1.8 40.1+ 1.6
35.6 ± 6.0 37.3 ± 3.3 27.9 + 3,3* 29.0_+ 9.5 26.5 + 1.4* 33.7_+4.6
35.6_+ 6.0 41.5 ± 5.1 36.0 ± 4.9 32.5 ± 9.0 27.2 _+ 1.6* 37.9±4.7
37.4 + 42.5 ± 39.5 ± 39.2 ± 4(I.1 ±
39.0 28.1 28.5 25.2 35.7
40.6± 35.6 ± 31.8 + 29.5 ± 46.0 ±
1.8 1.4 2.9 1.8 1.6
± + ± + ±
5.9 2.8* 5.8 3.4* 4.3
5.6 3.8 7.0 4.4* 5.1
5 3 6 6
43.9 ± 1.6 34.3 34.8 +_ 2.5 33.2 ± 2.4
45.4 ± 1.5 39.8 40.7 + 3.1 39.9 + 3.2
29.6 ± 4.0* 31.0 33.2 + 6.7 28.2 ± 4.4
33.0 + 5.7 35.3 39.(I ± 7.5 34.5 _+ 6.2
5 3 6 6
43.9 +_: 1,6 34.3 34.8 -+ 2.5 33.2 _+ 2.4
45.4 + 1.5 39.8 40.7 + 3.1 39.9 + 3.2
32.6 _+ 2.2* 31.0 33.3 + 7.3 33.0 ± 2.6
37.2 _+ 3.5 39.3 38.3 + 8.3 37.2 _+ 4.7
~' Baseline values are the mean of the intakes recorded during the two days preceding dorsal and ventral perfusions, therefore baseline values are identical for AD and AV and also for PD and PV perfusions for each perfused solution. *P < 0.05 compared to the baseline intake.
100 m m o l m a n n i t o l s o l u t i o n ( s o d i u m c o n c e n t r a t i o n
the commencement
o f local p e r f u s i o n s d i d n o t s h o w
150 m M , o s m o l a l i t y 400 m o s m o l / k g ) in 4 s h e e p , t h e
any difference from baseline water intakes. Perfu-
r e s u l t s o f w h i c h a r e n o t listed in t h e t a b l e s . P e r f u s i o n
sion o f h y p e r o s m o t i c s o l u t i o n s d i d n o t i n d u c e w a t e r
of ouabain,
drinking
s h e e p at A D
10 _6 M , r e d u c e d t h e s o d i u m i n t a k e o f a n d A V p e r f u s i o n sites, b u t d i d n o t
different
from
baseline
in t h e
sodium-
d e p l e t e s h e e p b e f o r e t h e a c c e s s to s o d i u m . I n g e s t i o n
i n f l u e n c e i n t a k e at t h e P D a n d P V p e r f u s i o n sites.
of sodium
C o r r e c t i o n of s o d i u m deficit of 378 + 30 a n d 339 +
perfusions different from baseline intake. For exam-
66 m m o l w a s 6 3 % at A D , 6 7 % at A V at 60 rain, a n d
ple, s h e e p p e r f u s e d w i t h artificial csf at t h e A D
65%
all
d r a n k a m e a n o f 2.0 + 2.0 d e l i v e r i e s , a n d at A V a
baseline.
m e a n o f 4.8 + 1.7 d e l i v e r i e s of w a t e r by t h e e n d o f
at
AD
significantly,
and P
<
78£0
at
0.05,
different
Perfusion of monensin,
AV
at
120 rain, from
10 5 M , d i d n o t i n f l u e n c e
s o d i u m i n t a k e at e i t h e r p e r f u s i o n sites. T h e r a n g e o f
perfusion.
did n o t s t i m u l a t e w a t e r i n t a k e d u r i n g
Sheep
perfused
with
200
mM
Na-csf
d r a n k m e a n s o f 5.9 + 2.3 l A D ) , 7.1 + 2.7 ( A V ) , 6.0
s o d i u m deficit p r e c e d i n g p e r f u s i o n o f m o n e n s i n was
+ 2.1 ( P D ) a n d 7.2 + 1.7 ( P V ) d e l i v e r i e s . P e r f u s i o n
370 t o
of h y p e r o s m o t i c g l u c o s e - c s f , o u a b a i n a n d m o n e n s i n
389 m m o l ,
s i m i l a r to
the
deficit
before
perfusion of ouabain. W a t e r i n t a k e r e c o r d e d at 1 0 - m i n i n t e r v a l s f r o m
solutions coincided with water intakes within the above ranges.
356 TABLE I1 Sodium, potassium concentration (raM) and osmolality (mOsm/kg) of the infused (push) and withdrawn (pull) solutions during local perfusion
Values are mean _+S.E.M. Perfusate
n
Push [Na]
Pull [Na]
Push [K]
Pull [K]
Push Osm
Pull Osm
Artificial csf i.c.v. 200 mM Na-csf~ 200mM Na-csf Hyperosmotic Glucose-csf Ouabain 10 6M Monensin 10-.5 M
8 6 30 13 24 26
146.9 + 0.6 201.2 + 2.3 149.7 + 1.5 148.1+0.7 149.3_+0.7
147.4 + 0.6 159.0 + 0.7 184.4 + 4.3* 150.9 + 1.4" 148.6_+0.6 149.8_+0.8
2.74 + 0.03 2.79 + 0.04 2.87 + 0.02 2.82+0.05 2.83_+0.05
2.73 + 0.04 2.62 + 0.09 2.75 + 0.05 2.78 + 0.02* 2.84_+0.05 2.81_+0.05
285.3+ 0.4 381.2+ 5.2 396.6 _+2.8 288.3_+1.4 294.6_+2.1
288.7 +_ 1.3 311.8 + 1.7 352.4 + 7.8* 364.2 _+9.2" 288.9 _+ 1.2 297.8 + 2
csf sampled at the end of i.c.v, infusion. *P < 0.05.
The composition of infused and withdrawn solutions is p r e s e n t e d in Table II. The calculated loss from the perfused solutions was between 20 and 30%. Perfusion of o u a b a i n or monensin did not influence the composition of the csf. DISCUSSION Local perfusion of 200 m M Na-csf caused a reduction in the sodium intake of s o d i u m - d e p l e t e sheep. This result confirms earlier findings that i.c.v. infusion of h y p e r o s m o t i c Na-csf caused a decrease in the sodium intake of sodium-deficient sheep 23. The specificity of the effects of increased NaCl concentration as o p p o s e d to increased osmolality alone was tested by perfusion of h y p e r o s m o t i c glucose solution: increased osmolality of the perfusate failed to influence the sodium intake of s o d i u m - d e p l e t e sheep. Localisation of the sodium-sensitive elements which influence the sodium intake of s o d i u m - d e p l e t e sheep 4 was a t t e m p t e d using the local perfusion technique e m p l o y e d in earlier studies 3'18. Local perfusion of 200 m M Na-csf caused a reduction in sodium intake at all 4 perfusion sites tested. The local change in csf sodium concentration at the 4 sites m a y influence structures close to the wall of the third cerebral ventricle, including the whole of lamina terminalis, the anterior, dorsal and posterior h y p o t h a l a m u s , the medial thalamus, the h a b e n u l a r structures. The loss during peffusion of a b o u t 25% of infused solution might raise the sodium concentration of the csf d o w n s t r e a m from the perfused
sites, if the csf production rate is assumed to be 7.5 ml/h, by about 2 mM. During infusion of 200 m M Na-csf into the lateral ventricle the sodium concentration of the csf increased by 7.3 + 2.8 m M (from the pre-infusion level of 151.7 + 1.0 to 159.0 + 0.7 m M at the end of infusion), and this did not influence the sodium intake of sheep. T h e r e f o r e we have to conclude that delivery of 200 m M Na-csf by local peffusion increased t h e sodium concentration of the ecf, and p r e s u m a b l y the icf of brain, at a discrete area m o r e than delivery of the same solution by i.c.v, infusion. T h e local increase in sodium concentration was sufficient to induce changes in the correction of sodium deficit. O u a b a i n has b e e n d e m o n s t r a t e d to increase the intracellular neuronal sodium concentration in the cerebral cortical slices of the guinea-pig brain at concentrations of 10 -6 M or higher 17, and specific binding of o u a b a i n to the brain tissue of several other species has b e e n r e p o r t e d m. Local perfusion of o u a b a i n at the concentration of 10 -6 M caused reduction of sodium intake only when perfused at the anterior sites. O u a b a i n influenced sodium intake p r o b a b l y by reducing the icf sodium concentration in a neuronal p o p u l a t i o n close to the site of perfusion. It may be assumed that in the sheep the neural elements specifically sensitive to increased icf sodium concentration and participating in the regulation of sodium intake are located in the neuropil surrounding the anterior part of the third cerebral ventricle. T h e exact location of these hypothetical sensors, however, is unknown. A b l a t i o n of the lamina terminalis or A V 3 V a r e a did not disrupt the
357 replacement of sodium deficit in sheep 6. In rats, lesion of the AV3V area was reported to decrease the daily sodium intake 1, while lesion of the ventral nucleus medianus increased daily sodium consumption 1~, and both lesions failed to interfere with the increased sodium intake stimulated by s.c. injection of formalin or polyethylene glycol. The discrepancy between the more localised effect of ouabain and the widespread effective area of increased local sodium concentration may indicate that increased csf and ecf and presumably icf sodium concentration, due to the perfusion of 200 mM Na-csf, interferes with neuronal activity in other brain areas not specifically sensitive to changes in icf sodium concentration. These areas may include some parts of the neuronal network involved in the regulation of motivation and may decrease the drive for drinking sodium solution in response to sodium deficit. This may be true in sheep, but not in other species, e.g. rats and wild rabbits, in which increased csf sodium concentration failed to decrease the sodium intake stimulated by sodium deficiency7~9. Another explanation for the more localised effect of ouabain might be the following: the proposed sodium-sensitive neurones extend along the whole lateral wall of the third ventricle, but they are closer to the ventricular surface at the anterior than at the posterior half and sodium may move further into the neuropil than ouabain. It is interesting, that the effect of ouabain could not be reproduced by monensin, at the reported 10-5 M, and in pilot studies at 5 times higher concentrations. Monensin was demonstrated to increase intracellular sodium concentration in mouse neuroblastoma-rat glioma hybrid cells at a concentration of 20 glM~2~ and inhibit plasma membrane proteolipid synthesis in cultured embryonic rat neurones at 1 /~M 16. Monensin, however, was not tested in neurones in vivo and may not influence the neuronal sodium transport at all. Alternately, monensin may REFERENCES 1 Bcalcr. S.L. and Johnson, A.K., Sodium consumption following lesions surrounding the anteroventral third ventricle, Brain Res. Bull., 4 (1979) 287-290. 2 Bruning, J.L. and Kintz, B.L., Computational Handbook of Statistics, Scon-Foresman, Glenview, IL, 1977. 3 Cox, P.S., Denton, D.A., Mouw, D.R. and Tarjan, E., Natriuresis induced by localized perfusion within the third
not penetrate into the neuropil from the csf. Simultaneous administration of ouabain, 10 6 M , and monensin, 10-5 M, did not cause any changes in the sodium intake of sodium-deficient sheep different from that of ouabain alone (unpublished observations). The effect of local perfusion of 200 mM Na-csf on sodium intake was short, it lasted only during the perfusion. During the second hour of sodium access after the cessation of perfusion sheep drank further amounts of sodium and corrected their sodium deficit accurately. In contrast, the effect of local perfusion of ouabain persisted after the termination of perfusion. This is probably due to the slow release of ouabain from its specific binding sites in the brain ~9 and the prolonged specific effect of ouabain. Sheep showed no reaction at all to local perfusion of ouabain, and in contrast to studies where ouabain was infused into the carotid artery 5"2°, no change in water intake was observed. The changes in the sodium intake of the sodiumdeplete sheep in the present study were not preceded or accompanied by changes in water intake. This suggests that the sodium sensors involved in the regulation of the sodium intake of the sodiumdeplete sheep are distinct from, and probably more sensitive to changes in ecf and icf sodium concentration than those involved in the regulation of thirst ~3. ACKNOWLEDGEMENTS This work was supported by grants from the National Health and Medical Research Council of Australia. The excellent technical assistance of Mr. Robert J. Plenter and Mr. Brett Purcell is gratefully acknowledged. Preliminary results were presented at the NATO Advanced Research Workshop in Camerino, Italy, 1984, and at the Centennial Meeting of the Polish Physiological Society, Warsaw, Poland, 1987. cerebral ventricle of sheep, Am. J. Physiol.. 252 (1987) R1-R6. 4 Denton, D.A., The Hunger for Salt-- An Anthropological, Physiological and Medical Analysis, Springer, Berlin, 1982. 5 Denton, D.A., Kraintz, F.W. and Kraintz, L., The inhibition of salt appetite of sodium-deficient sheep by intracarotid infusion of ouabaim Commun. Behav. Biol., 4 (1969) 183-194. 6 Denton, D.A, McKinley, M.J., Tarjam E. and Weisinger,
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7
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15
R.S., Sodium appetite: some elements in an overview, Appetite, 7 (1986) 250-251 (Abstr.). Denton, D.A., Nelson, J.E and Tarjan, E., Water and salt intake of wild rabbits (Oryctolagus cuniculus (L)) following dipsogenic stimuli, J. Physiol. (Lond.), 362 (1985) 285301. Denton, D.A., Orchard, E. and Weller, S., The effect of rapid change of sodium balance and expansion of plasma volume on sodium appetite of sodium deficient sheep, Comp. Behav. Biol., 6 (1971) 245-258. Epsteih, A.N., Zhang, D., Schultz, J., Rosenberg, M., Kupsha, R and Stellar, E., The failure of ventricular sodium to control sodium appetite in the rat, Physiol. Behav., 32 (1984) 683-686. Evans, C.A., Reynolds, J.M., Reynolds, M.L., Saunders, N.R. and Segal, N., The development of a blood-brain barrier mechanism in foetal sheep, J. Physiol. (Lond.), 238 (1974) 371-386. Gardiner, T.W., Jolley, J.R., Vagnucci, A.H. and Stricker, E.M., Enhanced sodium appetite in rats with lesions centered on nucleus medianus, Behav. Neurosci., 100 (1986) 531-535. Lichtshtein, D., Dunlop, K., Kaback, H.R. and Blume, A.J., Mechanism of monensin-induced hyperpolarization of neuroblastoma-glioma hybrid NG 108-15, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 2580-2584. McKinley, M.J., Denton, D.A. and Weisinger, R.S., Sensors for antidiuresis and thirst - - osmoreceptors or CSF sodium detectors, Brain Research, 141 (1978) 80-103. Mouw, D.R., Abraham, S.E and McKenzie, J.S., Use of ventriculo cisternal perfusion in conscious sheep, Lab. Anim. Sci., 24 (1974) 505-509. Osborne, P.G., Denton, D.A. and Weisinger, R.S., The role of taste in rapid sodium satiation by sodium-deficient
sheep, Appetite, 8 (1987) 91-99. 16 Shea, T.B., Fischer, I. and Sapirstein, V., Expression ol plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture, J. Neurochem., 47 (1986) 697-706. 17 Swanson, P.D. and Ullis, K., Ouabain-induced changes in sodium and potassium content and respiration of cerebral cortex slices, J. Pharmacol. Exp. Ther.. 153 (1966) 321328. 18 Tarjan, E., Cox, P., Denton, D.A., McKinley, M.J. and Weisinger, R.S., The effect of local change in CSF [Na] in the anterior third ventricle on salt appetite, L~]'e Sci., 105 (1986) 473-478. 19 Tobin, T. and Brody, T.M., Rates of dissociation of enzyme-ouabain complexes and Ko.5 values in (Na + + K + ) adenosine triphosphatase from different species, Biochem. Pharmacol., 21 (1972) 1553-1560. 20 Weisinger, R.S., Denton, D.A. and McKinley, M.J., Inhibition of water intake by ouabain administration in sheep, Pharmacol. Biochem. Behav., 7 (1977) 121-128. 21 Weisinger, R.S., Denton, D.A., McKinley, M.J., Muller, A.E and Tarjan, E., Cerebrospinal fluid sodium concentration and salt appetite. Brain Research, 326 (1985) 95-105. 22 Weisinger, R.S., Denton, D.A., Di Nicolantonio, R., McKinley, M.J., Muller, A.E and Tarjan, E., Role of angiotensin in sodium appetite of sodium-deplete sheep, Am. J. Physiol., 253 (1987) R483-R488. 23 Weisinger, R.S., Considine, P., Denton, D.A., LekseU, L., McKinley, M.J., Mouw, D.R., Muller, A.F. and Tarjan, E., Role of sodium concentration of the cerebrospinal fluid in the salt appetite of sheep, Am. J. Physiol., 242 (1982) R51-R63.
352
s'~ i~h, 5()ti ~ i~'~:-;~ ~52--",:',,'-
BRES 14876
Cerebral sodium sensors in the sodium-deplete sheep Eva Tarjan, Derek A. Denton and Richard S. Weisinger Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Vic. (Australia) (Accepted 14 March 1989)
Key words. Monensin; Ouabain; Push-pull perfusion; Sheep; Sodium appetite; Thirst
The sodium intake of sodium deplete sheep was studied during local, push-pull perfusion of different solutions within the third cerebral ventricle. Sheep were made sodium deplete by continuous loss of parotid saliva, and were allowed access to 0.6 M NaHCO 3 solution for 2 h daily. Local perfusion within the third cerebral ventricle was performed before and during the access to sodium solution. Four perfusion sites were used: anterior dorsal and ventral, and posterior dorsal and ventral. Perfusion of 200 mM Na-csf caused a decrease in sodium intake at each perfusion site. Perfusion of ouabain, 10 -6 M, caused a reduction in sodium intake only during perfusions within the anterior portion of the third ventricle. The results may indicate that specific neuronal elements sensitive to changes in intracellular sodium concentration are located around the anterior portion of the third cerebral ventricle. These neurones, however, are not exclusive sites from where sodium intake of sodium deplete sheep can be influenced.
INTRODUCTION Studies on sodium deficiency d e m o n s t r a t e d that sheep m a d e sodium deplete by losing sodium-rich saliva through chronic p a r o t i d fistula drink amounts of sodium solution c o m m e n s u r a t e with their deficit s . T h e exact mechanism of accurate correction of body sodium deficit is unknown, but several contributing factors have been studied. Interference with the renin-angiotensin system by systemic administration of converting enzyme inhibitor disrupted the correction of sodium deficit in sheep 22. A d a p t a t i o n to the taste of a given sodium concentration also plays an i m p o r t a n t role in thc precise r e m e d y of sodium deficit 15. O t h e r studies d e m o n s t r a t e d that changes in the cerebrospinal fluid (csf) sodium concentration induced by infusions o f different substances into the lateral cerebral ventricle (i.c.v.) also modify the sodium i n t a k e of sodium-deficient sheep 23. In these e x p e r i m e n t s sodium concentration was increased by infusion o f artificial csf with a sodium concentration of 0.5 M (hyperosmotic Na-csf), and resulted in a decrease in sodium intake. W h e n the csf sodium concentration was decreased by i.c.v, infusion of
hyperosmotic manuitol solution it caused an increase in sodium intake 23. B a s e d on the above and later observations, utilising saccharides with different abilities to p e n e t r a t e into cells and cross the b l o o d brain barrier, it was suggested that there are specific areas in the brain, accessible by i,c.v, infusion but some distance into the neuropil which, having sensed the change in the sodium concentration of the brain extracellular fluid (e.c.f.), will act to modify sodium intake 21. The aim of the present e x p e r i m e n t s was to investigate w h e t h e r these sodium sensors can be localized to any discrete brain a r e a surrounding the third cerebral ventricle. Local push-pull perfusion was used to create a m i c r o e n v i r o n m e n t of localised change in a discrete area within one of the 4 quadrants of the third cerebral ventricle. Earlier this technique was found successful in localising a brain area, surrounding the anterior dorsal q u a d r a n t of the third cerebral ventricle, responsible for the natriuresis elicited by increased csf sodium concentration 3. The effect on sodium appetite of the local increase in csf sodium concentration caused by perfusion of 200 m M Na-csf was c o m p a r e d to that of the
Correspondence: E. Tarjan, Howard Florey Institute, University of Melbourne, Parkville, Vic. 3052, Australia. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
353 generalised increase in csf sodium concentration induced by i.c.v, infusion of 200 mM Na-csf, and local perfusion with glucose solution of equal osmolality. In addition, the effect on sodium appetite of local perfusion of ouabain, a Na+-K + ATPase inhibitor, and monensin, a Na+/H ÷ ionophore, both known to increase intracellular sodium concentration, were evaluated. MATERIALS AND METHODS Twenty-one crossbred Merino ewes, b. wt. 30-40 kg, were used. The animals were surgically prepared in two stages, both under general inhalation anaesthesia (Fluothan/oxygen mixture) after induction with i.v. sodium thiopentone. In the first stage oophorectomy was performed and a permanent unilateral parotid fistula was made. In the second stage a 16-gauge stainless steel guide cannula was implanted above the anterior portion of the third cerebral ventricle and another guide cannula was placed either above the lateral ventricle or above the posterior portion of the third cerebral ventricle. Placement of the guide cannulae was aided by intraoperative ventriculography using iopamidol (Iopamino, Schering AG.) and verified by postmortem examination. The animals were held in individual metabolism cages and were fed 0.8-0.9 kg oaten-lucerne chaff (sodium content 50-150 mmol/kg, potassium content 200-300 mmol/kg) at 16.30 h daily. The chaff was supplemented with 10 g KH2PO 4 daily to replace salivary phosphate loss. Sheep were trained to press one pedal in order to obtain delivery of sodium solution into a drinking cup (15 ml of 0.6 M N a H C O 3 = 9 mmoi/delivery). Pressing a second pedal caused delivery of 50 ml of water into a second drinking cup. The animals had 2 h access daily (12.00-14.00 h) to sodium after the sounding of a tone and learned rapidly to replace daily salivary sodium loss during 2 h TM. They had continuous access to water. All deliveries were consumed. The number of deliveries was counted and recorded continuously by computer (IBM Series I). Local push-pull perfusions were performed at 4 different sites in the third cerebral ventricle: anterior dorsal lAD) at or 1 mm above the level of the anterior commissure, anterior ventral (AV) at the
level of the optic recess, posterior dorsal (PD) at or 1 mm below the level of the pineal recess and posterior ventral (PV) 1 mm above the entrance to the aquaeduct cerebri. A sterile double-barrelled needle (push-pull cannula, Plastic Products, Roanoke, VA) of appropriate length was inserted via the guide tube into the anterior or posterior portion of the third cerebral ventricle. The inner needle tip was 1 mm longer than the outer one. The inner needle was used for infusion and the outer one for withdrawal, both at the same 1.2 ml/h rate with a Harvard infusion pump (model 55-5557) 3. The perfusion solutions used were artificial cerebrospinai fluid (composition in mM: Na 150; K 2.8; Ca 1.2; Mg 0.9; C1 135; HCO~ 22; PO 4 0.5) 13. 200 mM Na-csf, 100 mmol D-glucose (hyperosmotic glucose-csf, sodium concentration 150 raM, osmolality 400 mosmol/kg). Ouabain, 10" M, and monensin, 10 5 M, solutions were prepared immediately before perfusion in artificial csf. The pH of the perfusate was adjusted to 7.3-7.4 before the experiment. Perfusion commenced 1 h before the access to sodium solution and continued during the first hour of access period. After the termination of perfusion the sheep had one more hour access to sodium. Six sheep received i.c.v, infusion with a 2()-gauge needle of appropriate length attached to a metal luer-lok cap inserted through the guide tube into the lateral ventricle. The needle was connected via a polyethylene cannula to a 10-ml syringe held in an infusion pump (Perfusor, Braun). A 3 h infusion (1 ml/h) was begun 1 h prior to and continued until the end of the sodium access period. Urine and saliva were collected daily before the access to sodium solution. Daily sodium loss was measured two days before and one day after the experiment. The sodium concentration of saliva and urine was measured with a Technicon autoanalyser. The sodium and potassium concentration of perfusate samples were measured by Corning flame photometer (model 450). Osmolality of the perfusates was determined by freezing point depression on an Advanced Cryomatic osmometer (model 3CII). Statistical analysis. The number of deliveries of sodium solution counted at each 10-min interval during the second hour of perfusion and the following 1-h of sodium access was compared with the
354 mean of the number of deliveries during baseline days by analysis of variance (repeated measures design) with a subsequent t-test 2. The same statistical analysis was used to compare water intake at each 10-rain interval during the 2 h of perfusion and the following 1 h of sodium access between the experimental day and the baseline days. The mean of preceding baseline days was calculated as the mean of the two days preceding the perfusion of a given solution at the A D (or PD) site and the mean of the two days preceding the perfusion of the same solution at the AV (or PV) site. In no instances were there differences between the intakes during the baseline days. Daily sodium loss was calculated as the sum of salivary and urinary sodium loss. Correction of sodium deficit was calculated as the ratio of intake (number of deliveries counted multiplied by 9 mmol/delivery) to sodium loss during the 22 h preceding the access period. The sodium and potassium concentration and osmolality of the infused and withdrawn solutions were compared by paired ttests. RESULTS The time course of repletion of sodium deficit during the 2-h access to sodium solution for animals perfused at the AD3V with artificial csf, 200 mM Na-csf or ouabain is presented in Fig. 1. During the first 10 min of access, sheep drank amounts of sodium equivalent to 50-60% replacement of their deficit. During the following 10 min the intake of sodium during perfusions of 200 mM Na-csf and
LOCAL PERFUSION WITHIN THE A D 3 V
f n ~z u,t
START BAR PRESS
.!!
oz :f m© :ici
•
~
~. =. :-:
;
•
oo,.
"
20
"=P, ,o -6'0
-30
0 r mu
30
80
9'0
120
mifll
Ik"dd
Fig. l. Cumulative number of deliveries of 0.6 M NaHCO 3 drank during local perfusion of artificial csf (n = 5), 200 mM Na-csf (n = 10) and ouabain, 10 .6 M (n = 6), within the anterior dorsal third ventricle. *P < 0.05 compared to artificial csf.
ouabain solutions were significantly lower than the intakes during perfusion of artificial csf. At 20 rain sheep perfused with artificial csf drank 34.4 ± 6.1 deliveries, those perfused with 200 mM Na-csf drank 26.6 + 2.6 deliveries (P < 0.05 compared to csf) and those perfused with ouabain consumed 23.7 _ 1.~ deliveries (P < 0.05 compared to csf). Intake remained lower throughout the first 60 min of access while perfusion of 200 mM Na-csf or ouabain was maintained. After the end of perfusion, sheep previously perfused with 200 mM Na-csf drank further amounts of sodium and at the end of the 2-h access period intake was not different from those perfused with csf. Sheep perfused with ouabain, however, did not correct their sodium deficit after the end of perfusion. Sodium intake at the end of perfusion (60 min) and at the end of sodium access period (120 min) for the different perfused solutions at the 4 perfusion sites is detailed in Table I. Perfusion of artificial csf did not influence the sodium intake of the animals compared to the intake during the preceding baseline, control days. This is in agreement with previous findings: i.c.v, infusion of artificial csf did not influence the sodium intake of sodium-deplete sheep 23. Sheep corrected their sodium deficit incurred during the 22 h before access to sodium by 96% with the A D perfusion (loss was 335 ± 48 mmol), and by 83% with the AV perfusion (loss was 422 + 47 mmol) at 60 min. Infusion of 200 mM Na-csf for 3 h into the lateral cerebral ventricle (presented among the A D perfusions in Table I) did not influence sodium intake. In contrast, local perfusion of 200 mM Na-csf decreased the sodium intake at all 4 peffusion sites during perfusion, but intake increased to baseline levels during the second hour of access with the exception of perfusion at PD. The sodium deficit ranging from 339 to 411 mmol incurred during the 22 h prior to access to sodium was corrected at 65% at AD, 75% at AV and PD, and 71% at PV perfusion sites during the first 60 min of perfusion (all significantly, P < 0.05, different from baseline). Perfusion of hyperosmotic glucose-csf did not influence the sodium intake of sodium-depleted sheep: at every time interval and each perfusion site it was not different from the intake during baseline. The same results were obtained during perfusion of
355
TABLE I Number of deliveries o f O.6 M NaHCO ~(15 ml = 9 retool~delivery) consumed after 60 and 120 rain o f access to sodium solution by 22 h sodium-deficient sheep during local perfusion of different solutions in the 4 quadrants of the third cerebral ventricle and during the preceding baseline days Values arc mean _+ S.E.M. Number of deliveries drank
Perfl~sate
Baseline" O0 rain Anterior dorsal third ventricle Artificial csf i.c.v. 200 mM Na-csf 200 mM Na-csf Hyperosmotic glucose-csf Ouabain 10 ~M Monensin 10 5 M Anterior ventral third ventricle Artificial csf 200 mM Na-csf Hyperosmotic glucose-csf Ouabain 10-~' M Monensin 10 5 M Posterior dorsal third ventricle 2(10 mM Na-csf Hyperosmotic glucose-csf Ouabain 10 o M Monensin 10 s M Posterior ventral third ventricle 200 mM Na-csf H ype rosmotic glucose -csf Ouabain 10 aM Monensin 10 s M
5 6 10 4 6 7
35.0 ± 40.0 + 38.9 ± 35.4 ± 37.0 ± 36.9±
5 10 4 6 7
35.0 38.9 35.4 37.0 36.9
1.8 2.3 1.3 2.8 1.7 1.4
+ 1.8 ± 1.3 +_ 2.8 +_ 1.7 _+ 1.4
Perfusion 120 min
60 min
120 min
37.4 ± 1.8 40.8_+ 2.4 42.5 ± 1.4 39.5 ± 2.9 39.2 +_ 1.8 40.1+ 1.6
35.6 ± 6.0 37.3 ± 3.3 27.9 + 3,3* 29.0_+ 9.5 26.5 + 1.4* 33.7_+4.6
35.6_+ 6.0 41.5 ± 5.1 36.0 ± 4.9 32.5 ± 9.0 27.2 _+ 1.6* 37.9±4.7
37.4 + 42.5 ± 39.5 ± 39.2 ± 4(I.1 ±
39.0 28.1 28.5 25.2 35.7
40.6± 35.6 ± 31.8 + 29.5 ± 46.0 ±
1.8 1.4 2.9 1.8 1.6
± + ± + ±
5.9 2.8* 5.8 3.4* 4.3
5.6 3.8 7.0 4.4* 5.1
5 3 6 6
43.9 ± 1.6 34.3 34.8 +_ 2.5 33.2 ± 2.4
45.4 ± 1.5 39.8 40.7 + 3.1 39.9 + 3.2
29.6 ± 4.0* 31.0 33.2 + 6.7 28.2 ± 4.4
33.0 + 5.7 35.3 39.(I ± 7.5 34.5 _+ 6.2
5 3 6 6
43.9 +_: 1,6 34.3 34.8 -+ 2.5 33.2 _+ 2.4
45.4 + 1.5 39.8 40.7 + 3.1 39.9 + 3.2
32.6 _+ 2.2* 31.0 33.3 + 7.3 33.0 ± 2.6
37.2 _+ 3.5 39.3 38.3 + 8.3 37.2 _+ 4.7
~' Baseline values are the mean of the intakes recorded during the two days preceding dorsal and ventral perfusions, therefore baseline values are identical for AD and AV and also for PD and PV perfusions for each perfused solution. *P < 0.05 compared to the baseline intake.
100 m m o l m a n n i t o l s o l u t i o n ( s o d i u m c o n c e n t r a t i o n
the commencement
o f local p e r f u s i o n s d i d n o t s h o w
150 m M , o s m o l a l i t y 400 m o s m o l / k g ) in 4 s h e e p , t h e
any difference from baseline water intakes. Perfu-
r e s u l t s o f w h i c h a r e n o t listed in t h e t a b l e s . P e r f u s i o n
sion o f h y p e r o s m o t i c s o l u t i o n s d i d n o t i n d u c e w a t e r
of ouabain,
drinking
s h e e p at A D
10 _6 M , r e d u c e d t h e s o d i u m i n t a k e o f a n d A V p e r f u s i o n sites, b u t d i d n o t
different
from
baseline
in t h e
sodium-
d e p l e t e s h e e p b e f o r e t h e a c c e s s to s o d i u m . I n g e s t i o n
i n f l u e n c e i n t a k e at t h e P D a n d P V p e r f u s i o n sites.
of sodium
C o r r e c t i o n of s o d i u m deficit of 378 + 30 a n d 339 +
perfusions different from baseline intake. For exam-
66 m m o l w a s 6 3 % at A D , 6 7 % at A V at 60 rain, a n d
ple, s h e e p p e r f u s e d w i t h artificial csf at t h e A D
65%
all
d r a n k a m e a n o f 2.0 + 2.0 d e l i v e r i e s , a n d at A V a
baseline.
m e a n o f 4.8 + 1.7 d e l i v e r i e s of w a t e r by t h e e n d o f
at
AD
significantly,
and P
<
78£0
at
0.05,
different
Perfusion of monensin,
AV
at
120 rain, from
10 5 M , d i d n o t i n f l u e n c e
s o d i u m i n t a k e at e i t h e r p e r f u s i o n sites. T h e r a n g e o f
perfusion.
did n o t s t i m u l a t e w a t e r i n t a k e d u r i n g
Sheep
perfused
with
200
mM
Na-csf
d r a n k m e a n s o f 5.9 + 2.3 l A D ) , 7.1 + 2.7 ( A V ) , 6.0
s o d i u m deficit p r e c e d i n g p e r f u s i o n o f m o n e n s i n was
+ 2.1 ( P D ) a n d 7.2 + 1.7 ( P V ) d e l i v e r i e s . P e r f u s i o n
370 t o
of h y p e r o s m o t i c g l u c o s e - c s f , o u a b a i n a n d m o n e n s i n
389 m m o l ,
s i m i l a r to
the
deficit
before
perfusion of ouabain. W a t e r i n t a k e r e c o r d e d at 1 0 - m i n i n t e r v a l s f r o m
solutions coincided with water intakes within the above ranges.
356 TABLE I1 Sodium, potassium concentration (raM) and osmolality (mOsm/kg) of the infused (push) and withdrawn (pull) solutions during local perfusion
Values are mean _+S.E.M. Perfusate
n
Push [Na]
Pull [Na]
Push [K]
Pull [K]
Push Osm
Pull Osm
Artificial csf i.c.v. 200 mM Na-csf~ 200mM Na-csf Hyperosmotic Glucose-csf Ouabain 10 6M Monensin 10-.5 M
8 6 30 13 24 26
146.9 + 0.6 201.2 + 2.3 149.7 + 1.5 148.1+0.7 149.3_+0.7
147.4 + 0.6 159.0 + 0.7 184.4 + 4.3* 150.9 + 1.4" 148.6_+0.6 149.8_+0.8
2.74 + 0.03 2.79 + 0.04 2.87 + 0.02 2.82+0.05 2.83_+0.05
2.73 + 0.04 2.62 + 0.09 2.75 + 0.05 2.78 + 0.02* 2.84_+0.05 2.81_+0.05
285.3+ 0.4 381.2+ 5.2 396.6 _+2.8 288.3_+1.4 294.6_+2.1
288.7 +_ 1.3 311.8 + 1.7 352.4 + 7.8* 364.2 _+9.2" 288.9 _+ 1.2 297.8 + 2
csf sampled at the end of i.c.v, infusion. *P < 0.05.
The composition of infused and withdrawn solutions is p r e s e n t e d in Table II. The calculated loss from the perfused solutions was between 20 and 30%. Perfusion of o u a b a i n or monensin did not influence the composition of the csf. DISCUSSION Local perfusion of 200 m M Na-csf caused a reduction in the sodium intake of s o d i u m - d e p l e t e sheep. This result confirms earlier findings that i.c.v. infusion of h y p e r o s m o t i c Na-csf caused a decrease in the sodium intake of sodium-deficient sheep 23. The specificity of the effects of increased NaCl concentration as o p p o s e d to increased osmolality alone was tested by perfusion of h y p e r o s m o t i c glucose solution: increased osmolality of the perfusate failed to influence the sodium intake of s o d i u m - d e p l e t e sheep. Localisation of the sodium-sensitive elements which influence the sodium intake of s o d i u m - d e p l e t e sheep 4 was a t t e m p t e d using the local perfusion technique e m p l o y e d in earlier studies 3'18. Local perfusion of 200 m M Na-csf caused a reduction in sodium intake at all 4 perfusion sites tested. The local change in csf sodium concentration at the 4 sites m a y influence structures close to the wall of the third cerebral ventricle, including the whole of lamina terminalis, the anterior, dorsal and posterior h y p o t h a l a m u s , the medial thalamus, the h a b e n u l a r structures. The loss during peffusion of a b o u t 25% of infused solution might raise the sodium concentration of the csf d o w n s t r e a m from the perfused
sites, if the csf production rate is assumed to be 7.5 ml/h, by about 2 mM. During infusion of 200 m M Na-csf into the lateral ventricle the sodium concentration of the csf increased by 7.3 + 2.8 m M (from the pre-infusion level of 151.7 + 1.0 to 159.0 + 0.7 m M at the end of infusion), and this did not influence the sodium intake of sheep. T h e r e f o r e we have to conclude that delivery of 200 m M Na-csf by local peffusion increased t h e sodium concentration of the ecf, and p r e s u m a b l y the icf of brain, at a discrete area m o r e than delivery of the same solution by i.c.v, infusion. T h e local increase in sodium concentration was sufficient to induce changes in the correction of sodium deficit. O u a b a i n has b e e n d e m o n s t r a t e d to increase the intracellular neuronal sodium concentration in the cerebral cortical slices of the guinea-pig brain at concentrations of 10 -6 M or higher 17, and specific binding of o u a b a i n to the brain tissue of several other species has b e e n r e p o r t e d m. Local perfusion of o u a b a i n at the concentration of 10 -6 M caused reduction of sodium intake only when perfused at the anterior sites. O u a b a i n influenced sodium intake p r o b a b l y by reducing the icf sodium concentration in a neuronal p o p u l a t i o n close to the site of perfusion. It may be assumed that in the sheep the neural elements specifically sensitive to increased icf sodium concentration and participating in the regulation of sodium intake are located in the neuropil surrounding the anterior part of the third cerebral ventricle. T h e exact location of these hypothetical sensors, however, is unknown. A b l a t i o n of the lamina terminalis or A V 3 V a r e a did not disrupt the
357 replacement of sodium deficit in sheep 6. In rats, lesion of the AV3V area was reported to decrease the daily sodium intake 1, while lesion of the ventral nucleus medianus increased daily sodium consumption 1~, and both lesions failed to interfere with the increased sodium intake stimulated by s.c. injection of formalin or polyethylene glycol. The discrepancy between the more localised effect of ouabain and the widespread effective area of increased local sodium concentration may indicate that increased csf and ecf and presumably icf sodium concentration, due to the perfusion of 200 mM Na-csf, interferes with neuronal activity in other brain areas not specifically sensitive to changes in icf sodium concentration. These areas may include some parts of the neuronal network involved in the regulation of motivation and may decrease the drive for drinking sodium solution in response to sodium deficit. This may be true in sheep, but not in other species, e.g. rats and wild rabbits, in which increased csf sodium concentration failed to decrease the sodium intake stimulated by sodium deficiency7~9. Another explanation for the more localised effect of ouabain might be the following: the proposed sodium-sensitive neurones extend along the whole lateral wall of the third ventricle, but they are closer to the ventricular surface at the anterior than at the posterior half and sodium may move further into the neuropil than ouabain. It is interesting, that the effect of ouabain could not be reproduced by monensin, at the reported 10-5 M, and in pilot studies at 5 times higher concentrations. Monensin was demonstrated to increase intracellular sodium concentration in mouse neuroblastoma-rat glioma hybrid cells at a concentration of 20 glM~2~ and inhibit plasma membrane proteolipid synthesis in cultured embryonic rat neurones at 1 /~M 16. Monensin, however, was not tested in neurones in vivo and may not influence the neuronal sodium transport at all. Alternately, monensin may REFERENCES 1 Bcalcr. S.L. and Johnson, A.K., Sodium consumption following lesions surrounding the anteroventral third ventricle, Brain Res. Bull., 4 (1979) 287-290. 2 Bruning, J.L. and Kintz, B.L., Computational Handbook of Statistics, Scon-Foresman, Glenview, IL, 1977. 3 Cox, P.S., Denton, D.A., Mouw, D.R. and Tarjan, E., Natriuresis induced by localized perfusion within the third
not penetrate into the neuropil from the csf. Simultaneous administration of ouabain, 10 6 M , and monensin, 10-5 M, did not cause any changes in the sodium intake of sodium-deficient sheep different from that of ouabain alone (unpublished observations). The effect of local perfusion of 200 mM Na-csf on sodium intake was short, it lasted only during the perfusion. During the second hour of sodium access after the cessation of perfusion sheep drank further amounts of sodium and corrected their sodium deficit accurately. In contrast, the effect of local perfusion of ouabain persisted after the termination of perfusion. This is probably due to the slow release of ouabain from its specific binding sites in the brain ~9 and the prolonged specific effect of ouabain. Sheep showed no reaction at all to local perfusion of ouabain, and in contrast to studies where ouabain was infused into the carotid artery 5"2°, no change in water intake was observed. The changes in the sodium intake of the sodiumdeplete sheep in the present study were not preceded or accompanied by changes in water intake. This suggests that the sodium sensors involved in the regulation of the sodium intake of the sodiumdeplete sheep are distinct from, and probably more sensitive to changes in ecf and icf sodium concentration than those involved in the regulation of thirst ~3. ACKNOWLEDGEMENTS This work was supported by grants from the National Health and Medical Research Council of Australia. The excellent technical assistance of Mr. Robert J. Plenter and Mr. Brett Purcell is gratefully acknowledged. Preliminary results were presented at the NATO Advanced Research Workshop in Camerino, Italy, 1984, and at the Centennial Meeting of the Polish Physiological Society, Warsaw, Poland, 1987. cerebral ventricle of sheep, Am. J. Physiol.. 252 (1987) R1-R6. 4 Denton, D.A., The Hunger for Salt-- An Anthropological, Physiological and Medical Analysis, Springer, Berlin, 1982. 5 Denton, D.A., Kraintz, F.W. and Kraintz, L., The inhibition of salt appetite of sodium-deficient sheep by intracarotid infusion of ouabaim Commun. Behav. Biol., 4 (1969) 183-194. 6 Denton, D.A, McKinley, M.J., Tarjam E. and Weisinger,
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