Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands ... using both a peripheral and a central route of administration of these extracts ...
Brain Research, 113 (1976)499-516
499
~' Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF S U B F O R N I C A L O R G A N B A L A N C E 1N T H E R A T
EXTRACTS
ON
SALT-WATER
JOAN Y. SUMMY-LONG*, ISAAC L. CRAWFORD** and WALTER B. SEVERS Department of Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State Unive;sit),, College of Medicine, Hershey, Pa. 17033 (U.S.A.)
(Accepted February 4th, 1976)
SUMMARY The subfornical organ (SFO) is regarded as a neurosecretory structure but no information is available on the nature or biological effects of the secretory product(s). Supernatants of water homogenates of rat SFO were lyophilized and reconstituted in artificial cerebrospinal fluid (CSF). lntracerebroventricular (IVT), but not subcutaneous, administration of this material to rats produced diuresis, natriuresis and kaliuresis in the following 8 h daylight period. During the overnight cycle, consummatory behavior and excretion of sodium and potassium were reduced. Similar responses were obtained after 1VT administration of cerebellar cortex (CB) or large amounts of plasma. SFO, CB and cerebral cortex (CC) were incubated in potassiumenriched CSF to enhance release of secretory products. Urine volume was increased 8 h after 1VT injection of SFO media; in the overnight cycle, food consumption, absolute urinary sodium and potassium, and [Na +] were reduced. These effects were not produced by IVT injection of CC or CB media, or equal amounts of plasma proteins. Additional experiments demonstrated that choroid plexi and SFO effects were similar and that the active SFO material was dialyzable and thermal stable. These data suggest that SFO contains a water-soluble substance which is released into a potassium-enriched medium. The material is heat stable, has a relatively low molecular weight, and alters salt-water balance after injection into ventricular cerebrospinal fluid.
* These data are part of a thesis to be submitted as a partial requirement for the Doctor of Philosophy degree to The Pennsylvania State University. ** Present address: Departments of Neurology and Pharmacology, Southwestern Medical School, The University of Texas Health Science Center, Dallas, Texas 75215, U.S.A.
500 1NTRODtJCTI()N The subfornical organ (SFO) is a circumventricular structure located al lhe junction of the lamina terminalis and the tela choroidea of the third cerebral ventricle:L This small nodular structure is anatomically characterized by close surface contact with ventricular and subarachnoid cerebrospinal fluid 29, and by the absence of an endothelial cell ultrastructure usually found in cerebral capillaries and indicative of a blood-brain barrier 7,''j,a~. Data from light '5.11.~1 scanning 12,t9 and electron transmission microscopy ~,4,1s,''' suggest the presence of neurosecretory material within the SFO perikarya. Two types of neurosecretory granules have been reported in neuronal cell bodies: type 1 granules are Gomori positive, electron dense and are apparently discharged into pericapillary spaces; type I1 granules contain colloid material of low electron density and are oriented for release into subpendymal spaces ".4,18. Scanning electron micrographs depict a presumed secretory material on the ventricular ependyma covering the SFO in the rabbit le and ratl:L On the basis of anatomic evidence, several investigators postulated that the SFO contains and releases neurosecretory substances 2,~1,5.~l .le,18,~.9,'.~l, although the chemical nature and physiological actions of such secretory products are unknown. However, increased secretory activity or number of type 1 granules has been observed in response to osmotic stimuli s,z',~, prolonged anesthesia and cerebral manipulations 4, and alcohol or estrogen treatment 15. Such responses may also occur for type 11 granules during pregnancy~L The SFO has been implicated in the regulation of salt-water balance. Pharmacologic evidence and lesion studies suggest that this structure is necessary for the dipsogenic action of centrally or peripherally administered angiotensin I !~,"0,~'~.~7and intracerebroventricular carbachol '~8. In addition, ultrastructural changes characteristic of increased synthetic activity were observed within the SFO after water deprivation 6. Lesions of the SFO increased sodium intake "° and prevented the rise in sodium excretion usually observed after intracarotid infusion of hypertonic saline 3°. These and other reports ]6.17,e6 support the hypothesis that the SFO is involved in body fluid homeostasis. Since morphologic evidence suggests that the SFO contains neurosecretory products and pharmacologic data indicate that this structure may function in body fluid regulation, we investigated the effect of SFO extracts on salt-water balance in the rat. Because of the unique anatomic characteristics of the SFO and the possibility of neurosecretion into either vascular or ventricular compartments, we began our study using both a peripheral and a central route of administration of these extracts. Additional experiments were designed to characterize some physicochemical properties of the SFO extract. METHODS Salt-water balance studies Adult male Sprague-Dawley rats (275-465 g) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). A cannula 24 was positioned in the left lateral cerebro-
501 ventricle and fixed to the skull with acrylic cement and an anchor screw. After 4 days the rats were weighed and housed in individual metabolism cages under environmentally controlled conditions. The light cycle was 12 h light:12 h dark; lights on at 7 a.m. Powdered rat chow and water were avialable ad libitum. In each experiment there were no statistical differences (P ~ 0.05, t test) in body weight of the groups of animals to be given various treatments. The 2-day salt-water balance study began on the next day. The experimental protocol was similar each day and consisted of injections at 9 a.m. and 1 p.m. Water, but not food, was available during the daytime observation period (9 a.m.-5 p.m.). Both food and water were available during the overnight interval (5 p.m.-9 a.m.). Food and water intake and urinary parameters were measured separately for these time periods. Urine sodium and potassium concentrations were determined by flame photometry using an IL 143 flame photometer. On the first experimental day all rats received 10 ~ul of artificial cerebrospinal fluid (CSF 13) intracerebroventricularly (IVT) to determine inherent variability in the measured parameters. On the second experimental day, groups of rats (6-8/group) received 10/zl of CSF, SFO preparation, or another similarly prepared tissue control. Tissue controls, either from the cerebellar cortex (CB), cerebral cortex (CC) or choroid plexus (CP), were tested simultaneously in all experiments to assess the specificity of any observed SFO activity. CSF treated rats served as a control for any vehicle, injection or handling effects. Data were analyzed using the permutated Student's t test. Effects of SFO injections were considered 'specific' if 3 statistical criteria were simultaneouly met: (1) SFO differed from CSF (P < 0.05), (2) SFO differed from tissue control (P < 0.05), and (3) CSF and tissue control were similar (P ~ 0.05). In some experiments the relationship between overnight food and water intake was estimated by linear regression analysis. Microdissection Male Sprague-Dawley rats in normal salt-water balance were sacrificed by decapitation. The brains were removed and kept in ice. In the microdissection procedure, ventral hypothalamic tissue was aspirated until the third ventricle was exposed and the convergence of the choroid plexus onto the subfornical organ area was visualized (Fig. 1A) with a stereomicroscope. Fig. 1 also illustrates the macro- and microscopic anatomy of this region prepared from a tissue block dissected from fixed brain using this approach. The SFO was removed by transecting the hippocampal commissure just above and below the SFO protrusion, and by lateral cuts bordering the fornix. The SFO tissue section thus contained a small amount of choroid, hippocampal commissure and fornix. In some experiments SFO activity was studied without CP by careful excision of the plexus before SFO removal. Tissue samples from the temporal region of the cerebral cortex, the cerebellar cortex, and choroid plexi from the lateral and third ventricles were also used in various experiments. Approximately 1.5 mg of individual tissue samples were removed from each rat. Microdissections were completed within 5 min after sacrifice.
502
Fig. 1. Morphology of the rat subfornical organ (SFO). A: ventral exposure of the ros~ral surface ~:,~ the third ventricle ( : 5: as seen in microdissection procedure) reveals a protruskm, the SFO. b ~ m d anteriorly b~, the columns of the l\~rnix (Fx) and posteriorly by the dentate gyri of he h p p o c a m m ~ (H). The highly vascular choroid plexus (CP) converges onto the area: CP also is seen in the Ic|t lateral ventricle. B: in the low power photomicrograph microdissected in a similar manner I ~t;: thin Epon section with Azure II and toluidine blue), SFO parenchyma lies subjacent to the hip!~,> campal commissure (HC), connects direclly to CP and contains large sinusoidal ,~cssels (SV;~: ~ts ependymal surlace is bathed by ventricular cerebrospinal fluid (CSFL (7: the cell type seen mosl oflcn in light micrographs ( 400; same Epon section as B) is moderate in size wilh a rounded or a\.oid nucleus containing a prominent nucleolus, and has occasional lipid droplets and dense microbodies in the cytoplasm (arrow). Note the enlarged adventitial space surrounding the capillaries (c). 1): m the electron micrograph ( 23,000; Epon section from (7, glutaraldehyde osmium fixation, uranyJ acetate stain), the SFO ventricular surface is covered by ependymal cells with canaliculi (arrow) ~ hich provide extensive connections between CSF and SFO parenchyma. Small ovoid or elongated mit,,~chondria are interspersed in the cytoplasm. The capillary membranes are fenestrated and con~ai~ pinocytotic vesicles.
503
Extraction Pooled samples of individual tissues were homogenized in cold deionized water (1:100, w/v) and centrifuged at 40,000 × g for 30 min; supernatants were lyophilized to dryness. Residues were resuspended in CSF or saline so that the protein contents 14 of the final injections were approximately equal. Extracts of tissue from 3 to 4 rats were eventually used for injection into each test rat. In a thermal stability experiment, SFO water homogenates were centrifuged and the supernatant divided in half: one portion remained on ice, the other was heated to 100 °C for 10 min. Heat-formed precipitate was removed by centrifugation; then both supernatants were lyophilized. In a separate experiment the effect of dialysis on SFO activity was determined by placing half of the water-extracted supernatant (2.5 ml) inside Fisher (No. 3787-D12, 0.25 in.) tubing which was then dialyzed twice against 60 ml deionized water for 6 and 12 h at 4 °C. The two dialysates were combined and lyophilized to dryness. The other half of the SFO supernatant was refrigerated for 6 h before lyophilization. Incubations After each tissue (SFO, CB or CC) was removed, it was bisected and immersed (23 °C, 5-10 rain) in 0.1-0.2 ml of CSF containing either 5.6 mM K + and 2.2 m M Ca 2+, or 56 m M K~ and 2.2 m M Ca 2+, made isotonic by appropriate reductions of sodium and magnesium molarity. Elevations in K + and Ca 2+ have been reported to increase the release of secretory material in vitro 9,10. Multiple tissue samples (4-5) were incubated for each rat injected IVT. The media were centrifuged, then either injected immediately or frozen overnight for administration the following day. In one experiment incubation medium and control 56 m M K + CSF were lyophilized for a short time to concentrate the sample. The total volume was adjusted to 0.2 ml with deionized water before individual injections. This procedure was necessary to adjust the dose which was standardized by protein content. Plasma studies To evaluate the effect of IVT injections of plasma, trunk blood from normal animals was collected into heparinized tubes (100 U/tube), centrifuged and the plasma frozen until used. Thawed plasma was diluted to appropriate protein concentrations with CSF before lVT injections. The salt-water balance protocol was then followed. Peripheral injections An experimental protocol similar to that used in the above salt-water balance studies was followed except IVT cannulae were not implanted. Subcutaneous (s.c.) injections of either 0.5 ml saline or a water extract (SFO, CB) were administered. RESULTS The day-to-day variability of the measured parameters was evaluated in the various experiments. No differences (P > 0.05) occurred in individual groups of rats receiving CSF on two successive experimental days. Differences (P < 0.05) in urinary
504 TABLE I Effects of water extracts of the subfornical organ area on consummatory behavior and urinary parameters: intraventricular and subcutaneous administration Subfornical organ (SFO) and cerebellar cortex (CB) tissues were microdissected, homogenized in cold deionized water and centrifuged at 40,000 × g for 30 rain. Supernatants were lyophilized and resuspended in either artificial cerebrospinal fluid (CSF) for injection into the left lateral cerebroventricle (IVT) or in 0.9 ~ saline for subcutaneous administration (s.c.). Similar protein concentrations of SFO and CB extracts, 179/~g/10/d 1VT, and 166/tg/0.5 ml s.c., were administered in each experiment at 9 a.m. and 1 p.m. Consummatory behavior and urine parameters were measured during the initial 8 h (9 a.m.-5 p.m.) and the overnight period (5 p.m.-9 a.m.). Values presented are means ± S.E.; probabilities are from Student's t test. Number of animals are shown in parentheses. NS -- nonsignificant differences (P > 0.05). Treatment
Consummatory behavior
Urine data
0-8 h
Overnight
0-8 h
Drinking (ml)
Drinking (ml)
Eating (g)
Volume (rot)
Sodium (mEquiv./l)
(mEquiv.)
IVT CSF (27) CB (19) SFO (24)
0.8±0.3 0.5±0.2 0.2 ± 0.1
36.8±1.2 31.4±1.7 19.2 ± 1.7
20.8±0.9 15.0±1.4 7.0 ± 1.0
6.3±0.4 10.9±0.7 13.8 ± 0.6
121±11 114±6 108 ± 3
0.75 ± 0.07 1.22 ± 0.09 1.48 ± 0.06
P values CSF vs. CB CSF vs. SFO SFO vs. CB
NS NS NS
< 0.02 < 0.001 < 0.001
< 0.001 < 0.001 < 0.001
< 0.001 < 0.001 < 0.005
NS NS NS
< 0.001 < 0.001 < 0.02
S.C. Route Saline (8) CB (8) SFO (8)
0.5 ± 0.2 0.8 ± 0.3 1.5 ± 0.3*
41.4 ± 2.8 42.0 ± 1.5 41.0 ± 1.9
22.5 ± 1.8 25.1 ± 1.7 21.9 ± 1.3
86 ± 13 107 ± 22 102 ± 10
0.61 ± 0.15 0.55 ± 0.10 0.45 ± 0.06
6.6 ± 0.7 5.2 ± 0.8 4.5 i 0.4*
* Significantly different from saline control (P < 0.05).
[ N a +] a n d [K +] in t h e 0 - 8 h p e r i o d w e r e o c c a s i o n a l l y o b s e r v e d a m o n g t h e g r o u p s o f rats o n the d a y w h e n all a n i m a l s r e c e i v e d C S F i n j e c t i o n s . O c c a s i o n a l l y , a n u n e x p e c t e d d e c r e a s e in o v e r n i g h t c o n s u m m a t o r y
b e h a v i o r o c c u r r e d , usually w h e n f l u c t u a t i o n s
in e n v i r o n m e n t a l c o n d i t i o n s ( t e m p e r a t u r e ,
h u m i d i t y a n d / o r light cycle) w e r e n o t
a d e q u a t e l y c o n t r o l l e d . T h e results f r o m these e x p e r i m e n t s w e r e d i s c a r d e d . E f f e c t s o f water extracts o f the subfornical organ area I n t r a v e n t r i c u l a r i n j e c t i o n s o f S F O w a t e r e x t r a c t s (179/~g p r o t e i n ) p r o d u c e d : an initial ( 0 - 8 h) i n c r e a s e in u r i n e v o l u m e , a b s o l u t e s o d i u m a n d p o t a s s i u m e x c r e t i o n , a n d a s i m u l t a n e o u s d e c r e a s e in p o t a s s i u m c o n c e n t r a t i o n ( T a b l e l). I n t h e o v e r n i g h t period consummatory behavior, urine volume, sodium and potassium concentrations and the absolute amounts
o f these c a t i o n s w e r e all d e c r e a s e d . W a t e r e x t r a c t s o f
c e r e b e l l a r c o r t e x (CB), a n o n - p e r i v e n t r i c u l a r tissue c o n t r o l , p r o d u c e d q u a l i t i v e l y similar effects o n s o m e , b u t n o t all, o f t h e p a r a m e t e r s affected by S F O ( T a b l e I). W a t e r e x t r a c t s o f t h e S F O p r o d u c e d 'specific' c h a n g e s as d e f i n e d e a r l i e r (i.e., S F O ¢ C S F
505
Overnight Potassium (mEquiv./1) (mEquiv.)
Volume (ml)
Sodium
Potassium
(mEquiv,/l)
(mEquiv.)
(mEquiv./l)
(mEquiv.)
130±7 109±9 82±4
0.79±0.06 1.13 ~ 0 . 0 8 l.ll ±0.05
13,5 i 0.6 11,2±0.5 9,2 ± 0.5
151 ± 7 132±9 8 4 ± 10
1.98±0.09 1.48±0.12 0.76±0.10
259± 11 242± 13 194~ 13
3.38 ~ 0.10 2.66 ~ 0.15 1.74i 0.12
NS < 0.001 • 0.01
< 0.001 < 0.001 NS
< 0.01 < 0.001 < 0.01
NS < 0.001 < 0.005
< 0.005 < 0.001 < 0.001
NS - 0.001 • 0.02
~. 0.001 < 0.001 0.001
84 :t- 14 1 0 0 i 17 118 ~ 11
0.61 i 0.14 0.54±0.12 0.53i0.07
13,6 i 1.7 14,6±1.6 13,2±1.4
162 ± 12 162±11 166±7
2.19 i 0.31 2.35±0.27 2.2110.27
276 ± 19 2705-15 269±10
3.62 ± 0.43 3.87 i 0.33 3.54£0.39
a n d CB; CB ~ CSF). These included a decrease in 0-8 h a n d overnight u r i n a r y [K +] a n d o v e r n i g h t [Na+]. S u b c u t a n e o u s a d m i n i s t r a t i o n of S F O p r o d u c e d a small increase in water intake a n d a slight decrease in urine v o l u m e in the 0-8 h interval when c o m p a r e d to saline, b u t n o t the CB, treated rats (Table 1). Since parameters were n o t affected in a 'specific' m a n n e r by s.c. SFO, all further experiments utilized the central (IVT) route of administration.
Effects o f dialyzed and heated subfornical organ extracts Because the volume of dialysate which had to be lyophilized was large, the S F O water extract a n d the dialysate prepared from a p o r t i o n of it were tested in separate groups of rats with separate controls. The S F O dialysate, c o n t a i n i n g material with an a p p r o x i m a t e molecular weight < 40,000, p r o d u c e d effects different from C S F injections. These effects were qualitatively similar to those observed with the non-dialyzed S F O water extract except s o d i u m a n d potassium c o n t e n t of the 0-8 h urine did n o t
506 TABLE 1I Eff~cts of dialyzed or heated sublornical organ water extracts on consummatory behavior and urinary parameters : intraventricnlar admhfistration
Water homogenates of subfornical organs were centrifuged (40,000 × g, 30 rain); part of the supernatant was lyophilized, and the remainder dialyzed then lyophilized as described in the text. The undiatyzed supernatant and the dialysate were assayed in separate experiments. Temperature stability was studied by heating supernatants of SFO ho;nogenates (100 'C, 10 rain); flocculates were removed by centrifugation and the material lyophilized. All material was reconstituted in CSF prior to use; injections (10/d) were made at 9 a.m. and 1 p.m. Protein content of SFO (supernatant) and SFO dialysate injections was approximately 55/~g; heated SFO (SFO, 100 C ) injections contained 48/~g protein. Consummatory ~ehavior and urir,e parameters were measured during the initial 8 h (9 a.m.-5 p.m.) and the overnight period (5 p.m. - 9 a.m.). Values presented are means ± S.E.; probabilities are from Student's t test. Number of animals shown in parentheses. Treatment
CSF (8) SFO (8) CSF (8) SFO Dialysate (8) CSF (7) SFO--100 ~C (8)
Consummatory behavior
Urine data
0-8 h
Overnight
08h
Drhtking (ml)
Drinking (ml)
EathTg (g)
Volume (ml;
1.0 ± 0.3 0.5 ± 0.3 0.5 ± 0.2 0.8 ± 0.4 1.4 -- 0.5 1.4±0.8
35.1 ~ 3.2 23.8 ~ 3.2* 30.8 -~-4.1 10.4 ± 3.4** 34.3 ± 5.0 23.1 ~-4.4
20.4 ± 1.4 8.8 ± 1.4"* 17.5 ± 2.3 3.6 ± 1.6"* 16.8 ± 2.9 8.6±2.1"
5.6 12.7 ± 9.4 ± 14.8 ± 10.1 13.0 ±
Sodium
0.7 0.6"* 1.3 1.5" 0.9 1.0"
(mEquiv./I)
(mEquiv.)
136 4- 14 111___8 143 ± 18 122 ± 6 96 + 9 117 ~ 9
0.72 ± 0.09 1.42 ± 0.13"* 1.30 _-4-0.19 1.78 ± 0.13 0.98 ± 0.15 1.49 ± 0.12"
* Significantly different from appropriate CSF control (P < 0.05). ** P
