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c-Fos protein expression in the rat subfornical organ following osmotic stimulation
:Veuro.sciencc l, ellct,s'. 139 (1992) I 6 , 1992 Elsex, ier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940,'92/$ 05.00
1
NSL 08573
c-Fos protein expression in the rat subfornical organ following osmotic stimulation Lisa G i o v a n n e l l i ~' and F l o y d E. B l o o m h ~l)~partmcHI o/P/tarHlacolok,)', (,)Hvt'rsit) O/ Florence, Florence i llall ) aml hDepartment olNeuropharntacolo,w, .S'cripl~
The subfornical organ (SFO) is one of the circumventricular structures of the brain involved in the regulation of body fluids [13, 28]. Together with other structures of the lamina terminalis region, the SFO, in particular, is thought to mediate drinking behavior induced by osmotic stirnuli or Angiotensin 1I (ANG II [15, 29, 30]). The efferents pathways of the SFO which have so far been identified project to the preoptic hypothalamic region and to the magnocellular neurosecretory hypothalamic nuclei [20, 22]. The latter have previously been shown to mediate the increase of both vasopressin (VP) and oxytocin (OXY) release induced by A N G II [7, 14]. We and others have previously used c-Fos protein expression as a marker of activation of magnocellular ncurons in the hypothalamus following hypertonic stimulation [3, 9]. As a result of such an activation, wlsopressin and oxytocin are released both centrally and in the plasma to restore the normal body fluid homeostasis by means of water retention and Na excretion [17,311. The aim of the present study was to investigate whether osmotic challenge induces c-Fos protein expression in the SFO component of the neural circuitry responsive to osmotic stimulation. Preliminary evidence (',Jrre,vJomtcm'e: 1,. Giovannelli. Department of Pharmacology, University of Florence. giale Morgagni 65, Florence 50134, haly. Fax: (39) 55 4361613
that this was the case led us to study the time course of this effect in order to define tile sequence of activation o[" circumventricular and hypothalamic neurons following this kind of stimulation. We also subsequently imposed electrolytic lesions on the SFO to clarify whether the SFO efl'erent pathway to the magnocellular nuclei is essential for the activation of vasopressinergic and oxytocinergic neurons in response to hypertonic stimulation. The male Sprague-Dawley rats used in these experiments weighed 250-300 g. The animals were given Ik)od and water ad libitum and maintained under a normal light dark cycle. During the week before the experiment, the animals were handled daily for about 15 rain. On the 8th day, the experimental groups (at least 4 animals per group) were injected i.p. with 1 ml per 100 g body weight of hypertonic saline solution (1.5 M NaCI) and sacriliced at different times (15-180 mint after the injection. The control groups were treated in the same way except lk~r being injected with isotonic saline solution (0.9% NaC1). At the time of sacrifice, the animals were anesthetized with chloral hydrate (35 mg/100 g b.wt.) and perfused through the ascending aorta with 50 ml of 0.9% NaCI followed by 300 ml of ice-cold 2% paraformaldehydc in phosphate-buffered saline (PBS). The brains were subscquenlly taken out of the skull, cut in blocks and postfixed in the same fixative for an additional 2 h. They were then cryoprotccted by incubation in 30% sucrose overnight, and cut in a cryostat. Coronal sections 20 # m thick were
obtained, mounted on gelatin-coated slides and processed for immunohistochemistry. A sheep polyclonal antibody raised against the N-terminal peptide of the c-Fos protein (Cambridge Research Biochemicals) was used at the dilution of 1:1000 in PBS. After a blocking step (incubation with 3% normal horse serum and 0.3% Triton X-100 in PBS), the sections were incubated overnight at room temperature with the primary antibody in the presence of Triton and 1% horse serum. On the following day the peroxidase-avidin- biotin procedure (Vector ABC kit) was used to localize the primary antibody. The peroxidase color reaction was allowed to proceed for about 4 min in the presence of diaminobenzidine and 0.04% nickel chloride. The slides were finally washed in PBS, dried, coverslipped and analyzed under a Zeiss Axiophot microscope. Lesions of the subfornical organ were performed two weeks before the experiment. The animals were anaesthetized with halothane, placed on a stereotaxic apparatus and maintained under halothane and oxygen for the duration of the surgery. A 1 mA current was passed for 10 s in each of 4 different rostro-caudal locations ( 1.1,
1.2, 1.3 and 1.4 m m posterior to bregma, depth 6.2, ~. !. 5.2 and 5.2 m m from the skull) between the top of an insulated stainless steel electrode and a rectal anode. The sham-operated animals were treated in the same way with no current passed through the electrode. The location of the lesion was checked after the experiment, by staining coronal sections through the SFO, prepared as described above, with Richardson's stain. The extent of the lesion was estimated by comparing corresponding sections of the SFO in the lesioned and sham-operated animals. Nuclear staining for c-Fos protein was identified in numerous cells in the subfornical organ as early as 30 min alter hypertonic saline injection (Figs. lb and 2b), No difference between the hypertonica]ly stimulated and the control animals was found 15 min after the injection (not shown) and only very few c-Fos-stained nuclei were found in the SFO of animals injected with isotonic saline 30 rain betbre sacrifice (Figs. l a and 2a). The expression o]" c-Fos protein in the SFO was still present at 1 h alter osmotic stimulation (Figs. lc and 2c) and declined by 3 h (Figs. ld and 2d). Stained nuclei were found throughout
Fig. 1. c-Fos-immunoreactivenuclei in the rat rostral SFO (bregma -0.60 mm [25]) 30 min alter isotonic saline (a), and 30, 60 and 180 min alter hypertonic saline injection (b. c. d. respectively).Arrows indicate examples of labelled nuclei. Bar -- 100 #m,
the whole rostro-caudal extension of the SFO (Figs. 1 and 2). The electrolytic lesion of the SFO area induced a loss of about 75% of the SFO in 3 out of 8 animals, while 30 50% of the SFO resulted in damage in the remaining 5 rats. Also in the animals with 75% damage of the SFO, the very anterior part of the SFO (rostral to bregma 0.60 mm [25]) was undamaged. Only 2 of the lesioned animals lost weight after surgery as compared to the sham-operated. No correlation was found between weight loss and extent of the lesion. In none of the lesioned animals, did damage of the SFO prevent c-Fos protein expression in the hypothalamus 30 min after hypertonic stimulation. As reported previously [9], such expression was concentrated in the paraventricular (PVN, Fig. 3) and supraoptic (SON) nuclei, and scattered throughout the anteriot and lateral hypothalamus (not shown). Hypertonic stimulation did not induce c-Fos expression in the nucleus tractus solitarius (NTS), where only a few c-Fos-immunoreactive nuclei were found in both the isotonic (n - 4) and hypertonic (n = 4) saline injected
animals after 30 min from the stimulation (Fig. 4). No difference in c-Fos expression between osmotically stimulated and control animals was found in the NTS 15 min after injection, nor in the ventrolateral medulla (VLM) or the locus coeruleus (LC) after 15 or 30 rain (not shown). We have shown that osmotic stress induces c-Fos protein expression in the rat SFO, and that such an effect is evident at 30 min after stimulation and lasts for about 3 h. While the present paper was submitted, a report was published that showed induction of c-Fos immunoreactivity in the SFO following intravenous hypertonic saline administration [23]. c-Fos gene expression has been widely used in the last few years as a marker of neuronal activation in response to a w~riety of stimuli [2, 6, 12, 26] because of its rapid onset [27]. The fact that a population of neurons in the SFO responds to osmotic stimulation is not surprising in view of the well known role of this circumventricular structure in the regulation of body fluid homeostasis in response to osmotic and hypow)lemic stimuli [13, 28, 29]. In contrast, we found no effect of os-
a
¢
II
Fig. _. c-Fos immunoreactivity in tile rat caudal SFO (brcgma 1.40 mm [2511 30 rain after isotonic saline (a), and 30, 60 and 18(1 rain after hypcrtonic saline injection (b, c. d, respectively). Arrm~s indicate examples of labelled nuclei. Bar I00 Ira1. :
Fig. 3. Microphotographs of rat caudal SFO stained with Richardson's solution (a, c) and of'the PVN of the same animals inmmnostained for c-Fos protein (b, d). a, b: sham-operated animal, c, d: animal with electrolytic lesion of the SFO t75% organ loss). III. Ihird ventricle. Bar 100/.q'n.
motic challenge on c-Fos expression in the brainstem catecholaminergic nuclei which have also been reported to relay baroreceptive informations from the periphery to the CNS, that is the NTS, LC and VLM [5, 19, 24]. The hypothalamus is the other major brain structure which, along with the circumventricular organs, has long since been implicated in the response to osmotic stimula-
Fig. 4. c-Fos-immunoreactive nuclei in the rat NTS (bregma 13.80 mm [25]) 30 min after injection of isotonic (a) or hypertonic saline solution (b). AP, area postrema; Gr. nucleus gracilis. Arrows indicate examples of labelled nuclei. Asterisks indicate the central canal. Bar I00/am.
tion [1, 10, 21]. Induction o f c - F o s protein immunoreactivity in the paraventricular and supraoptic nuclei of the hypothalamus after hypertonic saline rejections has been demonstrated by this lab and others [3, 9, 27]. We have evidence that the onset of the magnocellular nuclei activation is at about 30 min after osmotic stimulation (Giovannelli et al., manuscript submitted), which is the same time at which the SFO starts to express c-Fos. In view of the existence of a pathway connecting the SFO to the PVN and SON [20, 22], it could be hypothesized that the hypothalamic response to osmotic stimuli is mediated by the SFO, as is the response to A N G II [7, 14, 29]. In our hands, however, animals with a 75% lesion of the SFO showed no reduction of PVN and SON activation after hypertonic injection, as measured by c-Fos immunostaining. A lesion of similar extent has been shown by Weisinger et al. [32] to decrease sodium appetite in rats, The same authors have shown that SFO lesions do not alter water intake in response to hypertonic saline injection. Furthermore, Knepel et al. [16] demonstrated that SFO lransection does not modify.' vasopressin release induced
by hypertonic saline injection. Even though we have not performed a total ablation of the SFO, leaving the very rostral portion intact in each of the lesioned animals, these data suggest that at least the central and caudal portion of the SFO and the hypothalamus are separately and simultaneously activated by osmotic stimuli. More complete SFO lesions will be necessary in order to extend such a conclusion to the whole SFO. It has been shown that an intact SFO is essential t\)r A N G ll-mediated VP and OXY release [7, 14, 16]. Our experiments show that most of the SFO is not necessary for magnocellular hypothahtmic neurons to respond to hypertonic stimulation, leading to the hypothesis that the SFO neuronal population which responds to osmotic stimuli by expressing c-Fos is probably not the same which is involved in the response to A N G II, nor does it project to the PVN and SON. Such a popuhttion could consist of osmosensitive neurons projecting to other brain areas, like the anterior third ventricular region (AV3V [13,201). In fact, the regulation of VP and OXY release upon osmotic stress appears to be a complex one, involving projections n o t e l f l y f r o m the S F O but a l s o fronl o t h e r hypothalamic areas, as the AV3V and the median preoptic nucleus (MnPO [4, 10, 11]). Lesions of both the AV3V and the MnPO have been shown to impair fluid balance and VP and OXY release in response to osmotic stimuli [8, 13] and the AV3V is thought to contain osmosensitive neurons itself [11]. Furthermore, magnocellular hypothalamic neurons have been shown to be osmosensitive [18, 21]. Thus, it can be hypothesized that after the impairment of the pathway conveying inlk~rmation fl'om the SFO to the hypothalamus, VP and OXY neurons can still be activated by blood hypertonicity either directly or through other pathways. We thank Dr. George F. Koob tbr fruitful discussion and Robert Lintz for technical help during animal surgery. This study was supported by Grant AA 06420. This is Scripps Research Institute paper n. 7096. 1 Abe, H. and Ogata, N., Ionic illcchanJsnls for the osmotically-induced depolarization in neurones of the guinea-pig suprm)ptic nuc l e u s q n \ , i t r o ' , J . Physiol.. 327 (1982) 157 171. 2 Aronin. N., Sagar, S.M.. Sharp, F.R. and Schwartz, W.J., Light rcgulatcs expression of a Vos-rchlted protein in rat suprachiasmalic nuclei, Prec. Natl. Acad. Sci. USA, 87 (1990) 5959 5962. 3 ('cccatclli. S,. Villar, M.J.. Goldstcm, M.J. and H6kfelt, T., Expression of c-Fos inlmunoreaclivity in transmitter-characterized ilCtlrons after stress, Proc. Natl. A c a d S c i . USA, 8611989} 9569 9573. 4 Chaudry, M.,,\., Dyball, R.E.J., Honda, K. and Wright, N.C., Fhe role of interconnection between supraoptic nucleus and anterior lhird vcntricular region in osmoreguhition in the rat, J. Physiol.. 411) (19891 123 145, 5 Cunninghanl Jr.. E.T. and Sawchcnko, P.E.. Anatonlical spccilici D
of noradrencrgic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus, J. Getup, Neurol., 274 [1988) 60 76.
6 Dragunow. M. and Robertson. H.A.. Kindling stimulation induces c-Fos protein(s) m granule cells of the rat dentate gyms, Nalure, 329(1987) 441 442. 7 Ferguson, A.V. and Kastmg, N.W., Angiotcnsm acts al the subfornical organ to increase plasma OXylochl concentrations in the roll. Rcgul. Pcpt.. 23 (19b;S) 343 352. 8 (icu'diner. T.W.. Vcrbalis, J.G. and Stl-ickcr, E.M., lmpah-cd secretion of ,.asopl'cSSill and oxytocin in rats ariel+ Icsiolis of Ihe llucleus rncdianus, A m . J . Physiol.,2491191'¢S)R681 688. O Gio~annclli. L.. Shh'omani, P.J.. ,lirikm~ski. G.F. and Bloom. ICE., Ox\ teem not.trollS in the rat hypothahnnus exhibit c-Fos inlnlunoreactivity LlpoI1 osnlotic stress, llrain Rcs., 531 (1990) 299 303. 10 Honda, K., Ncgoro. H., tliguchi. T. alld Tadokoro, Y., Acii~,ation of net.lroseclclof\ cells b~ osnlotic slhlltlldiioi1 o1" anlel'O~.cnlral thirdvcntricle, Am.J. Ph',siol..252(1987)R1030 ItJ45. I1 HolIda, K.. ~CgOfO, tt.. Dyball, R.E.,I., Higuchi. 1. cilld Takano, S.. The osmoreccptor conlplcx in the ial: cxidcncc for mtorciclions bclwccn /he stipraoptic ~llld otl'ler dicnccphalic nuclei..1. Physiol., 431 (199fl)225 241. 12 Hunl, S.P., Phli, i\. and EVClll, (7.. Indtlction ot'c-Fosqikc pfolchl hi spinal cord IlCLIfons I\~llowing SCllsor\ stimulation. NalLil-C, 3_~ (1987) 632 634. 13 ,Iohnson. A.K.. "Fhc perivcntricular anlcrovcntra[ lhird vcn/riclc (AV3V): it> rchllionship ~ith the >ubfornical organ and neural systems inxol\cd in maintahmlg both fluid homeostclsis, Brain Rcs. Bull., 15 119<<'45)595 601. 14 lo~ino. M. clnd Sleaido, L.. Vasoprcssin I'elca~,e io ccnlral and pcriphcrcll ailgiotcnsin 11 in l'tlls with lesions of the scibli)rnical orgl.ln, Bl'Clin Re,>.. 322 119841 365 368. 15 Ishihashi, S i. ()t)l]}LII'{I, 5".. (;ucgucn. B. and Nicolaidis, S.. Neuronal responses in stibl'ornical organ and other regions Io angiotcnsin I1 applied b) various fotllC~,, l/ram Rcs. Bull.. 14 (1985) 307 314. 16 Knepcl, W.. Ntlt[o. D. and Me}or. I).K.. El'feel of transeclion of subl\~rnica[ organ ell'trent projections on \asoprcssin release illduced b} angio[cnsin or isoprcnalinc in the ral, Brain Rcs., 248 [1982) 180 184. 17 Landgraf. R., Ncumann. I. and Schwarzbcrg. H.. Central and pcriphcl'al release of \asoprcssin and oxytocin ill the consciotls iLll :.i['tCl osmotic slimulation, Brain Rcs.. 457 ( 19Sb;i 219 225. 18 l,cng. G.. Mason, W.T, and D~ci-, R.(i,. The supraoptic nucleus as an osmorcccptor. Ncurocndocrmologx, 34 119821 75 82. 19 Lightmun. S.I,., ]'odd, K. and E\erilt, B.J., Ascending noradrcnergic projcclions fron'l brainsicnl: e'~idcncc t'or Cl m~ljor roic in the regulation of blood pressure and \'nsoprcssin secretion, Exp. Brain Res..55(1984i 145 151. 2(1 l.ind. R.W.. Van Hocscn. G.W. and ,lohnson, A.K.. An H R P study of the connections of the subfornica] organ of the roll, J. (k)nlp. Nctirol., 210 11982) 265 277. 21 Mason. w . r . . Supraoptic llOUrOllS of the rat hypolhalaFnus arc osnloscnsiti\c. ,'~CI[LII'C,287 [It)SOl 154 156. 22 Misclis. R.P,., The efferent projeclions of Ihc subfornical organ of the iq.tl: a circumventricular el-Still \\ilhm a neural network subserving warm- bahmcc, Brain Res.. 230 (1981) I 23. 23 Oldflcld. B.J.. Bickncll, R.J.. McAIIcn, R.M.. Wcisingcr, R.S. and McKinley. M.,I., lntravcnot.is hypcrionic saline induces Fos inlmunorcactivity in neurons throughout the lamina tcrminalis. Brain Rcs..561 11991i 151 156. 24 Palko\vitz. M. and ZclbOl-szk}, l.., NctlFo:.nl{itolllv of central cardio-
25 26
27
28
vascular control. Nucleus tractus solitarius: afferent and efferent connections in relation to the baroreceptor reflex arc. In W. de Jong, A.R Provoost and A.R Shapiro (Eds.), Hypertension and Baroception Mechanisms, Elsevier, Amsterdam. 1977, pp. 9 34. Paxinos, G, and Watson. C.. The Rat Brain in Stercotaxic ('oordinates, 3rd edn., Academic Press, Orlando, 1988. Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-Eos protein in the brain: melabolic mapping at the cellular level, Science. 240 (1988) 1328-1331. Sharp, F.R., Sagar, S.M., Hicks, K., Lowenstein, D. and Hisanaga, K,, c-Fos mRNA, los, and los-related antigen induction by hypertonic saline and stress, J. Neurosci., 11 (t991t 2321 2331. Sibbald, J.R., Sirett, N.E. and Hubbard. ,1.1., Osmosensilivc neurons in the rat subfornical organ, Proc. Univ. Otago Mcd. Sch., 62 (19841 93 95.
29 Simpson, J.B., The circumvemricular organs and tile central acliotl~ of angiolensin, Neurocndocrinology, 32 ( 19~:I ) 248 256. 30 Van Houten, M., Mangiapanc, M.L.. RcM, I.A. and Ganong~ W,F., [Sar I, AlaS]-angiotensin II m cerebrospmal fluid blocks the binding of blood-borne [~>l]-angiotensin 11 Io ihe drcumventricul~ r organs, Neuroscicnce, 10 11983) 1421 1426 3t Verbalis, J.G., Mangione, M.P. and Strickcr, E.M., Oxytocin produces natriuresis in rats at physiological plasma concentrations. Endocrinology, 128(19911 1317 1322. 32 Weisinger. R.S., Denton, D.A., Di Nicolantonio, R.. Hards, D,K., McKinley. M.,I., Oldfield, B. and Osborne, P.G.. Sublornical organ lesion decreases sodium appetite in the sodium-depleted rat, Brain Res.. 526 (1990t 23 30.
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NSL 08573
c-Fos protein expression in the rat subfornical organ following osmotic stimulation Lisa G i o v a n n e l l i ~' and F l o y d E. B l o o m h ~l)~partmcHI o/P/tarHlacolok,)', (,)Hvt'rsit) O/ Florence, Florence i llall ) aml hDepartment olNeuropharntacolo,w, .S'cripl~
The subfornical organ (SFO) is one of the circumventricular structures of the brain involved in the regulation of body fluids [13, 28]. Together with other structures of the lamina terminalis region, the SFO, in particular, is thought to mediate drinking behavior induced by osmotic stirnuli or Angiotensin 1I (ANG II [15, 29, 30]). The efferents pathways of the SFO which have so far been identified project to the preoptic hypothalamic region and to the magnocellular neurosecretory hypothalamic nuclei [20, 22]. The latter have previously been shown to mediate the increase of both vasopressin (VP) and oxytocin (OXY) release induced by A N G II [7, 14]. We and others have previously used c-Fos protein expression as a marker of activation of magnocellular ncurons in the hypothalamus following hypertonic stimulation [3, 9]. As a result of such an activation, wlsopressin and oxytocin are released both centrally and in the plasma to restore the normal body fluid homeostasis by means of water retention and Na excretion [17,311. The aim of the present study was to investigate whether osmotic challenge induces c-Fos protein expression in the SFO component of the neural circuitry responsive to osmotic stimulation. Preliminary evidence (',Jrre,vJomtcm'e: 1,. Giovannelli. Department of Pharmacology, University of Florence. giale Morgagni 65, Florence 50134, haly. Fax: (39) 55 4361613
that this was the case led us to study the time course of this effect in order to define tile sequence of activation o[" circumventricular and hypothalamic neurons following this kind of stimulation. We also subsequently imposed electrolytic lesions on the SFO to clarify whether the SFO efl'erent pathway to the magnocellular nuclei is essential for the activation of vasopressinergic and oxytocinergic neurons in response to hypertonic stimulation. The male Sprague-Dawley rats used in these experiments weighed 250-300 g. The animals were given Ik)od and water ad libitum and maintained under a normal light dark cycle. During the week before the experiment, the animals were handled daily for about 15 rain. On the 8th day, the experimental groups (at least 4 animals per group) were injected i.p. with 1 ml per 100 g body weight of hypertonic saline solution (1.5 M NaCI) and sacriliced at different times (15-180 mint after the injection. The control groups were treated in the same way except lk~r being injected with isotonic saline solution (0.9% NaC1). At the time of sacrifice, the animals were anesthetized with chloral hydrate (35 mg/100 g b.wt.) and perfused through the ascending aorta with 50 ml of 0.9% NaCI followed by 300 ml of ice-cold 2% paraformaldehydc in phosphate-buffered saline (PBS). The brains were subscquenlly taken out of the skull, cut in blocks and postfixed in the same fixative for an additional 2 h. They were then cryoprotccted by incubation in 30% sucrose overnight, and cut in a cryostat. Coronal sections 20 # m thick were
obtained, mounted on gelatin-coated slides and processed for immunohistochemistry. A sheep polyclonal antibody raised against the N-terminal peptide of the c-Fos protein (Cambridge Research Biochemicals) was used at the dilution of 1:1000 in PBS. After a blocking step (incubation with 3% normal horse serum and 0.3% Triton X-100 in PBS), the sections were incubated overnight at room temperature with the primary antibody in the presence of Triton and 1% horse serum. On the following day the peroxidase-avidin- biotin procedure (Vector ABC kit) was used to localize the primary antibody. The peroxidase color reaction was allowed to proceed for about 4 min in the presence of diaminobenzidine and 0.04% nickel chloride. The slides were finally washed in PBS, dried, coverslipped and analyzed under a Zeiss Axiophot microscope. Lesions of the subfornical organ were performed two weeks before the experiment. The animals were anaesthetized with halothane, placed on a stereotaxic apparatus and maintained under halothane and oxygen for the duration of the surgery. A 1 mA current was passed for 10 s in each of 4 different rostro-caudal locations ( 1.1,
1.2, 1.3 and 1.4 m m posterior to bregma, depth 6.2, ~. !. 5.2 and 5.2 m m from the skull) between the top of an insulated stainless steel electrode and a rectal anode. The sham-operated animals were treated in the same way with no current passed through the electrode. The location of the lesion was checked after the experiment, by staining coronal sections through the SFO, prepared as described above, with Richardson's stain. The extent of the lesion was estimated by comparing corresponding sections of the SFO in the lesioned and sham-operated animals. Nuclear staining for c-Fos protein was identified in numerous cells in the subfornical organ as early as 30 min alter hypertonic saline injection (Figs. lb and 2b), No difference between the hypertonica]ly stimulated and the control animals was found 15 min after the injection (not shown) and only very few c-Fos-stained nuclei were found in the SFO of animals injected with isotonic saline 30 rain betbre sacrifice (Figs. l a and 2a). The expression o]" c-Fos protein in the SFO was still present at 1 h alter osmotic stimulation (Figs. lc and 2c) and declined by 3 h (Figs. ld and 2d). Stained nuclei were found throughout
Fig. 1. c-Fos-immunoreactivenuclei in the rat rostral SFO (bregma -0.60 mm [25]) 30 min alter isotonic saline (a), and 30, 60 and 180 min alter hypertonic saline injection (b. c. d. respectively).Arrows indicate examples of labelled nuclei. Bar -- 100 #m,
the whole rostro-caudal extension of the SFO (Figs. 1 and 2). The electrolytic lesion of the SFO area induced a loss of about 75% of the SFO in 3 out of 8 animals, while 30 50% of the SFO resulted in damage in the remaining 5 rats. Also in the animals with 75% damage of the SFO, the very anterior part of the SFO (rostral to bregma 0.60 mm [25]) was undamaged. Only 2 of the lesioned animals lost weight after surgery as compared to the sham-operated. No correlation was found between weight loss and extent of the lesion. In none of the lesioned animals, did damage of the SFO prevent c-Fos protein expression in the hypothalamus 30 min after hypertonic stimulation. As reported previously [9], such expression was concentrated in the paraventricular (PVN, Fig. 3) and supraoptic (SON) nuclei, and scattered throughout the anteriot and lateral hypothalamus (not shown). Hypertonic stimulation did not induce c-Fos expression in the nucleus tractus solitarius (NTS), where only a few c-Fos-immunoreactive nuclei were found in both the isotonic (n - 4) and hypertonic (n = 4) saline injected
animals after 30 min from the stimulation (Fig. 4). No difference in c-Fos expression between osmotically stimulated and control animals was found in the NTS 15 min after injection, nor in the ventrolateral medulla (VLM) or the locus coeruleus (LC) after 15 or 30 rain (not shown). We have shown that osmotic stress induces c-Fos protein expression in the rat SFO, and that such an effect is evident at 30 min after stimulation and lasts for about 3 h. While the present paper was submitted, a report was published that showed induction of c-Fos immunoreactivity in the SFO following intravenous hypertonic saline administration [23]. c-Fos gene expression has been widely used in the last few years as a marker of neuronal activation in response to a w~riety of stimuli [2, 6, 12, 26] because of its rapid onset [27]. The fact that a population of neurons in the SFO responds to osmotic stimulation is not surprising in view of the well known role of this circumventricular structure in the regulation of body fluid homeostasis in response to osmotic and hypow)lemic stimuli [13, 28, 29]. In contrast, we found no effect of os-
a
¢
II
Fig. _. c-Fos immunoreactivity in tile rat caudal SFO (brcgma 1.40 mm [2511 30 rain after isotonic saline (a), and 30, 60 and 18(1 rain after hypcrtonic saline injection (b, c. d, respectively). Arrm~s indicate examples of labelled nuclei. Bar I00 Ira1. :
Fig. 3. Microphotographs of rat caudal SFO stained with Richardson's solution (a, c) and of'the PVN of the same animals inmmnostained for c-Fos protein (b, d). a, b: sham-operated animal, c, d: animal with electrolytic lesion of the SFO t75% organ loss). III. Ihird ventricle. Bar 100/.q'n.
motic challenge on c-Fos expression in the brainstem catecholaminergic nuclei which have also been reported to relay baroreceptive informations from the periphery to the CNS, that is the NTS, LC and VLM [5, 19, 24]. The hypothalamus is the other major brain structure which, along with the circumventricular organs, has long since been implicated in the response to osmotic stimula-
Fig. 4. c-Fos-immunoreactive nuclei in the rat NTS (bregma 13.80 mm [25]) 30 min after injection of isotonic (a) or hypertonic saline solution (b). AP, area postrema; Gr. nucleus gracilis. Arrows indicate examples of labelled nuclei. Asterisks indicate the central canal. Bar I00/am.
tion [1, 10, 21]. Induction o f c - F o s protein immunoreactivity in the paraventricular and supraoptic nuclei of the hypothalamus after hypertonic saline rejections has been demonstrated by this lab and others [3, 9, 27]. We have evidence that the onset of the magnocellular nuclei activation is at about 30 min after osmotic stimulation (Giovannelli et al., manuscript submitted), which is the same time at which the SFO starts to express c-Fos. In view of the existence of a pathway connecting the SFO to the PVN and SON [20, 22], it could be hypothesized that the hypothalamic response to osmotic stimuli is mediated by the SFO, as is the response to A N G II [7, 14, 29]. In our hands, however, animals with a 75% lesion of the SFO showed no reduction of PVN and SON activation after hypertonic injection, as measured by c-Fos immunostaining. A lesion of similar extent has been shown by Weisinger et al. [32] to decrease sodium appetite in rats, The same authors have shown that SFO lesions do not alter water intake in response to hypertonic saline injection. Furthermore, Knepel et al. [16] demonstrated that SFO lransection does not modify.' vasopressin release induced
by hypertonic saline injection. Even though we have not performed a total ablation of the SFO, leaving the very rostral portion intact in each of the lesioned animals, these data suggest that at least the central and caudal portion of the SFO and the hypothalamus are separately and simultaneously activated by osmotic stimuli. More complete SFO lesions will be necessary in order to extend such a conclusion to the whole SFO. It has been shown that an intact SFO is essential t\)r A N G ll-mediated VP and OXY release [7, 14, 16]. Our experiments show that most of the SFO is not necessary for magnocellular hypothahtmic neurons to respond to hypertonic stimulation, leading to the hypothesis that the SFO neuronal population which responds to osmotic stimuli by expressing c-Fos is probably not the same which is involved in the response to A N G II, nor does it project to the PVN and SON. Such a popuhttion could consist of osmosensitive neurons projecting to other brain areas, like the anterior third ventricular region (AV3V [13,201). In fact, the regulation of VP and OXY release upon osmotic stress appears to be a complex one, involving projections n o t e l f l y f r o m the S F O but a l s o fronl o t h e r hypothalamic areas, as the AV3V and the median preoptic nucleus (MnPO [4, 10, 11]). Lesions of both the AV3V and the MnPO have been shown to impair fluid balance and VP and OXY release in response to osmotic stimuli [8, 13] and the AV3V is thought to contain osmosensitive neurons itself [11]. Furthermore, magnocellular hypothalamic neurons have been shown to be osmosensitive [18, 21]. Thus, it can be hypothesized that after the impairment of the pathway conveying inlk~rmation fl'om the SFO to the hypothalamus, VP and OXY neurons can still be activated by blood hypertonicity either directly or through other pathways. We thank Dr. George F. Koob tbr fruitful discussion and Robert Lintz for technical help during animal surgery. This study was supported by Grant AA 06420. This is Scripps Research Institute paper n. 7096. 1 Abe, H. and Ogata, N., Ionic illcchanJsnls for the osmotically-induced depolarization in neurones of the guinea-pig suprm)ptic nuc l e u s q n \ , i t r o ' , J . Physiol.. 327 (1982) 157 171. 2 Aronin. N., Sagar, S.M.. Sharp, F.R. and Schwartz, W.J., Light rcgulatcs expression of a Vos-rchlted protein in rat suprachiasmalic nuclei, Prec. Natl. Acad. Sci. USA, 87 (1990) 5959 5962. 3 ('cccatclli. S,. Villar, M.J.. Goldstcm, M.J. and H6kfelt, T., Expression of c-Fos inlmunoreaclivity in transmitter-characterized ilCtlrons after stress, Proc. Natl. A c a d S c i . USA, 8611989} 9569 9573. 4 Chaudry, M.,,\., Dyball, R.E.J., Honda, K. and Wright, N.C., Fhe role of interconnection between supraoptic nucleus and anterior lhird vcntricular region in osmoreguhition in the rat, J. Physiol.. 411) (19891 123 145, 5 Cunninghanl Jr.. E.T. and Sawchcnko, P.E.. Anatonlical spccilici D
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