hypothalamic activity during altered salt and water balance in the snake bothrops jararaca
TRANSCRIPT
J Comp Physiol B (2008) 178:57–66
DOI 10.1007/s00360-007-0199-7ORIGINAL PAPER
Hypothalamic activity during altered salt and water balance in the snake Bothrops jararaca
Leonardo Zambotti-Villela · Camila Eduardo Marinho · Rafaela Fadoni Alponti · Paulo Flavio Silveira
Received: 25 April 2007 / Revised: 20 July 2007 / Accepted: 28 July 2007 / Published online: 17 August 2007© Springer-Verlag 2007
Abstract The eVects of water and salt overload on theactivities of the supraoptic and paraventricular nuclei andthe adjacent periventricular zone of the hypothalamus ofthe snake Bothrops jararaca were investigated by measure-ments of Fos-like immunoreactivity (Fos-ir). Both waterand salt overload resulted in changes in body mass, plasmaosmolality, and plasma concentrations of sodium, potas-sium, and chloride. Hyper-osmolality increased Fos immu-noreactivity in the rostral supraoptic nucleus (SON), theparaventricular nucleus (PVN), and adjacent periventricularareas. Both hyper- and hypo-osmolality increased Fosimmunoreactivity in the intermediate SON, but not in otherareas of the hypothalamus. Immunostaining was abundantin cerebrospinal Xuid (CSF)-contacting tanycyte-like cellsin the ependymal layer of the third ventricle. These datahighlight some features of regional distribution of Fosimmunoreactivity that are consistent with vasotocin func-tioning as a hormone, and support the role of hypothalamicstructures in the response to disruption of salt and waterbalance in this snake.
Keywords Fos · Body Xuid and electrolyte homeostasis · Hypothalamus · Supraoptic nucleus · Paraventricular nucleus · CSF-contacting cells
AbbreviationsAC Anterior commissureC Control groupCSF Cerebrospinal XuidFos-ir Fos-like immunoreactivityOC Optic chiasmaOD Optical densityOT Optic tractPD Population densityPeV Periventricular areas along the rostrocaudal extent
of PVN and SON adjacent to these nucleiPVN Paraventricular nucleusR1 Region located caudally at 3,200 �m from the ros-
tral end of the olfactory bulbR2 Region located caudally at 200 �m from R1R3 Region located caudally at 560 �m from R1R4 Region located caudally at 780 �m from R1RCN Suprachiasmatic nucleusSL Salt-overloaded groupSON Supraoptic nucleusWL Water-overloaded group
Introduction
Hypothalamo-neurohypophysial system is a major compo-nent of the neuroendocrine regulation of hydrodynamic bal-ance in all vertebrates, which consists of neurones and glialcells located in the paraventricular (PVN) and supraoptic(SON) nuclei (Rodríguez 1984). In mammals, these neuro-nes synthesize the peptides arginine vasopressin and oxyto-cin and release them into the blood circulation (Ludwiget al. 1997) and the extracellular space of hypothalamictissue, whereas glial cells regulate the extracellular environ-ment of these neurones (Walz and Hertz 1983).
Communicated by I.D. Hume.
L. Zambotti-Villela · C. E. Marinho · R. F. Alponti · P. F. Silveira (&)Laboratory of Pharmacology, Instituto Butantan, Av. Vital Brazil, 1500, São Paulo, 05503-900 SP, Brazile-mail: [email protected]
123
58 J Comp Physiol B (2008) 178:57–66
In mammals, it is well-known that the secretion of vaso-pressin is due to volume and osmotic changes of extracellu-lar Xuids (Zemo and McCabe 2002), while the secretion ofoxytocin is stimulated mainly by parturition and suckling(Neumann et al. 1995), as well as by systemic osmoticstimulation (Leng et al. 1988). Immunolabeling of Fosprotein, a product of the immediate early gene c-fos, madepossible the investigation of osmosensitive pathways regu-lating the activity of vasopressin and oxytocin neurones(Hamamura et al. 1992). However, the relationship of Fosto water and salt balance has not yet been examined in rep-tiles, although a few studies have reported the presence ofFos protein in lizards (Ahboucha and Gamrani 2001; Berto-lucci et al. 2000; Blasco-Ibanez et al. 1992; ChieY et al.1997) and turtles (Greenway and Storey 2000; Yaqub et al.1995).
The challenges to body Xuid homeostasis have onlyrarely been demonstrated to result in changes in the amountof neurosecretory material in the hypothalamus (Philibertand Kamemoto 1965; Mancera et al. 1990) or in the blood-stream of reptiles (Ladyman et al. 2006; Rice 1982). In thebloodstream, the vasopressin homolog vasotocin has beenmeasured to date in only two snake species: Bothrops jara-raca by Silveira et al. (1998) and Notechis scutatus byLadyman et al. (2006).
When the lizard Varanus gouldii (Rice 1982) and thesnake N. scutatus (Ladyman et al. 2006) were submittedto hydrosaline challenges, the result was a range ofplasma vasotocin concentrations that were linearly corre-lated with plasma osmolality. In similar studies with thesnake B. jararaca, Silveira et al. (1998) found that theexpected linear correlation between plasma osmolalityand vasotocin concentration was positive in chronic andacute salt-overloaded and vasotocin-injected groups but,surprizingly, was negative in water-deprived and water-overloaded groups (WL). Consequently, when these treat-ment groups were pooled and analyzed as one group, therewas no correlation between plasma osmolality and vasoto-cin concentration (Silveira et al. 1998). Since a close posi-tive correlation between plasma osmolality and vasopressinconcentrations has been demonstrated in mammals undervarious states of hydration and salt regimens (Robertson1976), these Wndings of Silveira et al. (1998) raised thehypothesis that the vasotocin/mesotocin system of B. jara-raca could be insensitive to osmotic and/or volumetricstimuli.
To test this hypothesis, the present study examined thedistribution of Fos protein, and also checked whetherwater- and salt-overloading act directly to change Fos-likeimmunoreactivity (Fos-ir) in cells along the length of theparaventricular and supraoptic nuclei as well as in the peri-ventricular zone adjacent to these nuclei in the hypothala-mus of the snake B. jararaca.
Materials and methods
Animals and treatments
Adult snakes (B. jararaca WIED, 1824, Squamata, Serpen-tes, and Viperidae) (about 180 g in mass and 103 cm inlength) were collected from the wild in south and southeast-ern Brazil. They were identiWed by the Laboratory of Her-petology, Instituto Butantan, and then individually housedin cages (inside length £ width £ height 35 £ 26 £ 22cm3) in a restricted-access room where they were accli-mated for a minimum of 15 days to a controlled tempera-ture of 25°C, relative humidity of 65.3 § 0.9%, and 12-hlight:12-h dark photoperiod (lights on at 06:00 h). SexualidentiWcation was made by gently pressing the tail basebelow the cloaca, with the consequent exposure of one orboth hemipenises characterizing a male. The macroscopicexamination of the oviduct through a ventral incision onanesthetized females permitted the exclusion of pregnantsnakes from experimental procedures. The animals wereprovided with adequate food (one Swiss mouse weighing10% of the snake’s body mass to each snake every 18 days)and had free access to tap water.
Female and male snakes (normally hydrated and fastedfor 18 h) were randomly assigned to hydrosaline challenges(Silveira and Mimura 1996; Silveira et al. 1998; Alpontiet al. 2005). Animal care and procedures used were inaccordance with guidelines of the Brazilian Council Direc-tive (COBEA, São Paulo, SP, Brazil) and were approved bythe Ethics Committee of the Instituto Butantan (048/2002).
The control group (C) was oVered tap water ad libitumfor 16 days and received daily intrapleuroperitoneal bolus(ipl) administration of 1 mM NaCl (0.2 ml/100 g bodymass) at 07:30–08:30 h for two periods of Wve consecutivedays and a Wnal period of two consecutive days, with eachperiod separated by 2 days. The salt-overloaded group (SL)had free access to a 2.14 M NaCl drinking solution andreceived daily ipl injections of the same solution (0.2 ml/100 g body mass) during one period of four and another oftwo consecutive days separated by a 2-day interval. TheWL had free access to distilled water and received daily iplinjections of distilled water (5 ml/100 g body mass) at07:30–08:30 h for two periods of Wve consecutive days anda Wnal period of two consecutive days, with each periodseparated by 2 days. Snakes were weighed before and aftertheir respective treatments.
Blood and brain collections
The snakes were anesthetized with sodium pentobarbital(3 mg Nembutal per 100 g body mass, subcutaneously)between 10:00 and 12:30 h. They received an intracar-diac bolus injection of 100 IU sodium heparin in 0.1 ml
123
J Comp Physiol B (2008) 178:57–66 59
of Ringer’s solution (Silveira and Mimura 1999). Indi-vidual 1.0-ml blood samples were quickly obtained fromthe cardiac ventricle. Cardiac perfusion was thenperformed with the Ringer’s solution over a period of15 min and continued with Bouin’s Wxative for40–50 min at a Xow rate of 2.4–4.8 ml/min. The brainswere removed and Wxed for 48 h in Bouin’s Xuid thendehydrated and embedded in paraYn. Individual bloodsamples were used for haematocrit measurement andimmediately centrifuged at 10,000£g for 20 min at 5°Cto obtain plasma, which was stored at ¡80°C until thedetermination of osmolality, and protein and electrolyteconcentrations.
Analytical methods
Microhaematocrit values were obtained by centrifugation(microcentrifuge H-240, Hsiangtai Machinery Ind. Co.Ltd., Taipei, Taiwan) immediately after blood sampling.Osmolality was determined in duplicate in 10-�l samples offresh plasma (Osmette II cryoscopic osmometer, PrecisionSystems Inc., Natick, MA, USA).
Plasma protein concentration was measured at 630 nm intriplicate by the Bio-Rad protein assay (Bradford 1976) ona microplate reader PowerWave™ XS spectrophotometer(Bio-Tek, Winooski, VT, USA), using a standard curvewith bovine serum albumin (BSA) (Sigma, St. Louis, MO,USA).
Total sodium and potassium concentrations in plasmawere determined by atomic emission and total magnesiumand calcium by atomic absorption in appropriately dilutedsamples with a Zeiss atomic spectrophotometer PM-QIIM4QII. Total chloride was determined by a modiWed ultra-micro method (Schales and Schales 1941) with a Beckmanmicrotitrator model 153, where 10-�l samples were titratedwith mercuric nitrate solution in the presence of diphenylc-arbazone as indicator.
Fos-immunohistochemistry and microdensitometry
The identiWcation and nomenclature of the hypothalamicregions and nuclei were adopted from Silveira et al. (2001,2002) and Alponti et al. (2006). Four regions (R) were ana-lyzed through the entire rostrocaudal length of the PVN andSON: R1 at 3,200 �m from the rostral end of the olfactorybulb; R2 at 200 �m from R1; R3 at 560 �m from R1; R4 at780 �m from R1. We also analyzed all periventricular(PeV) areas adjacent to the PVN and SON along the entirerostrocaudal length of these nuclei.
A series of transverse sections (10-�m thick) werehydrated and immunostained by the peroxidase–antiper-oxidase method (Sternberger 1986) using a rabbit poly-clonal antiserum raised against a highly conserved peptide
from human c-fos p62 (c-fos K-25, sc-253, Santa CruzBiotech. Inc., Santa Cruz, CA, USA), which is identical tocorresponding sequences in mouse, rat, and chicken. Sec-tions were initially incubated for 15 min at 22°C in H2O2
(0.3% in Tris buVer) in order to remove endogenousperoxidase activity and then incubated for 20 h at 22°C inthe primary antiserum (1:100) (2 �g c-fos K-25/ml). Sec-tions were subsequently incubated in the second antise-rum (anti-rabbit IgG raised in goats, R2004, Sigma) at adilution of 1:40 for 45 min at 22°C, and then in the rabbitperoxidase–antiperoxidase complex (P2026, Sigma) at adilution of 1:75 for 45 min at 22°C. Sections were rinsedthrice in Tris buVer after H2O2, antisera and peroxidase–antiperoxidase incubation. All antisera and peroxidase–antiperoxidase complexes were diluted in Tris buVer, pH7.8, containing 0.7% non-gelling seaweed gelatin Type IVlambda carrageenan (Sigma), 0.5% Triton X-100 (Sigma),and 0.02% sodium azide (Merck, Hohenbrumn,Germany). As an electron donor, 0.04% 3.3�-diam-onibenzidine tetrahydrochloride (Sigma) and 0.04%ammoniun nickel sulfate hexahydrate (Sigma) in TrisbuVer, pH 7.8 with 0.007% H2O2 (Merck) were used forincubation and color development in darkness for 20 minat 22°C. In order to verify the immunohistochemical pro-cedure, some control sections were not treated with theprimary antiserum. No positive structures were found inany of these sections. For comparison among treatmentgroups (C, SL, and WL) under the same conditions ofimmunohistochemistry assay, this procedure was simulta-neously performed in sequence on representative adjacentsections of identical regions from animals submitted tothese treatments. Afterward those sections from the sameimunohistochemical procedure were visualized and ana-lyzed under bright-Weld illumination at the same contrastadjustment.
The Fos-ir was determined by examining the immuno-stained sections using a Nikon E600 microscope equippedwith a CoolSNAP-PRO® digital camera coupled to amicrocomputer system. The images were captured and den-sitometric analysis was performed by measurements of thenumber (staining score or population density) (PD) andintensity (staining intensity or optical density: transmit-tance in nm) (OD) of immunolabelings in delimitated andWxed locations of PVN, SON, and PeV, respectively, withWelds of view of 527, 822, and 230 �m2, using the imageprocessor Image-Pro-Plus 4.0® (Media Cybernetics, SilverSpring, MD, USA). These densitometric data from eachtreatment in each region were presented as mean § SEM ofpercentages of PD or OD relative to 100% (sum of absolutevalues of PD or OD in each treatment and region obtainedby the same immunohistochemical procedures and exam-ined under the same standardized conditions of bright-Weldillumination at the same contrast adjustment).
123
60 J Comp Physiol B (2008) 178:57–66
Statistical analysis
Data were analyzed statistically using GraphPad Prism®
and Instat® software. One-way analysis of variance(ANOVA) was performed on values of body mass, haemat-ocrit, plasma osmolality, protein, and electrolyte concentra-tions, as well as PD and OD among treatment groups orregions. When diVerences were detected, the Student–New-man–Keuls test was used to identify the diVerences amongmeans. Regression analysis was performed to obtain stan-dard curves for protein concentration. DiVerences wereconsidered statistically signiWcant at P < 0.05.
Results
As shown in Table 1, haematocrit and plasma concentra-tions of protein, magnesium, and calcium were similar inall treatment groups. Body mass was lower after the SLtreatment than WL. A fall in potassium concentrations wasassociated with the SL and WL treatments relative to C.Plasma concentrations of chloride and sodium were higherin SL than C or WL, and higher in C than WL. Plasmaosmolality was higher in SL than in C, and higher in C thanin WL.
Figure 1a shows the regions where Fos-ir was ana-lyzed. The supraoptic nucleus (SON) extends rostrocau-dally from a region limited by the caudal portion of theoptic chiasma (OC) and the anterior commissure (AC)(R1) to the level of the retrochiasmatic nucleus (R4),while the paraventricular nucleus (PVN) Wrst appears atthe level of the anterior commisssure (R2) and extendscaudally to R4. Fos-ir was examined from R1 to R4 in thePeV, but immunolabeling in the PeV in all studied situa-tions in the present work was positive only along thePVN (R2 to R4) (Fig. 1a). Figure 1b–e show the micro-scopic visualization of immunohistochemical labeling
for Fos antiserum in the C, SL, and WL groups: the label-ing was densitometrically analyzed to reveal the localiza-tion of Fos-like protein in the hypothalamus. TheseWgures show the typical appearance of increased Fos-ir inthe PVN and PeV (Fig. 1b) or the SON (Fig. 1d) and thatof basal Fos-ir in the PVN and PeV (Fig. 1c) or in theSON (Fig. 1e).
As shown in Fig. 2, PD values in the SON diVeredamong R1, R2, R3, and R4 only for the SL group, beinghigher in R1 than R4. Figure 2 also shows that OD valuesin the SON did not diVer signiWcantly among R1, R2, R3,and R4 within any of the treatment groups. Comparisonsamong diVerent treatments in each region of the SONshowed that SL animals had higher PD than C animals inR1. In R3, PD of both the SL and the WL groups washigher than that of C. There was no signiWcant diVerence inOD values in each region of the SON among the C, SL, andWL treatment groups.
As shown in Fig. 3, OD and PD values in the PeV didnot diVer among R2, R3, and R4 in any treatment group.Comparisons among diVerent treatments in each region ofthe PeV show that PD and OD values were higher in SLthan in C or WL in R3 and R4 (Fig. 3).
As shown in Fig. 4, OD and PD values in the PVN didnot diVer among R2, R3, and R4 in any treatment group.Comparisons among diVerent treatments in each region ofthe PVN show that PD values in R3 and R4 were higher inSL than in C or WL animals. When compared to C and WL,OD values of the SL group were higher in all regions of thePVN (R2, R3, and R4) (Fig. 4).
Values of PD and OD presented in Figs. 2, 3, and 4were also statistically compared within each treatmentgroup (C, SL, and WL) among SON, PVN, and PeV ineach examined region, where SON and PVN are simulta-neously present (R2 to R4) (see Fig. 1). There were nosigniWcant diVerences between any treatment groups inany region.
Table 1 Body mass, haematocrit and plasma osmolality, protein and electrolyte concentrations of B. jararaca submitted to normal hydration (C),or water (WL) or salt (SL) overloading
Values are mean § SEM. The number of animals is given in bracketsd Percentage of values before treatment. Values for diVerent parameters presented on the same line were measurements for the same animals. With-in a column of data, values marked with diVerent letters were signiWcantly diVerent (ANOVA P < 0.05, Student–Newman–Keuls P < 0.05)
Treatments Body massd
Haematocrit (%)
Osmolality (mOsm/kg)
Protein (mg/ml)
Na (mM) K (mM) Mg (mM) Ca (mM) Cl (mM)
C 98 § 3ab 28 § 2 270 § 4a 46 § 4 190 § 15a 6 § 0.1a 1.7 § 0.1 2.4 § 0.1 182 § 12a
(6) (6) (6) (6) (6) (6) (5) (5) (5)
WL 109 § 5a 28 § 2 170 § 18b 45 § 7 120 § 7b 5 § 0.2b 1.3 § 0.1 2.6 § 0.2 135 § 16b
(5) (3) (3) (3) (5) (5) (5) (5) (5)
SL 87 § 8b 25 § 1 360 § 25c 40 § 3 228 § 8c 5 § 0.3b 1.3 § 0.2 2.3 § 0.2 245 § 17c
(5) (5) (5) (5) (5) (5) (5) (5) (5)
123
J Comp Physiol B (2008) 178:57–66 61
Discussion
Our results demonstrate that salt- or water-overloading pro-moted changes in body mass, and levels of osmolality,sodium, chloride, and potassium in the plasma, as well as inFos-ir in the hypothalamic SON, PVN, and PeV of thesnake B. jararaca. Since all animals used here were killedat the same time of day and year, circadian and/or seasonaldiVerences could not be evaluated. The experiments werecarried out in spring when estradiol levels are low and thereare no diVerences in plasma cystyl aminopeptidase (vasoto-cinase) activity between reproductive and non-reproductiveCrotalus durissus terriWcus female snakes (Almeida-Santoset al. 2004). Unfortunately, the reproductive cycle ofB. jararaca is still unknown and, consequently, any eVectsof the stage of the reproductive cycle or of sexual dimor-phism could not be examined.
Salt overloading promoted a 10% decrease in body massof B. jararaca, while water overloading promoted a 10%increase. It is generally accepted that a 10% decrease inbody mass reXects a decrease in blood volume of the orderof 8–10% (usually referred to as 10% dehydration), whichstimulates vasopressin secretion (Robertson 1976). Plasma
osmolality followed plasma sodium and chloride concen-trations in B. jararaca, as observed in all investigated verte-brates, including the snake N. scutatus (Ladyman et al.2006). Despite the lack of information about the inXuenceof calcium and magnesium on physiological processes inreptiles, its balance seemed to be important as indicated bythe ability of B. jararaca to maintain unchanged plasmalevels of these electrolytes when osmolality was disturbed.In humans, calcium and magnesium functions are known tobe closely linked and abnormal plasma concentrations ofthese minerals often result in cardiovascular and neurologi-cal changes (Baker and Worthley 2002). In contrast, potas-sium is predominantly an intracellular ion that contributesto »50% of the intracellular Xuid osmolality and it islargely responsible for the resting membrane potential. Thelatter accounts for its inXuence on the excitability of muscleand nervous tissue. Consequently, hypokalaemia can causecardiovascular, neurological or skeletal muscle dysfunction(Glover 1999), but these dysfunctions seem unlikely at thelow level of alteration observed here in hyper- or hypo-osmotic B. jararaca. Similar hematocrit and plasma proteinconcentrations between salt- and water-overloaded B. jara-raca support data which have suggested a high capacity of
Fig. 1 a Schematic drawings of rostrocaudal transverse sections through hypothalamic regions R1 to R4 showing the paraventricular (PVN), supraop-tic (SON), and suprachiasmatic (RCN) nuclei, adjacent periventricular zone along the rostrocaudal extent of the PVN and SON (PeV), optic chiasma (OC), optic tract (OT), and ante-rior commissure (AC); typical appearance of increased Fos-ir in the PVN and PeV (b), and in the SON (d) of salt-overloaded animals; typical appearance of basal Fos-ir in the PVN and PeV (c), and in the SON (e) of control animals. Scale bar, 20 �m
123
62 J Comp Physiol B (2008) 178:57–66
snakes to restore blood volume and to transfer Xuidbetween the vascular and extravascular compartments(Smits and Lillywhite 1985).
We observed the staining for anti-Fos antiserum only inthe nuclei of hypothalamic cells of B. jararaca, which isindicative of the integrity of tissue preparation, since thepresence of Fos outside of the cellular nucleus appears to bea harbinger of neuronal death (Smeyne et al. 1993).Unstimulated hypothalamic neurones contained Fos-ir,indicating that this class of protein is expressed under nor-mal hydration conditions in this snake.
Our data show that, under the same applied stimulus, thenumber and intensity of Fos-ir labelings in common sec-tional regions (R2, R3, and R4) did not diVer signiWcantlyamong SON, PVN or PeV, suggesting homogeneousresponses of these structures to the same stimulus in each oftheir respective regions. However, comparisons of the samestimulus among the responses of diVerent regions of thesame structure (SON, PVN or PeV), as well as amongdiVerent treatment groups (SL, WL, or C) in each regionalportion of these structures (R1 to R4 for SON, and R2 to R4for PVN and PeV) revealed important diVerences in thenumber and/or intensity of Fos-ir labelings.
The hyper-osmotic stimulation increased the number andintensity of Fos-ir labelings in the longer axis of the caudalPVN and PeV, while hypo-osmolality had no eVect on Fos-ir
along the whole length of both the PVN and PeV. Althoughhyper-osmolality increased the number of Fos-ir labelingsin the rostral SON, in the intermediate SON both hypo-osmolality and hyper-osmolality had a common positivestimulatory eVect on the number of Fos-ir. These resultsdemonstrate that Fos immunohistochemistry labels at leastsome subset of activated cells in the SON, PVN, and PeVof B. jararaca. Thus, this activation was not a generalizedeVect of hyper- and hypo-osmotic stimuli, since Fos-ir wasnot detected in other hypothalamic areas. Furthermore,changes in the number (activated cells) or intensity (activitylevel) of Fos-ir were diVerent modalities of responses tovolume and osmotic stimuli, indicating that these two den-sitometric methods do not always produce equivalentresults. These results also agree with Wndings in rabbitsshowing that Fos-positive neurones in the PVN were muchmore variable in staining intensity than those in the SONafter the same eliciting stimulus (Li and Dampney 1994).
In B. jararaca, osmotic stimulation could be diVeren-tially sensed only in one focal zone of the SON, whileanother focal zone of this nucleus seems to be sensitive tovolume and osmotic stimuli. As observed in the presentstudy, both stimuli can promote an increase in the numberof activated cells only in the SON. The PVN and PeV couldbe insensitive to volume stimulus, while their osmorespon-siveness is reXected by increased numbers and intensity of
Fig. 2 Number (PD) (black bars) and intensity (OD) (white bars)values of Fos-like immunoreactivity densitometry from salt (SL) orwater (WL) overloading, or controls (C), in each region (R1 to R4) ofsupraoptic nucleus (see Fig. 1). Values are mean § SEM of percent-ages of PD or OD relative to 100% (sum of absolute values of PD orOD in each treatment and region presented in Fig. 1) obtained in thesame immunohistochemical procedure and examined under the samestandardized conditions of bright-Weld illumination at the same
contrast adjustment. Number of immunohistochemical procedures forsamples from each treatment is given in brackets. Comparisons weremade among diVerent treatments in the same region: *P < 0.05 or**P < 0.01 relative to PD of C (ANOVA P < 0.05, Student–Newman–Keuls t-test), and among diVerent regions in the same treatment: valuesmarked with diVerent letters were signiWcantly diVerent (ANOVAP < 0.05, Student–Newman–Keuls P < 0.05)
123
J Comp Physiol B (2008) 178:57–66 63
Fos-ir. In turn, the intermediate SON could represent aregion in which these two diVerent sensorial modalities areintegrated in the B. jararaca brain. In addition to the possi-bility that hyper-osmolality could be a prominent stimula-tion involved in the control of body Xuid volume andosmolality in B. jararaca, the PVN and PeV could exert amore prominent role than the SON in this control. Since theSON in B. jararaca is known to display only magnocellularneurones, whereas the PVN contains magnocellular andparvocellular neurones (Silveira et al. 2002), it is likely thatthis last cell type is less selective in discriminating betweenthese two stimuli but is more eVective in the osmoregula-tion than magnocellular neurones. In fact, our Wndings mayreXect the inability of certain cell types in the PVN, SON,and PeV of this snake to increase Fos-ir rather than ineVec-tiveness of hyper- and/or hypo-osmotic stimuli. As occursin rats, it can be hypothesized that direct stimulation ofmagnocellular SON neurones in B. jararaca increases theirWring rate and intra-SON peptide release but not c-fos geneexpression, which could be the result of other mechanismssuch as speciWc synaptic inputs and/or other types of stim-uli, needed to activate c-fos in these neurones (Ludwiget al. 1997). For example, apart from vasopressin and oxy-tocin-secreting neurones, the positive Fos-ir PVN neuronesmay have included those that secrete adrenocorticotropichormone (Lightman and Young 1987). The main popula-
tion of corticotropin-releasing hormone perikarya in thehypothalamus of B. jararaca was found in the PVN (Silveiraet al. 2001).
Similar to rats, the full expression of osmosensitivity byhypothalamic magnocellular neurones of this snake couldbe dependent on inputs from an osmoreceptor complexformed by periventricular structures, including the subfor-nical organ and organum vasculosum of the lamina termi-nalis (Hamamura et al. 1992; Ludwig et al. 1997). Thishypothesis is supported in the present study by the fact thathyper- and hypo-osmolality only altered the number ofactive cells but not their activity levels in the SON, as wellas by the presence of relatively homogeneous cell activityunder the same stimulatory conditions in the same sectionalregions among the SON, PVN, and PeV, and by the strongactivation of periventricular Fos-ir in the longer axis of cau-dal PVN. It is also known that hypertonic stimulation of therat SON increases the release within the nucleus not only ofvasopressin and oxytocin but also of several transmitters(Ludwig et al. 1997). In the snake B. jararaca, severaltransmitters, including mesotocin, could have increasedlocal release mediated by hypo-osmolality in that focal por-tion of SON sensitive to volume stimulus. It is also possiblethat a non-selective increase in transmitter release neutral-izes the stimulatory actions of otherwise eVective transmit-ters on the c-fos gene in those hypothalamic regions of this
Fig. 3 Number (PD) (black bars) and intensity (OD) (white bars)values of Fos-like immunoreactivity densitometry from salt (SL) orwater (WL) overloading, or controls (C), in each region (R2 to R4) ofadjacent periventricular zone along the rostrocaudal extent of theparaventricular nucleus (see Fig. 1). Values are mean § SEM of per-centages of PD or OD relative to 100% (sum of absolute values of PDor OD in each treatment and region presented in Fig. 1) obtained in thesame immunohistochemical procedure and examined under the same
standardized conditions of bright-Weld illumination at the same con-trast adjustment. Number of immunohistochemical procedures forsamples from each treatment is given in brackets. Comparisons weremade among diVerent treatments in the same region: **P < 0.01 rela-tive to PD and *P < 0.05 relative to OD of C or WL (ANOVAP < 0.05, Student–Newman–Keuls t-test), and among diVerent regionsin the same treatment, which revealed no diVerent values of PD or OD(ANOVA P < 0.05)
123
64 J Comp Physiol B (2008) 178:57–66
snake, where Fos-ir was not changed by hyper- or hypo-osmolality.
In reptiles, birds and mammals, magnocellular neurose-cretory cells form the SON and PVN. No cerebrospinal Xuid(CSF)-contacting dendrites were found in the SON. How-ever, intra-ependymal as well as distal neurones in the PVNsend dendrites to the third ventricle. This type of CSF-con-tacting neuron, like that of the median eminence and neuro-hypophysis, has also been demonstrated to represent aphylogenetically old form of non-synaptic transmission(Vigh et al. 2004). This form of intercellular communicationhas been named “volume” transmission, which is character-ized by signal diVusion in a three-dimensional fashionwithin the brain extracellular Xuid, in contrast to “wiring”transmission characterized by the presence of physicallyidentiWable communication channels within the neuronaland/or glial cell network, such as synaptic transmission andgap junctions (Agnati et al. 1995). The presence of Fos-irwas abundant in CSF-contacting cells embedded or lyingattached to the ependymal layer of the third ventricle ofB. jararaca brain. CSF-contacting cells could be tanycytes,a specialized form of ependymal bipolar cell, which bridgethe CSF to the portal capillaries and may link the CSF toneuroendocrine events (Rodríguez et al. 2005). The func-tions of these cells are still largely speculative. Besidespossible regulatory functions, tanycytes are considered to
guide hypothalamic axons and thought to be involved inbrain–blood barrier functions, i.e., transport mechanismsbetween ventricle and blood vessels of the portal system(Wittkowski 1998). However, CSF-contacting cells areknown to be osmosensitive in all studied vertebrates(Hamamura et al. 1992; Vigh et al. 2004). Thus, our datasuggest that CSF-contacting cells play a prominent role inneuroendocrine events of body Xuid homeostasis and that, inB. jararaca, the brain ependyma is not merely an inert liningbut may participate in these events.
Taken together, our data are consistent with vasotocinfunctioning as a hormone produced in hypothalamic nucleiunder the inXuence of volume and osmotic stimuli inB. jararaca. The apparent insensitivity to release vasotocin,which could be inferred from radioimmunoassays of thispeptide in B. jararaca plasma (Silveira et al. 1998) might beattributable to the hydrolysis of this circulating peptiderather than low sensitivity of the vasotocin/mesotocin neuro-secretory system, since B. jararaca presented high levels ofcirculating activity of oxytocinase/vasotocinase (Silveiraand Mimura 1996), which to a certain extent were positivelycorrelated with plasma osmolality (Alponti et al. 2005).
In conclusion, Fos protein is expressed under normalhydration conditions in B. jararaca and c-fos activation of asubset of cells in the SON, PVN, and PeV is dependent onthe type of stimulus. Although the mechanisms of c-fos
Fig. 4 Number (PD) (black bars) and intensity (OD) (white bars)values of Fos-like immunoreactivity densitometry from salt (SL) orwater (WL) overloading, or controls (C), in each region (R2 to R4)of the paraventricular nucleus (see Fig. 1). Values are mean § SEM ofpercentages of PD or OD relative to 100% (sum of absolute valuesof PD or OD in each treatment and region presented in Fig. 1) obtainedin the same immunohistochemical procedure and examined under thesame standardized conditions of bright-Weld illumination at the same
contrast adjustment. Number of immunohistochemical procedures forsamples from each treatment is given in brackets. Comparisons weremade among diVerent treatments in the same region: **P < 0.01 rela-tive to PD and *P < 0.05 relative to OD of C or WL (ANOVAP < 0.05, Student–Newman–Keuls t-test), and among diVerent regionsin the same treatment, which revealed no diVerent values of PD or OD(ANOVA P < 0.05)
123
J Comp Physiol B (2008) 178:57–66 65
induction by these stimuli in speciWc areas remain to be elu-cidated, induced changes in nuclear c-fos within the PVNand SON, as well as in CSF-contacting and ependymalcells in the PeV, demonstrate the participation and func-tional integrity of these neuroendocrine hypothalamicstructures to address challenges to the balance of extracel-lular body Xuid volume and osmolality in the snakeB. jararaca.
Acknowledgments We are very grateful to Prof. Dr. Ian Hume forhis invaluable comments and suggestions on the writing of this paper.The authors are indebted to the staV of the Laboratory of Herpetologyof the Instituto Butantan for the collection and identiWcation of thesnakes and to Dr. M. C. Breno for providing the snake’s skulls. Thanksare due to Mrs. F. Canhoto for her skilled technical assistance. Grantsponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP—Brazil; research grant number 03/13239-0 to P.F.S. and fel-lowship grant number 05/03745-0 to C.E.M; Grant sponsor: ConselhoNacional de Desenvolvimento CientíWco e Tecnológico—CNPq—Brazil; productivity grant number 306779/2003-0 to P.F.S. and fellow-ship grant numbers PIBIC/Instituto Butantan 102091/2004-8 to L.Z.Vand 108462/2003-0 to R.F.A.
References
Agnati LF, Zoli M, Stromberg I, Fuxe K (1995) Intercellular commu-nication in the brain: wiring versus volume transmission. Neuro-science 69:711–726
Ahboucha S, Gamrani H (2001) DiVerences in protein expression inthe subcommissural organ of normal and lordotic lizards (Agamaimpalearis). Metab Brain Dis 16:219–226
Almeida-Santos SM, Abdalla FM, Silveira PF, Yamanouye N, BrenoMC, Salomao MG (2004) Reproductive cycle of the NeotropicalCrotalus durissus terriWcus: I. Seasonal levels and interplay be-tween steroid hormones and vasotocinase. Gen Comp Endocrinol139:143–150
Alponti RF, Zambotti-Villela L, Murena-Nunes C, Marinho CE, OlivoRA, Silveira PF (2005) Cystyl aminopeptidase activity in theplasma, viscera and brain of the snake Bothrops jararaca. CompBiochem Physiol A Mol Integr Physiol 141:336–352
Alponti RF, Breno MC, Mancera JM, Martin-del-Rio MP, Silveira PF(2006) Distribution of somatostatin immunoreactivity in the brainof the snake Bothrops jararaca. Gen Comp Endocrinol 145:270–279
Baker SB, Worthley LI (2002) The essentials of calcium, magnesiumand phosphate metabolism: part II. Disorders. Crit Care Resusc4:307–315
Bertolucci C, Sovrano VA, Magnore MC, Foa A (2000) Role of sup-rachiasmatic nuclei in circadian and light-entrained behavioralrhythms of lizards. Am J Physiol Regul Integr Comp Physiol279:R2121–R2131
Blasco-Ibanez JM, Martinez-Guijarro FJ, Lopez-Garcia C, MellstromB, Naranjo JR (1992) Narine occlusion decreases basal levels ofFos in the cerebral cortex of the lizard Podarcis hispanica. Neu-roscience 50:647–654
Bradford MM (1976) A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of pro-tein-dye binding. Anal Biochem 72:248–254
ChieY P, Angelini F, Pierantoni R (1997) Proto-oncogene activity inthe testis of the lizard, Podarcis s. sicula, during the annual repro-ductive cycle. Gen Comp Endocrinol 108:173–181
Glover P (1999) Hypokalaemia. Crit Care Resusc 1:239–251
Greenway SC, Storey KB (2000) Mitogen-activated protein kinasesand anoxia tolerance in turtles. J Exp Zool 287:477–484
Hamamura M, Nunez DJ, Leng G, Emson PC, Kiyama H (1992) c-Fosmay code for a common transcription factor within the hypotha-lamic neural circuits involved in osmoregulation. Brain Res572:42–51
Ladyman M, Bradshaw D, Bradshaw F (2006) Physiological and hor-monal control of thermal depression in the tiger snake, Notechisscutatus. J Comp Physiol [B] 176:547–557
Leng G, Dyball RE, Russell JA (1988) Neurophysiology of body Xuidhomeostasis. Comp Biochem Physiol 90:781–788
Li YW, Dampney RAL (1994) Expression of Fos-like protein in brainfollowing sustained hypertension and hypotension in conciousrabbits. Neuroscience 61:613–634
Lightman SL, Young WS III (1987) Vasopressin, oxytocin, dynorphin,enkephalin and corticotropin-releasing factor mRNA stimulationin the rat. J Neuroendocrinol 1:35–46
Ludwig M, Johnstone LE, Neumann I, Landgraf R, Russel JA (1997)Direct hypertonic stimulation of the rat supraoptic nucleusincreases c-fos expression in glial cells rather than in magnocellu-lar neurones. Cell Tissue Res 287:79–90
Mancera JM, Fernandez-Llebrez P, Perez-Figares JM (1990) Immuno-cytochemical study of the hypophyseal neural lobe of Natrixmaura in dehydration. Rev Esp Fisiol 46:183–189
Neumann I, Pittman QJ, Landgraf R (1995) Release of oxytocin withinthe supraoptic nucleus. Mechanisms, physiological signiWcanceand antisense targeting. Adv Exp Med Biol 395:173–183
Philibert RL, Kamemoto FI (1965) The hypothalamo-hypophysealneurosecretory system of the ring-necked snake, Diadophispunctatus. Gen Comp Endocrinol 44:326–335
Rice GE (1982) Plasma arginine vasotocin concentrations in the lizardVaranus gouldii (Gray) following water loading, salt loading anddehydration. Gen Comp Endocrinol 47:1–6
Robertson GL (1976) The regulation of vasopressin function in healthand disease. Recent Prog Horm Res 33:333–385
Rodríguez EM (1984) Design and perspectives of peptide secretingneurones. In: NemeroV CB, Dunn AJ (eds) Hormones and behav-ior peptides. Spectrum Publishers, New York, pp 1–6
Rodríguez EM, Blazquez JL, Pastor FE, Pelaez B, Pena P, Peruzzo B,Amat P (2005) Hypothalamic tanycytes: a key component ofbrain-endocrine interaction. Int Rev Cytol 247:89–164
Schales A, Schales SS (1941) A simple and accurate method for thedetermination of choride in biological Xuids. J Biol Chem140:879–884
Silveira PF, Mimura OM (1996) L-cystine-di-beta-naphthylamidaseactivity in plasma of the snake Bothrops jararaca after chronicchallenges of water balance. Comp Biochem Physiol 114C:185–192
Silveira PF, Koike TI, Schiripa LN, Reichl AP, Magnoli FC, MimuraOM (1998) Plasma arginine-vasotocin and hydroosmotic status ofthe terrestrial pit viper Bothrops jararaca. Gen Comp Endocrinol109:336–346
Silveira PF, Mimura OM (1999) Concentrating ability of the Bothropsjararaca gallbladder. Comp Biochem Physiol 123:25–33
Silveira PF, Breno MC, Puorto G, Martin-del-Rio MP, Mancera JM(2001) Corticotropin-releasing hormone-like immunoreactivityin the brain of the snake Bothrops jararaca. Histochem J 33:685–694
Silveira PF, Breno MC, Martin-del-Rio MP, Mancera JM (2002) Thedistribution of vasotocin and mesotocin immunoreactivity in thebrain of the snake, Bothrops jararaca. J Chem Neuroanatomy24:15–26
Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Miao GG, SchillingK, Robertson LM, Curran T, Morgan JI (1993) Continuous c-fosexpression precedes programmed cell death in vivo. Nature363:166–169
123
66 J Comp Physiol B (2008) 178:57–66
Smits AW, Lillywhite HB (1985) Maintenance of blood volume insnakes: transcapillary shifts of extravascular Xuids during acutehemorrhage. J Comp Physiol B 155:305–310
Sternberger LA (1986) Imunocitochemistry. Willey-Liss, New YorkVigh B, Manzano-e-Silva MJ, Frank CL, Vincze C, Czirok SJ, Szabo
A, Lukats A, Szel A (2004) The system of cerebrospinal Xuid-contacting neurones. Its supposed role in the nonsynaptic signaltransmission of the brain. Histol Histopathol 19:607–628
Walz W, Hertz L (1983) Functional interactions between neurones andastrocytes. II. Potassium homeostasis at the cellular level. ProgNeurobiol 20:133–183
Wittkowski W (1998) Tanycytes and pituicytes: morphological andfunctional aspects of neuroglial interaction. Microsc Res Tech41:29–42
Yaqub A, Guimaraes M, Eldred WD (1995) Neurotransmitter modula-tion of Fos- and Jun-like proteins in the turtle retina. J Comp Neu-rol 354:481–500
Zemo D, McCabe J (2002) Transcriptional responses of the rat vaso-pressin gene to acute and repeated acute osmotic stress. NeurosciRes 44:45–50
123