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Effects of temperature and general anesthesia on the water gain and the inulin space of the brain of the toad, Bufo arenarum hensel
Camp. Biochem. PhysioL Vol. 88C, No. 2, pp. 331-334, 1987
0
Printed in Great Britain
0306~4492/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
EFFECTS OF TEMPERATURE AND GENERAL ANESTHESIA ON THE WATER GAIN AND THE INULIN SPACE OF THE BRAIN OF THE TOAD, BUFO ARENARUM HENSEL ENRIQUE
lnstituto
de Biologia
T.
SEGURA*,
y Medicina
ALICIA VARSAVSKY and SILVIA PETRIELLA? Experimental, Laboratorio de Fisiologia de1 Comportamiento, 2490, 1428 Buenos Aires, Argentina
(Received
14 January
Obligado
1987)
Abstract-l. The water gain in uifro and the inulin space of the brain of the toad were measured under different experimental conditions, 2. There exists a highly positive correlation between water uptake by the brain and the acclimation-incubation temperature, 7”-37°C. 3. Regional differences in the water gain and inulin space were also demonstrated when measured at 20°C. Higher gains were observed in hemispheres, mesodiencephalon and rhombencephalon. 4. The water gain was higher for whole brains obtained under deep anesthesia with ether or urethane but not under nembutal. The inulin space was also higher under ether but lower under urethane. Nembutal had no effect in this case either. 5. A hypothesis about the possible role of water and electrolyte movements in the mechanism of action of some general anesthetics is advanced.
INTRODUCTION
saturated with moisture at different acclimation temperatures. The brains were obtained after decapitation and dissected at room or acclimation temperature (see below) according to the technique previously described (Segura et al., 1971). The following three measurements were done in different groups of animals: (a) water gain (WG) of the incubated fraction of brain tissue, (b) the extracellular space (ECS) in terms of inulin space, and (c) the overall water content of the whole brain and of different regions. Brains were weighed in a precision balance before, during and after incubation in a RingerConway medium (3) plus 1% of inulin. The inuhn space was measured according to the technique described by Roe ef al. (1949), using resorcinol and thioruea as reagents. The inulin concentration correlated linearly with the absorbance in the range of the used concentrations (up to IO ng inulin/ml of solution). The optic density was estimated by means of DBG Beckman spectrophotometer. A series of experiments were performed.
Regional differences in water and electrolyte content and oxygen consumption of the brain associated with the electroencephalogram, thermal adaptation and behavior, have been described in some species of Anurans (Segura et al., 1971; Depaoli et al., 1973; Lascano et al., 1976). Moreover, drastic changes in the water economy produced by general anesthesia and dependent on adrenergic mechanisms, have also been observed in toads (Segura et al., 1982a,b). These effects appear to be related primarily to marked increases in skin permeability and to changes in urine production by the kidneys. The clear cut effects of general anesthesia on water storage and movements observed in peripheral tissues prompted us to investigate whether a similar phenomenon takes place in the CNS. This paper deals with: (a) effects of acclimation and bath temperature on the weight or water gain (WC) of incubated brains, (b) regional differences in the extracellular space (ECS) in terms of inulin space (IS), and (c) the effects of three different anesthetics on both WG and IS. MATERIAL
Experiment
AND METHODS
Experiments were done throughout the year in adult male toads (Bufo nrennrnm) of lO&lSOg body wt. They were used after 10 days of captivity in a windowless chamber, *Address all correspondence to Dr E. T. Segura, M.D., established investigator of the Consejo National de Investigaciones Cientificas y Tecnicas, Republica Argentina. tFellow of the Consejo National de lnvestigaciones Cientificas y Tecnicas, Republica Argentina. 331
I
Effect of temperature on water gain and extracellular space of the brain in vitro. (a) Animals acclimated at room temperature (20 + 0.5”C) and both measurements were also made at room temperature. The WG and IS were determined in whole brains (N = 6) or halves (N = 6) and compared. The same measurements were also done in the following areas of the bulbs, hemispheres, mesoolfactory encephalon, diencephalon and rhombencephalon separately. (b) Animals acclimated at different temperatures: 7, 20, 30 and 37°C. Measurements were also made at the acclimation temperature in brain halves. The acclimation period was always longer than 2 days. Brain extraction was done after decapitation without anesthesia. All measurements of WG and IS were made after at least 3&40 min of stabilization, according to the method of Zadunaisky and Curran (1963), who found that the IS of the frog’s brain remains stable after 30 min of incubation.
ENRIQUET. SEGURAet al.
332
Effects of different anesthetics on water gain, inulin space and overall water content of the brain. (a) The same procedures as Experiment I(a) were used, but brains were obtained from animals decapitated under deep anesthesia. The following anesthetics were used: sulphuric ether (N = IO), urethane (ethylcarbamate) I g/kg body wt subcutaneously (N = 6) and nembutal (sodium pentobarbital) 40 mg/kg body wt intracarotideal (N = 6). In the last case a carotid artery was cannulated very close to its entrance into the skull 24 hr before experiment. (b) Measurement of the water content of the whole brain during anesthesia: four further groups of 6 animals each were used. One group served as unanesthetized controls and the rest tested after administration of the same anesthetics and doses indicated previously. After decapitation the brains were rapidly taken out of the skull at 0°C wiped carefully with filter paper and weighed in a precision balance (0.1 mg). They were then dehydrated at 8o’C and weighed again until a constant weight, was reached. All differences were estimated as per cents of basal values. Statistical significances were established by means of the Student’s I-test or using the one-way ANOVA program of a HP 9815A calculator. RESULTS
Experiment
1
(a) No significant differences in the response to temperature either in the water gain or in the inulin space between whole brains or halves were observed (water gain: whole brain, 12.0 k 1.0; half brain, 12.2 + 0.8; inulin space: whole brain, 23.9 k 1.7; half brain, 22.4 + 0.8). For this reason the use of half brains in the rest of the experiments was adopted. (b) A highly significant correlation for a linear mode1 (r = 0.81 P < 0.001) between acclimationincubation temperature and water gain was observed according to the following equation: WG = 0.56 T + 0.63
16
1
I 6 U
a
EbP
Fig. 2. The weight gain (WC) of the brain of the toad at 20°C in vitro. (A) Regional differences in the water gain in vifro: WB, whole brain; H, halves; Hem, hemispheres; MD. midbrain and diencephalon; R, rhombencephalon. Ordinates: weight gain in per cent of basal values. (b) Weight gain by the brain of toads sacrificed under general anesthesia: C, unanesthetized controls; E, sulphuric ether; P, pentobarbital; U, urethane. Figures inside each column represent number of cases. ‘P c 0.05. “P < 0.01.
area were observed. So a significantly higher gain was noted for hemispheres (17.0 & 1.1, P < O.Ol), mesodiencephalon (17.1 It 1.0, P < 0.001) and rhombencephalon (15.8 Ifr 1.3, P < 0.005) but not for olfactory bulbs with respect to controls whole brains (12.0 _+ 1.0) or halves (12.2 F 0.8) (Fig. 2). Moreover, higher figures for inulin space were detected in olfactory bulbs (29.9+2.4%, N=6, P qO.01) and in rhombencephalon (28.3 + 0.9, N = 6, P < O.OOl), but not in hemispheres nor in mesodiencephalon (Fig. 3). Experiment
2
Effects of anesthetics. (a) The WG in vitro proved to be significantly higher for brains obtained under ether (15.2% & 0.8, N = IO, P < 0.001) or urethane (16.3 & 1.1, N = 6, ***
were WG is weight gain in per cent of basal value and T, temperature in “C (Fig. 1). Over 30°C the system appears to reach its saturation level. No significant effects of temperature on the inulin space were noted. The following are the actual values of IS obtained for different accIimation temperatures: 7“C, 22.7 f 1.O (N = 8); 20 C. 22.4 & 0.8 (N = 6); 3O”C, 21.9 f 1.1 (N = 7); 37 C. 22.9 & I .3 (N = 6). However, as it is clearly shown in Figs 2 and 3, significant differences in water gain and inulin space depending on the brain
R
2or
+’ 5
0
+-t
//
t 0
7
20
30 Toi
Fig. 1. Changes in water gain in per cent of basal values (ordinates) according to the temperature of the bath (abscisae). There exists a highly significant correlation for a linear model. Note the flattening of the curve beyond 30°C.
Fig. 3. The extracellular space (ECS) of the brain of toads. (a) Regional distribution: WB, whole brain controls; H, halves; 03, olfactory bulbs; Hem, hemispheres; MD, midbrain and diencephalon; R, rhomben~phalon. (b) Changes in the ECS due to general anesthesia: C, unanesthetized controls; E, ether; P, pentobarbital; U, urethane. Figures inside each column represent number of cases. ‘P i 0.05. “P < 0.01. “‘P<0.001.
Effects of temperature and general anesthesia P < 0.05) whereas no differences were noted with nembutal anesthesia, with respect to unanesthetized controls (12.2% k 0.2) (Fig. 2). The inulin space was also higher under ether (26.8 f 0.4%), N = 10, P < 0.001) but lower under urethane (19.2 + 1.0, N = 6, P < 0.05). Nembutal had no effect in this case either (Fig. 3). (b) No differences in the overall water content of the anesthetized brain were observed. DISCUSSION
Both, water compartmentation and water gain by the brain in vitro have been the subject of numerous studies in several species of mammalian and submammalian vertebrates (Bourke et al., 1965; Franck, 1970; Segura et al., 1971). Different techniques were applied to estimate the extracellular space and consequently a wide range of values has been observed (f&40% according to the findings of Fenstermacher et al., 1970). Thus the question about the most accurate method for this measurement remains open at present. Intimately associated with this point is the question about the functional meaning of the extracellular space of the brain broadly identified as the “brain cell microenvironment” (Schmitt and Samson, 1969). A relevant finding in this sense was that of Bourke et al. (1965) who observed a significant positive correlation between brain size and extracellular space along the vertebrate scale. Segura et nl. (1971) using the chloride space as an index defined an extracellular space of l&l 1% in the brain of the toad, in agreement with Bourke’s evolutionary hypothesis. Possible regional and time-dependent differences in the same brain have also been considered (Schmitt, 1969). With respect to factors influencing water uptake in vitro, Zadunaisky et al. (1965) conclude that the water gain by the isolated brain tissue is due to retention of salts largely by glia cells. Okamoto and Quastel (1970) classified the factors affecting electrolyte influx and thereby water uptake in two main categories: (1) those factors affecting permeability of membranes by structural alteration so allowing sodium migration down its electrochemical gradient. In this case the energetics of the cell would not be primarily affected; L-glutamate and electrical stimulation belong in this class, and (2) substances or procedures that modify the activity of the sodium pump, either by effect on Na+-K+ stimulated ATPase or on the synthesis or breakdown of ATP. Substances like ouabain, Cu2+ or 2,4- dinitrophenol or the absence of Na+ or K+ in the incubation medium might be included in this class. Several authors (Torack et al., 1965; Pate1 et al., 1971; Moller et al., 1974) agree that during incubation of brain slices, increases in extracellular space take place during the first 30 min, and then cease. For this reason a stabilization period of 40min before experimentation was chosen in the present study. A direct correlation between increases in extracellular space and significant extrusion of K+ by brain cells is generally accepted (Hertz, 1977). Moreover, increases in CNS excitability and migration of K+ from cells to the extracellular space appear to be closely associated. For instance, repetitive electrical
333
stimulations of the mammalian cortex or of afferent pathways or propagated seizures lead to a rise in the potassium concentration in the extracellular fluid of the brain or the spinal cord (Hofson et al., 1973; Moody et al., 1974; Singer and Lux, 1975). The highest extracellular content of K+ in the brain in vitro has been observed during “spreadingdepression” (Futamachi et al., 1974; Lothman et al., 1975). This phenomenon is normally accompanied by increases in oxygen consumption and swelling (Van Harreveld, 1958). Typical “spreading-depression” is obtained under moderate barbiturate anesthesia (Hertz, 1977). However, Katzman and Grossman (1975) reported no changes in the extracellular potassium in either the cat or rat cortex during ether or barbiturate anesthesia. Our own calculations made on brains of animals submitted to ether or urethane is in agreement with this last finding. However, since deep anesthesia causes depolarization of brain tissues, either water gain or increases in extracellular space might potentially be involved in its mechanism of production. The view that brain weight gain in vitro is primarily due to water uptake by cells is generally accepted (Hertz, 1977). This water gain is dependent on the osmotic difference between the inside and outside of the cells. Thus, a higher uptake would be indicative of a higher electrolyte concentration in the cells. This appears to be the case with ether but not with urethane. Of course, the possibility that anesthetics do affect the electrolyte distribution and the transmembrane gradient is open. As it was in the case for the distribution of water and different electrolytes, Nat, K+, Cl and Ca2+ (Segura et al., 1971) the brain of the toad proved to be inhomogeneous for water gain and inulin space. Thus, the WG was greater for telencephalon, mesodiencephalon and rombencephalon but lower for olfactory bulbs. Theoretical estimations of the electrolyte compartmentation using previous data (Segura et al., 1971) show significantly higher concentrations in the extracellular content of Nat and Clunder urethane only. In no case did intracellular electrolyte concentrations show regional differences. Moreover larger IS were measured in the olfactory bulb and rhombencephalon. However, no correlation between water content, water gain and extracellular space was found. The linear correlation between water gain and temperature of acclimation and incubation suggests a proportional increase of intracellular electrolytes, the bath becoming increasingly hypotonic with respect to the tissue with the corresponding increase in water gain. The stabilization in water gain observed between 30 and 37°C coincides with the marked decrease in oxygen consumption at similar temperatures reported previously in the same preparation (Depaoli et al., 1973). This indicates that temperature stress is able to affect the water uptake by the brain in vitro through the second mechanism mentioned by Okamoto and Quastel (1970). Since no significant differences in the IS were observed after incubation for either whole brains or halves, the harmfulness of surgical manipulation employed appears to be negligible. This is in agreement with Franck’s data (1970) but at variance with
ENRIQUE T. SECURAet al.
334
that of Kessey (1965), both working in mammalian brains. No effects on the IS due to changes in the bath temperature were detected. Similar results have been reported by Fenstermacher et al. (1970) working on the inulin space of the cat cortex in ho. They lowered the temperature of the brain to 15°C and no significant changes in tissue-specific resistivity nor in the inulin space were observed. On the basis of our results the following conclusions might be drawn: (1) Increases in the ECS of the brain consecutive to ether anesthesia would be due to solubility of the anesthetic in the lipids of the cells membrane. A significant increase in cationic release by lipidic vesicles of neurons under the effect of general anesthetics appears to be demonstated (Franks and Lieb, 1982). Decreases in the inulin space observed with urethane would be indicative of increases in water and ionic fluxes into the cells and swelling (Segura et al., 1982). The lack of effect of barbiturates on the ECS or on water gain might suggest that the anesthetic effects are mainly related to the selective disconnection of the reticular mechanisms they produce (Goodman and Gilman, 1980). (2) The perspective that water and electrolyte movements in the brain would be involved as primary components in the mechanism of action of some general anesthetics must be taken into account. research was aided by a grant of the Consejo National de Investigaciones Cientificas y Tecnicas, Republica Argentina. Acknowledgement-This
REFERENCES
Bourke R. S., Greemberg E. S. and Tower D. B. (1965) Variations of cerebral cortex fluid spaces in uivo as a function of species brain size. Am. J. Physiol. 208, 682692. Boyle P. J. and Conway E. J. (1941) Potassium accumulation in muscle and associated changes. J. Physiol. (Land.) 100, 163. Depaoli J. R., Segura E. T. and Lascano Elena C. (1973) Effects of acclimation on oxygen consumption (QO,) of the brain and its different areas in two genera of anurans. Camp. Biochem. Physiol. 46A, 725-733. Fenstermacher J. D., Li C. L. and Levin, V. A. (1970) Extracellular space of the cerebral cortex of normothermic and hypothermic cats. Exp. Neural. 26, 571-585. Franck G. (1970) Echanges cationiques au niveau des neurones et des cellules gliales du cerveau. Arch. Inr. Physiol. Biochim. 78, 6 13-866. Franks N. P. and Lieb W. R. (1982) Molecular mechanisms of general anaesthesia. Nature 300, 487493. Futamachi K., Mutani R. and Prince D. A. (1974) Potassium activity in rabbit cortex. Bruin Res, 75, 5-25. Goodman Gilman A., Goodman L. S. and Gilman A. (1980) The Pharmacological Basis of Therapeutics, 6th Edn. Macmillan Publishing Co., New York. Hertz L. (1977) Drug inducedalterations of ion distribution at the cellular level of the central nervous system Pharmar. Rev. 29, 35-65. Hotson J. R., Sypert G. W. and Ward A. A. (1973)
Extracellular potassium concentration changes during propagated seizures. E,rp. Neural. 38, 2g-26. Katzman R. and Grossman R. (1975) Neuronal activity and potassium movement. In Brain Work, AlJred Benzon Symposium (Edited by lngvar D. H. and Lassen N. A.). Vol. 8, pp. 149-166. MunKsgaard, Copenhagen. Keesey J. C.. Wallgreen H. and Mcllwain H. (1965) The sodium. potassium and chloride of cerebral tissues. Maintenance, change on stimulation and subsequent recovery. Biochem. J. 95, 289-300. Lascano Elena C.. Depaoli J. R. and Segura E. T. (1976) Influence of acclimation and determination temperature on the oxygen consumption of the brain in two species of anurans. J. Therm. Biol. 2, 209-212. Lothman E. W. and Somjen G. G. (1975) Extracellular potassium activity, intracellular and extracellular potential responses in the spinal cord. J. Physiol. (Land.) 252, 115-136. Mnller M.. Lund-Andersen H., Msllgard K. and Hertz L. (1974) Concordance between morphological and biochemical estimates of fluid spaces in rat brain cortex slices. Exp. Brain Res. 22, 299.-314. Moody W., Futamachi K. J. and Prince, D. A. (1974) Extracellular potassium activity during epileptogenesis. Exp. Neural. 42, 248-263. Okamoto K. and Quastel J. H. (1970) Water uptake and energy metabolism in brain slices from the rat. Biochem, J. 120, 25-36.
Pate1 K. K., Hartmann J. F. and Cohen M. M. (1971) Ultrastructural estimation of relative volume of extracellular space in brain slices. J. Neural. Sci. 12, 275-288. Roe J. H., Epstein J. H. and Goldstein N. P. (1949) A photometric method for the determination of inulin in plasma and urine. J. biol. Chem. 178, 839-845. Schmitt 0. F. (1969) Brain cell membranes and their microenvironment. Neurosci. Res. Prog. Bull. 7, 281-300. Schmitt 0. F. and Samson F. E. (1969) Brain cell microenvironment. Neurosci. Res. Prog. BUN. 7, 217417. Segura E. T. and Depaoli J. R. (1971) Topographic correspondence among electrographic pattern, water and electrolytes in the toad brain. Camp. Biochem. Physiol. 38A, 655-661. Segura E. T., Bandsholm Ulla C., Bronstein A. and Woscoboinik D. (1982a) Role of the CNS in the control of the water economy of the toad, Bufo arenarum Hensel. 1. Effects of handling, brain lesions, anesthesia and reversible coma upon water uptake, urine production and overall water balance. J. camp. Physiol. 146B. 95%100. Segura E. T., Bandsholm Ulla- C. and Bronstein A. M. (1982b) Role of the CNS in the control of the water economy of the toad Bufo arenarum Hensel. II. Adrenergic control of water uptake across the skin. J. camp. Physiol. 146B, 101-106. Singer W. and Lux H. D. (1975) Extracellular potassium gradients and visual receptive fields in the cat striate cortex. Brain Res. 96, 378-383. Torack R. M., Dufty M. L. and Haynes J. M. (1965) The effect of anisotonic media upon cellular ultrastructure in fresh and fixed rat brain. Z. Zellforsch Mikroskop. Anar. 66, 690-700. Van Harreveld A. (1958) Changes in the diameter of apical dendrites during spreading depression. Am. J. Physiol. 192, 457463.
Zadunaisky J. A. and Curran P. F. (1963) Sodium fluxes in isolated frog brain. Am. J. Physiol. 205, 949-956. Zandunaisky J. A., Wald Flora and De Robertis E. (1963) Osmotic behaviour and ultrastructural modifications in isolated frog brains. Exp. Neurol. 8, 29G-309.
0
Printed in Great Britain
0306~4492/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
EFFECTS OF TEMPERATURE AND GENERAL ANESTHESIA ON THE WATER GAIN AND THE INULIN SPACE OF THE BRAIN OF THE TOAD, BUFO ARENARUM HENSEL ENRIQUE
lnstituto
de Biologia
T.
SEGURA*,
y Medicina
ALICIA VARSAVSKY and SILVIA PETRIELLA? Experimental, Laboratorio de Fisiologia de1 Comportamiento, 2490, 1428 Buenos Aires, Argentina
(Received
14 January
Obligado
1987)
Abstract-l. The water gain in uifro and the inulin space of the brain of the toad were measured under different experimental conditions, 2. There exists a highly positive correlation between water uptake by the brain and the acclimation-incubation temperature, 7”-37°C. 3. Regional differences in the water gain and inulin space were also demonstrated when measured at 20°C. Higher gains were observed in hemispheres, mesodiencephalon and rhombencephalon. 4. The water gain was higher for whole brains obtained under deep anesthesia with ether or urethane but not under nembutal. The inulin space was also higher under ether but lower under urethane. Nembutal had no effect in this case either. 5. A hypothesis about the possible role of water and electrolyte movements in the mechanism of action of some general anesthetics is advanced.
INTRODUCTION
saturated with moisture at different acclimation temperatures. The brains were obtained after decapitation and dissected at room or acclimation temperature (see below) according to the technique previously described (Segura et al., 1971). The following three measurements were done in different groups of animals: (a) water gain (WG) of the incubated fraction of brain tissue, (b) the extracellular space (ECS) in terms of inulin space, and (c) the overall water content of the whole brain and of different regions. Brains were weighed in a precision balance before, during and after incubation in a RingerConway medium (3) plus 1% of inulin. The inuhn space was measured according to the technique described by Roe ef al. (1949), using resorcinol and thioruea as reagents. The inulin concentration correlated linearly with the absorbance in the range of the used concentrations (up to IO ng inulin/ml of solution). The optic density was estimated by means of DBG Beckman spectrophotometer. A series of experiments were performed.
Regional differences in water and electrolyte content and oxygen consumption of the brain associated with the electroencephalogram, thermal adaptation and behavior, have been described in some species of Anurans (Segura et al., 1971; Depaoli et al., 1973; Lascano et al., 1976). Moreover, drastic changes in the water economy produced by general anesthesia and dependent on adrenergic mechanisms, have also been observed in toads (Segura et al., 1982a,b). These effects appear to be related primarily to marked increases in skin permeability and to changes in urine production by the kidneys. The clear cut effects of general anesthesia on water storage and movements observed in peripheral tissues prompted us to investigate whether a similar phenomenon takes place in the CNS. This paper deals with: (a) effects of acclimation and bath temperature on the weight or water gain (WC) of incubated brains, (b) regional differences in the extracellular space (ECS) in terms of inulin space (IS), and (c) the effects of three different anesthetics on both WG and IS. MATERIAL
Experiment
AND METHODS
Experiments were done throughout the year in adult male toads (Bufo nrennrnm) of lO&lSOg body wt. They were used after 10 days of captivity in a windowless chamber, *Address all correspondence to Dr E. T. Segura, M.D., established investigator of the Consejo National de Investigaciones Cientificas y Tecnicas, Republica Argentina. tFellow of the Consejo National de lnvestigaciones Cientificas y Tecnicas, Republica Argentina. 331
I
Effect of temperature on water gain and extracellular space of the brain in vitro. (a) Animals acclimated at room temperature (20 + 0.5”C) and both measurements were also made at room temperature. The WG and IS were determined in whole brains (N = 6) or halves (N = 6) and compared. The same measurements were also done in the following areas of the bulbs, hemispheres, mesoolfactory encephalon, diencephalon and rhombencephalon separately. (b) Animals acclimated at different temperatures: 7, 20, 30 and 37°C. Measurements were also made at the acclimation temperature in brain halves. The acclimation period was always longer than 2 days. Brain extraction was done after decapitation without anesthesia. All measurements of WG and IS were made after at least 3&40 min of stabilization, according to the method of Zadunaisky and Curran (1963), who found that the IS of the frog’s brain remains stable after 30 min of incubation.
ENRIQUET. SEGURAet al.
332
Effects of different anesthetics on water gain, inulin space and overall water content of the brain. (a) The same procedures as Experiment I(a) were used, but brains were obtained from animals decapitated under deep anesthesia. The following anesthetics were used: sulphuric ether (N = IO), urethane (ethylcarbamate) I g/kg body wt subcutaneously (N = 6) and nembutal (sodium pentobarbital) 40 mg/kg body wt intracarotideal (N = 6). In the last case a carotid artery was cannulated very close to its entrance into the skull 24 hr before experiment. (b) Measurement of the water content of the whole brain during anesthesia: four further groups of 6 animals each were used. One group served as unanesthetized controls and the rest tested after administration of the same anesthetics and doses indicated previously. After decapitation the brains were rapidly taken out of the skull at 0°C wiped carefully with filter paper and weighed in a precision balance (0.1 mg). They were then dehydrated at 8o’C and weighed again until a constant weight, was reached. All differences were estimated as per cents of basal values. Statistical significances were established by means of the Student’s I-test or using the one-way ANOVA program of a HP 9815A calculator. RESULTS
Experiment
1
(a) No significant differences in the response to temperature either in the water gain or in the inulin space between whole brains or halves were observed (water gain: whole brain, 12.0 k 1.0; half brain, 12.2 + 0.8; inulin space: whole brain, 23.9 k 1.7; half brain, 22.4 + 0.8). For this reason the use of half brains in the rest of the experiments was adopted. (b) A highly significant correlation for a linear mode1 (r = 0.81 P < 0.001) between acclimationincubation temperature and water gain was observed according to the following equation: WG = 0.56 T + 0.63
16
1
I 6 U
a
EbP
Fig. 2. The weight gain (WC) of the brain of the toad at 20°C in vitro. (A) Regional differences in the water gain in vifro: WB, whole brain; H, halves; Hem, hemispheres; MD. midbrain and diencephalon; R, rhombencephalon. Ordinates: weight gain in per cent of basal values. (b) Weight gain by the brain of toads sacrificed under general anesthesia: C, unanesthetized controls; E, sulphuric ether; P, pentobarbital; U, urethane. Figures inside each column represent number of cases. ‘P c 0.05. “P < 0.01.
area were observed. So a significantly higher gain was noted for hemispheres (17.0 & 1.1, P < O.Ol), mesodiencephalon (17.1 It 1.0, P < 0.001) and rhombencephalon (15.8 Ifr 1.3, P < 0.005) but not for olfactory bulbs with respect to controls whole brains (12.0 _+ 1.0) or halves (12.2 F 0.8) (Fig. 2). Moreover, higher figures for inulin space were detected in olfactory bulbs (29.9+2.4%, N=6, P qO.01) and in rhombencephalon (28.3 + 0.9, N = 6, P < O.OOl), but not in hemispheres nor in mesodiencephalon (Fig. 3). Experiment
2
Effects of anesthetics. (a) The WG in vitro proved to be significantly higher for brains obtained under ether (15.2% & 0.8, N = IO, P < 0.001) or urethane (16.3 & 1.1, N = 6, ***
were WG is weight gain in per cent of basal value and T, temperature in “C (Fig. 1). Over 30°C the system appears to reach its saturation level. No significant effects of temperature on the inulin space were noted. The following are the actual values of IS obtained for different accIimation temperatures: 7“C, 22.7 f 1.O (N = 8); 20 C. 22.4 & 0.8 (N = 6); 3O”C, 21.9 f 1.1 (N = 7); 37 C. 22.9 & I .3 (N = 6). However, as it is clearly shown in Figs 2 and 3, significant differences in water gain and inulin space depending on the brain
R
2or
+’ 5
0
+-t
//
t 0
7
20
30 Toi
Fig. 1. Changes in water gain in per cent of basal values (ordinates) according to the temperature of the bath (abscisae). There exists a highly significant correlation for a linear model. Note the flattening of the curve beyond 30°C.
Fig. 3. The extracellular space (ECS) of the brain of toads. (a) Regional distribution: WB, whole brain controls; H, halves; 03, olfactory bulbs; Hem, hemispheres; MD, midbrain and diencephalon; R, rhomben~phalon. (b) Changes in the ECS due to general anesthesia: C, unanesthetized controls; E, ether; P, pentobarbital; U, urethane. Figures inside each column represent number of cases. ‘P i 0.05. “P < 0.01. “‘P<0.001.
Effects of temperature and general anesthesia P < 0.05) whereas no differences were noted with nembutal anesthesia, with respect to unanesthetized controls (12.2% k 0.2) (Fig. 2). The inulin space was also higher under ether (26.8 f 0.4%), N = 10, P < 0.001) but lower under urethane (19.2 + 1.0, N = 6, P < 0.05). Nembutal had no effect in this case either (Fig. 3). (b) No differences in the overall water content of the anesthetized brain were observed. DISCUSSION
Both, water compartmentation and water gain by the brain in vitro have been the subject of numerous studies in several species of mammalian and submammalian vertebrates (Bourke et al., 1965; Franck, 1970; Segura et al., 1971). Different techniques were applied to estimate the extracellular space and consequently a wide range of values has been observed (f&40% according to the findings of Fenstermacher et al., 1970). Thus the question about the most accurate method for this measurement remains open at present. Intimately associated with this point is the question about the functional meaning of the extracellular space of the brain broadly identified as the “brain cell microenvironment” (Schmitt and Samson, 1969). A relevant finding in this sense was that of Bourke et al. (1965) who observed a significant positive correlation between brain size and extracellular space along the vertebrate scale. Segura et nl. (1971) using the chloride space as an index defined an extracellular space of l&l 1% in the brain of the toad, in agreement with Bourke’s evolutionary hypothesis. Possible regional and time-dependent differences in the same brain have also been considered (Schmitt, 1969). With respect to factors influencing water uptake in vitro, Zadunaisky et al. (1965) conclude that the water gain by the isolated brain tissue is due to retention of salts largely by glia cells. Okamoto and Quastel (1970) classified the factors affecting electrolyte influx and thereby water uptake in two main categories: (1) those factors affecting permeability of membranes by structural alteration so allowing sodium migration down its electrochemical gradient. In this case the energetics of the cell would not be primarily affected; L-glutamate and electrical stimulation belong in this class, and (2) substances or procedures that modify the activity of the sodium pump, either by effect on Na+-K+ stimulated ATPase or on the synthesis or breakdown of ATP. Substances like ouabain, Cu2+ or 2,4- dinitrophenol or the absence of Na+ or K+ in the incubation medium might be included in this class. Several authors (Torack et al., 1965; Pate1 et al., 1971; Moller et al., 1974) agree that during incubation of brain slices, increases in extracellular space take place during the first 30 min, and then cease. For this reason a stabilization period of 40min before experimentation was chosen in the present study. A direct correlation between increases in extracellular space and significant extrusion of K+ by brain cells is generally accepted (Hertz, 1977). Moreover, increases in CNS excitability and migration of K+ from cells to the extracellular space appear to be closely associated. For instance, repetitive electrical
333
stimulations of the mammalian cortex or of afferent pathways or propagated seizures lead to a rise in the potassium concentration in the extracellular fluid of the brain or the spinal cord (Hofson et al., 1973; Moody et al., 1974; Singer and Lux, 1975). The highest extracellular content of K+ in the brain in vitro has been observed during “spreadingdepression” (Futamachi et al., 1974; Lothman et al., 1975). This phenomenon is normally accompanied by increases in oxygen consumption and swelling (Van Harreveld, 1958). Typical “spreading-depression” is obtained under moderate barbiturate anesthesia (Hertz, 1977). However, Katzman and Grossman (1975) reported no changes in the extracellular potassium in either the cat or rat cortex during ether or barbiturate anesthesia. Our own calculations made on brains of animals submitted to ether or urethane is in agreement with this last finding. However, since deep anesthesia causes depolarization of brain tissues, either water gain or increases in extracellular space might potentially be involved in its mechanism of production. The view that brain weight gain in vitro is primarily due to water uptake by cells is generally accepted (Hertz, 1977). This water gain is dependent on the osmotic difference between the inside and outside of the cells. Thus, a higher uptake would be indicative of a higher electrolyte concentration in the cells. This appears to be the case with ether but not with urethane. Of course, the possibility that anesthetics do affect the electrolyte distribution and the transmembrane gradient is open. As it was in the case for the distribution of water and different electrolytes, Nat, K+, Cl and Ca2+ (Segura et al., 1971) the brain of the toad proved to be inhomogeneous for water gain and inulin space. Thus, the WG was greater for telencephalon, mesodiencephalon and rombencephalon but lower for olfactory bulbs. Theoretical estimations of the electrolyte compartmentation using previous data (Segura et al., 1971) show significantly higher concentrations in the extracellular content of Nat and Clunder urethane only. In no case did intracellular electrolyte concentrations show regional differences. Moreover larger IS were measured in the olfactory bulb and rhombencephalon. However, no correlation between water content, water gain and extracellular space was found. The linear correlation between water gain and temperature of acclimation and incubation suggests a proportional increase of intracellular electrolytes, the bath becoming increasingly hypotonic with respect to the tissue with the corresponding increase in water gain. The stabilization in water gain observed between 30 and 37°C coincides with the marked decrease in oxygen consumption at similar temperatures reported previously in the same preparation (Depaoli et al., 1973). This indicates that temperature stress is able to affect the water uptake by the brain in vitro through the second mechanism mentioned by Okamoto and Quastel (1970). Since no significant differences in the IS were observed after incubation for either whole brains or halves, the harmfulness of surgical manipulation employed appears to be negligible. This is in agreement with Franck’s data (1970) but at variance with
ENRIQUE T. SECURAet al.
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that of Kessey (1965), both working in mammalian brains. No effects on the IS due to changes in the bath temperature were detected. Similar results have been reported by Fenstermacher et al. (1970) working on the inulin space of the cat cortex in ho. They lowered the temperature of the brain to 15°C and no significant changes in tissue-specific resistivity nor in the inulin space were observed. On the basis of our results the following conclusions might be drawn: (1) Increases in the ECS of the brain consecutive to ether anesthesia would be due to solubility of the anesthetic in the lipids of the cells membrane. A significant increase in cationic release by lipidic vesicles of neurons under the effect of general anesthetics appears to be demonstated (Franks and Lieb, 1982). Decreases in the inulin space observed with urethane would be indicative of increases in water and ionic fluxes into the cells and swelling (Segura et al., 1982). The lack of effect of barbiturates on the ECS or on water gain might suggest that the anesthetic effects are mainly related to the selective disconnection of the reticular mechanisms they produce (Goodman and Gilman, 1980). (2) The perspective that water and electrolyte movements in the brain would be involved as primary components in the mechanism of action of some general anesthetics must be taken into account. research was aided by a grant of the Consejo National de Investigaciones Cientificas y Tecnicas, Republica Argentina. Acknowledgement-This
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