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Stereological analysis of mitochondria from brains of temperature acclimated goldfish, Carassius auratus L. (5 and 30°C)
J. therm. Biol. Vol. 14, No. 4, pp. 187-190, 1989
0306-4565/89 $3.00+ 0.00 Copyright © 1989PergamonPress pk
Printed in Great Britain. All rights r--~erved
STEREOLOGICAL ANALYSIS OF MITOCHONDRIA FROM BRAINS OF TEMPERATURE ACCLIMATED GOLDFISH, CARASSIUS AURATUS L. (5 AND 30°C) KATHRYNGAEBELand BETTYI. ROOTS Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 1A1 (Received 11 December 1988; accepted 1 March 1989)
Abstract--l. The mitochondrial population in hypothalamic and hypophysial brain tissue from warm (30°C) and cold (5°C) acclimated goldfish (Carassius auratus L.) was analyzed using stereologieal techniques. 2. It was revealed that there is a significantly larger volume density (Vv) in the cold accfimated tissue, with no significant difference in either of the surface densities (Svext and Sv/nt) from either of the brain areas. 3. The hypothalamic brain tissue has a significantly lower specific surface (S/V) in the cold acclimated tissue but there is not a significant difference in this parameter for the hypophysial brain tissue. 4. The values for these three parameters (Vv, Svext and Sv/nt, and S/V) indieate that mitochondria from acclimated brain tissue undergo shape changes in response to thermal streu. 5. We suggest that the shape changes may be related to the change in the phospholipid composition of the inner mitochondrial membrane with acclimation temperature. Key Word Index--Temperature acclimation, mitochondria, quantitative, stereology, goldfish, hypothala-
mus, hypophysis, brain.
INTRODUCTION
Many poikilotherms encounter large fluctuations in their environmental temperature, therefore these organisms must possess physiological mechanisms that allow them to adapt to the new environment. Goldfish, Carassius auratus L., have the ability to acclimate to temperature changes in their environment. Cold acclimated brain tissue of goldfish (Freeman, 1950), as well as muscle tissue (Hochachka and Hayes, 1962), consumes more oxygen than the warm acclimated tissue. Significant differences in enzyme activities between cold and warm acclimated tissue have also been reported. Glycolyric enzymes in brain tissue have greater specific activities upon cold acclimation but muscle giycolytic enzymes show lower specific activities (Shakelee et al., 1977). The effect of environmental temperature o n the mitochondrial enzymes has been well documented, cytochrome c oxidase being one enzyme that exhibits a dramatic change in specific activity with acclimation temperature (Caldwell, 1969; Lehmann, 1970; Shakelee et al., 1977). The specific activity of cytochrome oxidase is greater upon cold acclimation and this holds true for both muscle and brain tissue (Sidell, 1977; Shakelee et al., 1977). Morphological changes that occur with temperature acclimation are not as well documented. It is known that there are twice as many oxidative fibres in epaxial muscle of 5°C acclimated goldfish compared with 25°C muscle tissue (Sidell, 1980). Also there are differences in the density of mitochondria in muscle tissue from goldfish acclimated to cold temperatures compared with warm acclimated animals (Tyler and Sidell, 1984).
The purpose of this study was to determine whether morphological changes occur during thermal acclimation in brain tissue of goldfish. The volume and surface densities of the mitochondrial population were determined using stereological techniques. METHODS Animals
Goldfish (C. auratus, L.) 10-13crn in length were obtained commercially (Hartz Can., Inc., Rexdale, Ont.). Fish were maintained for 4 weeks at acclimarion temperatures of 5 and 30°C in 68L tanks. There was a 12 h photoperiod in each of the environ. mental rooms kept at the respective acclimation temperatures. Fish were fed Purina Fish Chow twice a day at 30°C, and every other day at 5°C. Tissue preparation
Eight fish from each acclimation temperature were decapitated, brains removed and immediately immersed in 2.5% glutaraldehyde in Karlsson-Shultz (KS) phosphate buffer, pH 7.4 (Karlsson and Shultz, 1965). The anterior portion of the left lateral lobe of the hypothaiamus, and the entire hypophysis were removed, cut into approx. 1 mm cubes and placed in fresh fixative. After overnight fixation at 4°C, brains were post-fixed with 1% osmium tetroxide (OsO4) in KS buffer for 1.5 h and then dehydrated with a series of ethanol solutions in increasing concentrations (25, 50, 70, 90, 95 and 100%). Tissue was stained en bloc with 1% uranyl acetate in 100% ethanol and then embedded in epon-araldite (Mollenhauer, 1964) using
187
KATHRYN GAEBEL and BETTY I. ROOTS
188
propylene oxide as the infiltration medium. This entire procedure was repeated three times. Tissue blocks from 3 fish, were randomly chosen from each of the acclimation temperatures, resulting in final data collection from 18 animals, 9 from each temperature. Two blocks from the hypothalamus and four blocks from the hypophysis from each fish were sectioned using a Sorval MT-1 microtome, to produce three ribbons of ultrathin sections. These ribbons were placed on 300 mesh copper grids and stained to ensure that one grid of sections was usable (i.e. allowing for sections lost during staining process, sections that were too thick or wrinkled for photographing, over-staining, etc.). Grids were stained for 20min with 7% uranyl acetate in 70% methanol, washed with distilled water and then stained with Sato's lead citrate stain (Sato, 1967) for 5 rain.
Stereoiogy Ten areas of each section (one section per tissue block) were photographed twice, once at a low magnification (mag) and then at a higher mag, using a Philips 200 or 201 transmission electron microscope. To calculate the volume density of mitochondria (Vv) in brain tissue the low mag negatives were analyzed with a point lattice with 8.519]mm point-topoint spacing (which would be equivalent to 1 p m spacing within the tissue) such that each section was analyzed with 6480[5750] points covering 6172.815308.6]/~m 2. The numbers in square brackets refer to the measurements when the negatives were from the Philips 201 microscope. The lattice was placed over the screen of a Telex Caramite 4000 slide projector in which the negatives were placed for viewing and counting (Final mag 8580[8940] × ). The number of points which fell upon the mitochondria was recorded as well as the total number of points covering the test tissue. From this the volume density can be calculated (Williams, 1977): No. of points covering the mitochondria Vv ffi total No. of points covering the test tissue A mean volume density was then calculated for each of the two brain parts, from each acclimation temperature, in each of the three experiments. This yields means for sample sizes of 180 for the hypothalamus and 360 for the hypophysis per experiment. The volume density of mitochondria (Vv) reported is the mean of the three mean volume densities at each of the respective acclimation temperatures. The surface density for the outer mitochondrial membrane (Svext) and the inner mitochondrial
membrane including cristae (Sv/nt) was calculated using high mag negatives analyzed with a line lattice. The line lattice had 2.513]cm line-to-line spacings (again this is equivalent to 1 ~m spacing within the tissue) therefore each negative was analyzed with 167211881] nun of test line that covered an area of 487.91649.6]#m 2. Each negative was viewed twice. During the first viewing the number of times the test line intersected the outer mitochendrial membrane was recorded. The number of times the test line intersected the inner mitochondriai membrane including cristae was recorded during the second viewing. The entire length of test line covering the test tissue was also recorded. From these observations the surface density for the outer mitochondrial membrane, Svext, (or the inner mitochondrial membrane including cristae, Svint) can be calculated as follows (Williams, 1977): SV = 4 - × m
where m, the contour length is: No. of intersections × mag total length of test line × 1000 The final surface densities (Svext and Sv/nt) reported here are the mean of the mean surface densities of the three experiments. Each test area (10 per block) has a corresponding volume density (Vv) and two surface densities (Svext and Svim). From these parameters a specific surface density (S/V) can be calculated by simply dividing the Svext by the Vv. A mean of the mean specific surfaces from the three experiments can then be calculated. Statistical comparisons between sample sets were made using the Student's t-Test. n ×
m~-
2 ×
RESULTS
Stereological analyses (Table 1) show that there is a significant increase in mitochondrial volume density (Vv) with cold acclimation in both brain areas. The surface densities for both the internal mitochondriai membrane including cristae (Sv/m)and the external mitochondrial membrane (Svext) do not increase significantly with cold acclimation in either brain part. The specific surface, S/V is ~ c a n t l y ~ t between the two temperature extremes in the thalamic brain tissue but not in the hypophy~d brain tissue. An increase in mitochondrial volume density (Vv) can result from either (i) an increase in the number
Table 1. Stereological parameters for mitochondria in hypothalamic and hypophysial brain tissue from temperature accliamt~ 8oldflsb Hypothslamus parameter
5°C
30°C
Vv(%)
Hypophytis P t-Test
5°C
30°C
P t -Test
4.973 ± 0.000 4.067 ± 0,003 < 0.05 4.483 + 0.002 3.567 ± 0.000 < 0.02 0.239 ± 0.012 0.224 ± 0.008 NS 0.220 4- 0.014 0.185 + 0.026 NS Sv(/nt) 0.342 + 0.005 0.337 ± 0.009 NS 0.310 ± 0.030 0.2~8 + 0.044 NS S/V 4.758±0.005 5.781 +0,009 <0,02 4.931 4- 0.0~6. . . . 5 . Ill~ 0 . I6 9 3 NS I III t II II I [: I II [ Val .ue, are meant + ~ for I 8 blocks ft~m tlWhE~hslami¢ braintlmuland ~ ~ tlm~ tmsue at each temperature regime. P: St~tiati~ ~ p a r i s o n was ¢kaer~ined ueing S t ~ t ' s t-Test. Vv: volume density. Sv(ext): surface density for the outer mitochondrial membrane. Sv(/m): surface density for the inner mitocbondrial membrane including cristae. S/V: specific surface.
Sv(ext)
II
II
Stereological analysis of mitochondria from brains of temperature acclimated goldfish
189
concentration is enough to reduce the swelling caused by this fixative. As may be seen in Fig. 1 inset; the 'bulging' mitochondria are still present. This indicates that this morphological feature may not be an artifact caused by the fixative.
DISCUSSION
Fig. I. Electron micrograph of hypothalamic brain tissue from goldfish acclimated to 5°C showing mitochondria with dense matrices and areas that are empty and appear to 'bulge' out. Brain tissue from acclimated goldfish fixed with glutaraldehyde supplemented with 0.015% calcium (inset) also appear to 'bulge' out.
of mitochondria, (ii) an increase in the size of the individual mitochondria or (iii) a combination of both. Stereological methods do not allow the direct determination of numbers of organelles with irregular shapes such as mitochondria (Weibel, 1980). The specific surface (S/V) will give an indication of whether size or numbers are changing in the mitochondrial population. An increase in the volume density (Vv) and the surface density (Svext) and no change in the specific surface (S/V) suggest the changes are due to an increase in the number of mitochondria. An increase in both the volume and surface densities and a decrease in the specific surface suggests the mitochondria are getting larger. An increase in the volume density when the surface density does not change and the specific surface decreases, implies that the mitochondria must have changed shape. The specificsurface (S/V) is significantlyhigher in hypothalamic brain tissue from 5°C acclimated goldfish as compared with 30°C acclimated goldfishr However, there is no significantdifference in specific surface in hypophysial brain tissue from cold and warm acclimated goldfish. Mitochondria from temperature acclimated brain tissue do not appear to be the classicalmitochondria with the cristae folded into sheets. There are many organelles that exhibit an irregular pattern of inner membrane folding and increased matrical density while other mitochondria have regions within their matrix that bulge out and are empty (Fig. I). As this could have been an artifactas a consequence of using glutaraldehyde, the experiment was repeated using glutaraldehydc supplemented with 0.015% CaCI2. Busson-Mabillot (1971) showed that this calcium T.B. 14/4--B
Mitochondrial volume density increased in the brains of cold acclimated goldfish. This parallels the results found in cold acclimated muscle reported by Johnston (1982) and Tyler and Sidell (1984). No increase in either the external or internal surface densities was found in brain tissue, a result which is the same as that reported for white giycolytic muscle fibres (Tyler and Siddl, 1984). For red and pink muscle fibres, increases in both inner and outer surface densities have been found upon cold acclimation (Johnston, 1982; Tyler and Sidell, 1984). The results imply that brain mitochondria undergo shape changes in response to thermal stress. The change in mitochondrial shape rather than the number disagrees with previous findings of Jankowsky and Korn (1965) and Tyler and Sidell (1984). Jankowsky and Korn (1965) reported an increase in size and number of muscle mitochondria with cold acclimation, but this is only a qualitative observation and not substantiated with quantitative data. Other authors believe that an increase in the stereological parameters, in muscle tissue, are a result of a proliferation of mitochondria and not size changes (crosssectional diameter) (Johnston and Maitland, 1980; Johnston, 1982; Tyler and Sidell, 1984), but this does not preclude an increase in size through lengthening. The dissimilarity betw~n this study and the others may be due to fundamental differences in the tissues studied. Energy requirements and, consequently, muscle metabolism fluctuate depending upon the activity of the organism. Proliferation of mitochondria may be needed to increase the supply of ATP to active muscles (i.e. swimming musculature) to compensate for the decrease in metabolism caused by cold temperatures. Brain, an aerobic organ has a relatively constant metabolic state compared with that of muscle. Other studies have reported a significant increase in mitochondrial volumes with cold acclimation (Johnston and Maitland, 1980; Tyler and Sidell, 1984). It was inferred that such a change was to increase the capacity to supply ATP. However, shape change in the inner membrane may be the result of temperature induced lipid alterations that are known to occur in poikilotherms. The phospholipid composition in the mitochondrial inner membrane, from the brains of acclimated goldfish, is varied significantly when the acclimation temperature is lowered (Chang and Roots, 1988). The partial replacement of choline- by ethanolamine-glycerophospholipids at cold temperatures is expected to cause a disordering influence on the membrane structure since the overall molecular geometry will be changed. The restructuring of the lipid components to stabilize the bilayer conformation and to maintain optimal packing arrangements may lead to a shape change in the inner membrane (Chang and Roots, 1989).
190
KATHRYNGAEBELand BErrY I. ROOTS
Ubiquinone, a lipid soluble protein, is known to move within the bilayer during electron transfer (Robertson, 1983). Thus, changes reported in the lipid environment when acclimation temperature is altered might affect the dynamic movements of ubiquinone (Chang and Roots, 1985). The lipid compositional modification to mitochondrial inner membrane, providing the optimal selective microenvironment for ubiquinone and other membrane proteins is met, may therefore increase the flow of electrons to the cytochromes via complex III (Halestrap, 1982). The increase in mitochondrial matrix volume causes a stimulation of the oxidation of all substrates entering the respiratory chain before ubiquinone (i.e. coenzyme Q) (Halestrap et al., 1986) a process which would increase the supply of electrons entering the chain. Those electrons enter with no form of respiratory control (i.e. no additional coupling is needed). The greater abundance of electrons in turn increases the reduction of cytochromes. This may account for the increased cytochrome oxidase activity recorded in muscle and brain from cold acclimated fish. The increase in activity could keep the respiration rate and energy levels at that which is needed for cell function. Energy requirements may dictate what type of morphological changes take place within the mitochondrial population. The hypophysis is a more complex region of the brain compared with the hypothalamus. The adenohypophysis is made up of six unique nuclei, each responsible for a different hormone (Ball and Baker, 1969), This complexity of each individual nucleus may be responsible for the large standard error associated with the mean specific surface for the hypophysial brain tissue. The large standard error could be an indication that the response to thermal stress varies from nucleus to nucleus. Each individual nucleus would have to be analyzed to make concrete assumptions about the mitochondrial population in the hypophysis. We can state that there are shape changes occurring in the hypothalamic population in response to thermal stress. We suggest that the shape changes are associated with functional changes that allow mitochondria to supply the necessary ATP to keep physiological processes working at rates which can sustain cell function when environmental temperatures are lowered. Acknowledgements--This investigation was supported by a grant from the Natural Sciences and Engineering Research Council of Canada, No. A6052 to B.I.R. REFERENCES
Ball J. N. and Baker B. I. (1969) The pituitary gland: anatomy and histophysiologF. In Fish Physiology, Vol II. (Ed. by Hoar W. S. and Randall D. J.), pp. 1-110. Academic Press, New York. Busson-Mabillot S. (1971) Influence de la fixation chimique sur les ultrastructures. I. Etude sur les organites du foUicule ovarien d'un poisson teleosteen. J. Microscopie 17,, 317-348. Caidwell R. S. (1969) Thermal compensation of respiratory enzymes in tissues of goldfish (Carassius auratus L.). Comp. biochem. Physiol. 31, 79-93. Chang M. C. J. and Roots B. I. (1985) The effects of temperature and oxygen acclimation on phospholipids
of goldfish (Cara~sius auratus L.) brain mitochondria. Neurochem. Res. 10, I231-1246. Chang M. C. J. and Roots B. L (1988) Influence of environmental temperature on the phmpholipid composition of outer and inner mitochondrial ~ b r a n e from goldfish ( Carassius auratus L.) brain. J. therm. Biol. 13, 61-66. Chang M. C. J. and Roots B. I. (1989) The distribution of phospholipids on the mitochondriai inner membrane from the brains of goldfish acclimated at 5 and 30°C. J. therm. Biol. 14~ 195-204. Freeman J. A. (1950) Oxygen consumption, brain metabolism and respiratory movements of goldfish during temperature acclimatization, with special reference to lowered temperatures. Biol. Bull. 99, 416-424. Halestrap A. P. (1982) The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals. Biochem. J. 204, 37-47. Halestrap A. P., Quinlan p. T., Whippe D. E. and Armston A. E. (1986) Regulation of the mitochondrial matrix volume/n vivo and in vitro. The role of calcium: Biochem. J. 236, 779-787. Hochachka P. W. and Hayes F. R. (1962) The effect of temperature acclimation on pathways of glucose metabolism in the trout. Cdn. J. Zool. 40, 261-270. Jankowsky H. D. and Kom H. (1965) Der EinfluB der Adaptationstemperatur auf den Mitochondriengehait des Fischmuskels. Die Natur. Wiss. 52, 642-643. Johnston I. A. (1982) Capillarisation, oxygen diffusion distances and mitochondriai content of carp muscles following acclimation to summer and winter temperatures. Cell Tiss. Res. 222, 325-327. Johnston I. A. and Maitland B. (1980) Temperature acclimation in crucian carp, Carassius carassius L., morphometric analysis of muscle fibre ultrastructure. J. Fish Biol. 17, 113-125. Karlsson U. and Schultz R. L. (1965) Fixation of the CNS for electron microscopy by aldehyde perfusion. I. Preservation with aldehyde perfusates versus direct perfusion with OsO4 with specific reference to membranes and extracellular space. J. Ultrastruct. Res. 12, 160-186. Lehmann J. (1970) Ober die Abhingigkeit der Enzymaktivititen ira Rumpfmuskel der Goldoffe (ld~ ~ L.) yon der Vorbchandlungstemperatur. Int. Rev. Hydrobiol. 5~, 413-429. Mollenhauer H. H. (1964) Plastic embedding mixtures for use in Electron Microscopy. Stain Technol. 39, 111-114. Robcrtson R. N. (1983) The Lively Membrane. Cambridge University Press, Cambridge. Sato T. (1967) A modified method for lead staining for thin sections. J. Electron Microsc. 16, 193. Shakelee J. B., Chdstiansen J. A., Sidell B. D., Prosser C. U and Whir G. S. (1977) Molecular aspects of temperature acclimation in fish: contributions of changes in enzyme activities and isozyme patterns to metabolic reorglmization in the green sunfish. J. exp. Zool. 201, 1-20. Sideli B. D. (1977) Turnover of cytochrome c in skeletal muscle of green sunfish (Lepom/s cyanellus, R.) during thermal acclimation. J. exp. Zool. 199, 233-250. Sidell B. D. (1980) Responses of gold~h (CaraJsius auratus L.) muscle to acclimation temperature: altorations in biochemistry and proportions of different fibre types. Physiol. Zool. $3, 98--10% Tyler S. and Sidell B. D. (1984) Changes in the mitochondrial distribution and diffusion distances in muscle of goldfish upon acclimation to warm and cold temperature. J. exp. Zool. 232, 1-9. Weib¢l E. R. (1980) Stereological principles for morphometry in electron microscopic cytology. Int. R~. Cytol. 26, 235-302. Williams M. A. (1977) Practical Methods in Electron Microscopy, Vol 6. Quantitative Methods in Biology. (Ed. by Glauert A. M.). Elsevier, Amsterdam.
0306-4565/89 $3.00+ 0.00 Copyright © 1989PergamonPress pk
Printed in Great Britain. All rights r--~erved
STEREOLOGICAL ANALYSIS OF MITOCHONDRIA FROM BRAINS OF TEMPERATURE ACCLIMATED GOLDFISH, CARASSIUS AURATUS L. (5 AND 30°C) KATHRYNGAEBELand BETTYI. ROOTS Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 1A1 (Received 11 December 1988; accepted 1 March 1989)
Abstract--l. The mitochondrial population in hypothalamic and hypophysial brain tissue from warm (30°C) and cold (5°C) acclimated goldfish (Carassius auratus L.) was analyzed using stereologieal techniques. 2. It was revealed that there is a significantly larger volume density (Vv) in the cold accfimated tissue, with no significant difference in either of the surface densities (Svext and Sv/nt) from either of the brain areas. 3. The hypothalamic brain tissue has a significantly lower specific surface (S/V) in the cold acclimated tissue but there is not a significant difference in this parameter for the hypophysial brain tissue. 4. The values for these three parameters (Vv, Svext and Sv/nt, and S/V) indieate that mitochondria from acclimated brain tissue undergo shape changes in response to thermal streu. 5. We suggest that the shape changes may be related to the change in the phospholipid composition of the inner mitochondrial membrane with acclimation temperature. Key Word Index--Temperature acclimation, mitochondria, quantitative, stereology, goldfish, hypothala-
mus, hypophysis, brain.
INTRODUCTION
Many poikilotherms encounter large fluctuations in their environmental temperature, therefore these organisms must possess physiological mechanisms that allow them to adapt to the new environment. Goldfish, Carassius auratus L., have the ability to acclimate to temperature changes in their environment. Cold acclimated brain tissue of goldfish (Freeman, 1950), as well as muscle tissue (Hochachka and Hayes, 1962), consumes more oxygen than the warm acclimated tissue. Significant differences in enzyme activities between cold and warm acclimated tissue have also been reported. Glycolyric enzymes in brain tissue have greater specific activities upon cold acclimation but muscle giycolytic enzymes show lower specific activities (Shakelee et al., 1977). The effect of environmental temperature o n the mitochondrial enzymes has been well documented, cytochrome c oxidase being one enzyme that exhibits a dramatic change in specific activity with acclimation temperature (Caldwell, 1969; Lehmann, 1970; Shakelee et al., 1977). The specific activity of cytochrome oxidase is greater upon cold acclimation and this holds true for both muscle and brain tissue (Sidell, 1977; Shakelee et al., 1977). Morphological changes that occur with temperature acclimation are not as well documented. It is known that there are twice as many oxidative fibres in epaxial muscle of 5°C acclimated goldfish compared with 25°C muscle tissue (Sidell, 1980). Also there are differences in the density of mitochondria in muscle tissue from goldfish acclimated to cold temperatures compared with warm acclimated animals (Tyler and Sidell, 1984).
The purpose of this study was to determine whether morphological changes occur during thermal acclimation in brain tissue of goldfish. The volume and surface densities of the mitochondrial population were determined using stereological techniques. METHODS Animals
Goldfish (C. auratus, L.) 10-13crn in length were obtained commercially (Hartz Can., Inc., Rexdale, Ont.). Fish were maintained for 4 weeks at acclimarion temperatures of 5 and 30°C in 68L tanks. There was a 12 h photoperiod in each of the environ. mental rooms kept at the respective acclimation temperatures. Fish were fed Purina Fish Chow twice a day at 30°C, and every other day at 5°C. Tissue preparation
Eight fish from each acclimation temperature were decapitated, brains removed and immediately immersed in 2.5% glutaraldehyde in Karlsson-Shultz (KS) phosphate buffer, pH 7.4 (Karlsson and Shultz, 1965). The anterior portion of the left lateral lobe of the hypothaiamus, and the entire hypophysis were removed, cut into approx. 1 mm cubes and placed in fresh fixative. After overnight fixation at 4°C, brains were post-fixed with 1% osmium tetroxide (OsO4) in KS buffer for 1.5 h and then dehydrated with a series of ethanol solutions in increasing concentrations (25, 50, 70, 90, 95 and 100%). Tissue was stained en bloc with 1% uranyl acetate in 100% ethanol and then embedded in epon-araldite (Mollenhauer, 1964) using
187
KATHRYN GAEBEL and BETTY I. ROOTS
188
propylene oxide as the infiltration medium. This entire procedure was repeated three times. Tissue blocks from 3 fish, were randomly chosen from each of the acclimation temperatures, resulting in final data collection from 18 animals, 9 from each temperature. Two blocks from the hypothalamus and four blocks from the hypophysis from each fish were sectioned using a Sorval MT-1 microtome, to produce three ribbons of ultrathin sections. These ribbons were placed on 300 mesh copper grids and stained to ensure that one grid of sections was usable (i.e. allowing for sections lost during staining process, sections that were too thick or wrinkled for photographing, over-staining, etc.). Grids were stained for 20min with 7% uranyl acetate in 70% methanol, washed with distilled water and then stained with Sato's lead citrate stain (Sato, 1967) for 5 rain.
Stereoiogy Ten areas of each section (one section per tissue block) were photographed twice, once at a low magnification (mag) and then at a higher mag, using a Philips 200 or 201 transmission electron microscope. To calculate the volume density of mitochondria (Vv) in brain tissue the low mag negatives were analyzed with a point lattice with 8.519]mm point-topoint spacing (which would be equivalent to 1 p m spacing within the tissue) such that each section was analyzed with 6480[5750] points covering 6172.815308.6]/~m 2. The numbers in square brackets refer to the measurements when the negatives were from the Philips 201 microscope. The lattice was placed over the screen of a Telex Caramite 4000 slide projector in which the negatives were placed for viewing and counting (Final mag 8580[8940] × ). The number of points which fell upon the mitochondria was recorded as well as the total number of points covering the test tissue. From this the volume density can be calculated (Williams, 1977): No. of points covering the mitochondria Vv ffi total No. of points covering the test tissue A mean volume density was then calculated for each of the two brain parts, from each acclimation temperature, in each of the three experiments. This yields means for sample sizes of 180 for the hypothalamus and 360 for the hypophysis per experiment. The volume density of mitochondria (Vv) reported is the mean of the three mean volume densities at each of the respective acclimation temperatures. The surface density for the outer mitochondrial membrane (Svext) and the inner mitochondrial
membrane including cristae (Sv/nt) was calculated using high mag negatives analyzed with a line lattice. The line lattice had 2.513]cm line-to-line spacings (again this is equivalent to 1 ~m spacing within the tissue) therefore each negative was analyzed with 167211881] nun of test line that covered an area of 487.91649.6]#m 2. Each negative was viewed twice. During the first viewing the number of times the test line intersected the outer mitochendrial membrane was recorded. The number of times the test line intersected the inner mitochondriai membrane including cristae was recorded during the second viewing. The entire length of test line covering the test tissue was also recorded. From these observations the surface density for the outer mitochondrial membrane, Svext, (or the inner mitochondrial membrane including cristae, Svint) can be calculated as follows (Williams, 1977): SV = 4 - × m
where m, the contour length is: No. of intersections × mag total length of test line × 1000 The final surface densities (Svext and Sv/nt) reported here are the mean of the mean surface densities of the three experiments. Each test area (10 per block) has a corresponding volume density (Vv) and two surface densities (Svext and Svim). From these parameters a specific surface density (S/V) can be calculated by simply dividing the Svext by the Vv. A mean of the mean specific surfaces from the three experiments can then be calculated. Statistical comparisons between sample sets were made using the Student's t-Test. n ×
m~-
2 ×
RESULTS
Stereological analyses (Table 1) show that there is a significant increase in mitochondrial volume density (Vv) with cold acclimation in both brain areas. The surface densities for both the internal mitochondriai membrane including cristae (Sv/m)and the external mitochondrial membrane (Svext) do not increase significantly with cold acclimation in either brain part. The specific surface, S/V is ~ c a n t l y ~ t between the two temperature extremes in the thalamic brain tissue but not in the hypophy~d brain tissue. An increase in mitochondrial volume density (Vv) can result from either (i) an increase in the number
Table 1. Stereological parameters for mitochondria in hypothalamic and hypophysial brain tissue from temperature accliamt~ 8oldflsb Hypothslamus parameter
5°C
30°C
Vv(%)
Hypophytis P t-Test
5°C
30°C
P t -Test
4.973 ± 0.000 4.067 ± 0,003 < 0.05 4.483 + 0.002 3.567 ± 0.000 < 0.02 0.239 ± 0.012 0.224 ± 0.008 NS 0.220 4- 0.014 0.185 + 0.026 NS Sv(/nt) 0.342 + 0.005 0.337 ± 0.009 NS 0.310 ± 0.030 0.2~8 + 0.044 NS S/V 4.758±0.005 5.781 +0,009 <0,02 4.931 4- 0.0~6. . . . 5 . Ill~ 0 . I6 9 3 NS I III t II II I [: I II [ Val .ue, are meant + ~ for I 8 blocks ft~m tlWhE~hslami¢ braintlmuland ~ ~ tlm~ tmsue at each temperature regime. P: St~tiati~ ~ p a r i s o n was ¢kaer~ined ueing S t ~ t ' s t-Test. Vv: volume density. Sv(ext): surface density for the outer mitochondrial membrane. Sv(/m): surface density for the inner mitocbondrial membrane including cristae. S/V: specific surface.
Sv(ext)
II
II
Stereological analysis of mitochondria from brains of temperature acclimated goldfish
189
concentration is enough to reduce the swelling caused by this fixative. As may be seen in Fig. 1 inset; the 'bulging' mitochondria are still present. This indicates that this morphological feature may not be an artifact caused by the fixative.
DISCUSSION
Fig. I. Electron micrograph of hypothalamic brain tissue from goldfish acclimated to 5°C showing mitochondria with dense matrices and areas that are empty and appear to 'bulge' out. Brain tissue from acclimated goldfish fixed with glutaraldehyde supplemented with 0.015% calcium (inset) also appear to 'bulge' out.
of mitochondria, (ii) an increase in the size of the individual mitochondria or (iii) a combination of both. Stereological methods do not allow the direct determination of numbers of organelles with irregular shapes such as mitochondria (Weibel, 1980). The specific surface (S/V) will give an indication of whether size or numbers are changing in the mitochondrial population. An increase in the volume density (Vv) and the surface density (Svext) and no change in the specific surface (S/V) suggest the changes are due to an increase in the number of mitochondria. An increase in both the volume and surface densities and a decrease in the specific surface suggests the mitochondria are getting larger. An increase in the volume density when the surface density does not change and the specific surface decreases, implies that the mitochondria must have changed shape. The specificsurface (S/V) is significantlyhigher in hypothalamic brain tissue from 5°C acclimated goldfish as compared with 30°C acclimated goldfishr However, there is no significantdifference in specific surface in hypophysial brain tissue from cold and warm acclimated goldfish. Mitochondria from temperature acclimated brain tissue do not appear to be the classicalmitochondria with the cristae folded into sheets. There are many organelles that exhibit an irregular pattern of inner membrane folding and increased matrical density while other mitochondria have regions within their matrix that bulge out and are empty (Fig. I). As this could have been an artifactas a consequence of using glutaraldehyde, the experiment was repeated using glutaraldehydc supplemented with 0.015% CaCI2. Busson-Mabillot (1971) showed that this calcium T.B. 14/4--B
Mitochondrial volume density increased in the brains of cold acclimated goldfish. This parallels the results found in cold acclimated muscle reported by Johnston (1982) and Tyler and Sidell (1984). No increase in either the external or internal surface densities was found in brain tissue, a result which is the same as that reported for white giycolytic muscle fibres (Tyler and Siddl, 1984). For red and pink muscle fibres, increases in both inner and outer surface densities have been found upon cold acclimation (Johnston, 1982; Tyler and Sidell, 1984). The results imply that brain mitochondria undergo shape changes in response to thermal stress. The change in mitochondrial shape rather than the number disagrees with previous findings of Jankowsky and Korn (1965) and Tyler and Sidell (1984). Jankowsky and Korn (1965) reported an increase in size and number of muscle mitochondria with cold acclimation, but this is only a qualitative observation and not substantiated with quantitative data. Other authors believe that an increase in the stereological parameters, in muscle tissue, are a result of a proliferation of mitochondria and not size changes (crosssectional diameter) (Johnston and Maitland, 1980; Johnston, 1982; Tyler and Sidell, 1984), but this does not preclude an increase in size through lengthening. The dissimilarity betw~n this study and the others may be due to fundamental differences in the tissues studied. Energy requirements and, consequently, muscle metabolism fluctuate depending upon the activity of the organism. Proliferation of mitochondria may be needed to increase the supply of ATP to active muscles (i.e. swimming musculature) to compensate for the decrease in metabolism caused by cold temperatures. Brain, an aerobic organ has a relatively constant metabolic state compared with that of muscle. Other studies have reported a significant increase in mitochondrial volumes with cold acclimation (Johnston and Maitland, 1980; Tyler and Sidell, 1984). It was inferred that such a change was to increase the capacity to supply ATP. However, shape change in the inner membrane may be the result of temperature induced lipid alterations that are known to occur in poikilotherms. The phospholipid composition in the mitochondrial inner membrane, from the brains of acclimated goldfish, is varied significantly when the acclimation temperature is lowered (Chang and Roots, 1988). The partial replacement of choline- by ethanolamine-glycerophospholipids at cold temperatures is expected to cause a disordering influence on the membrane structure since the overall molecular geometry will be changed. The restructuring of the lipid components to stabilize the bilayer conformation and to maintain optimal packing arrangements may lead to a shape change in the inner membrane (Chang and Roots, 1989).
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KATHRYNGAEBELand BErrY I. ROOTS
Ubiquinone, a lipid soluble protein, is known to move within the bilayer during electron transfer (Robertson, 1983). Thus, changes reported in the lipid environment when acclimation temperature is altered might affect the dynamic movements of ubiquinone (Chang and Roots, 1985). The lipid compositional modification to mitochondrial inner membrane, providing the optimal selective microenvironment for ubiquinone and other membrane proteins is met, may therefore increase the flow of electrons to the cytochromes via complex III (Halestrap, 1982). The increase in mitochondrial matrix volume causes a stimulation of the oxidation of all substrates entering the respiratory chain before ubiquinone (i.e. coenzyme Q) (Halestrap et al., 1986) a process which would increase the supply of electrons entering the chain. Those electrons enter with no form of respiratory control (i.e. no additional coupling is needed). The greater abundance of electrons in turn increases the reduction of cytochromes. This may account for the increased cytochrome oxidase activity recorded in muscle and brain from cold acclimated fish. The increase in activity could keep the respiration rate and energy levels at that which is needed for cell function. Energy requirements may dictate what type of morphological changes take place within the mitochondrial population. The hypophysis is a more complex region of the brain compared with the hypothalamus. The adenohypophysis is made up of six unique nuclei, each responsible for a different hormone (Ball and Baker, 1969), This complexity of each individual nucleus may be responsible for the large standard error associated with the mean specific surface for the hypophysial brain tissue. The large standard error could be an indication that the response to thermal stress varies from nucleus to nucleus. Each individual nucleus would have to be analyzed to make concrete assumptions about the mitochondrial population in the hypophysis. We can state that there are shape changes occurring in the hypothalamic population in response to thermal stress. We suggest that the shape changes are associated with functional changes that allow mitochondria to supply the necessary ATP to keep physiological processes working at rates which can sustain cell function when environmental temperatures are lowered. Acknowledgements--This investigation was supported by a grant from the Natural Sciences and Engineering Research Council of Canada, No. A6052 to B.I.R. REFERENCES
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