Seasonal differences in brain gangliosides of the djungarian hamster (Phodopus sungorus)

J. therm. Biol. Vol. 10, No. 2, pp. 119-124, 1985

0306-4565/85 $3.00+0.00 Copyright © 1985 Pergamon Press Ltd

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SEASONAL DIFFERENCES IN BRAIN GANGLIOSIDES OF THE DJUNGARIAN HAMSTER

(PHODOPUS SUNGOR US) M. MUHLEISEN,* R. HILBIG, J. MARX and H. RAHMANN'~ Zoological Institute, University of Stuttgart-Hohenheim, 7000 Stuttgart 70 (Hohenheim), F.R.G. (Received 7 June 1984; accepted in revised form 26 September 1984)

Abstraet--l. The concentration of proteins, sialo-glycoproteins and gangliosides and the ganglioside composition of eight brain regions from summer- and winter-acclimated Djungarian hamsters (Phodopus sungorus) were investigated. 2. In addition to seasonal variations in the concentration of proteins, sialo-glycoproteins and gangliosides significant differences in the ganglioside composition occurred; the amounts of the more polar compounds (Grl b in some structures and GQIb in all brain regions) were higher in winter than in summer. 3. Most striking was the complete absence of the less-polar ganglioside N,O-acetylated Gr~b during winter. 4. The results are discussed with regard to general scheme of brain ganglioside implication in the process of modulation of the synaptic membrane in response to thermal challenge. These data on changes in polarity of the brain gangliosides are in agreement with physico-chemical in vitro results using ganglioside monolayers. Key Word Index--Brain gangliosides; seasonal differences; Djungarian hamster; Phodopus sungorus.

INTRODUCTION

temperature. For this purpose the Djungarian hamster (Phodopus sungorus), a small mammal living in the Siberian steppe, seemed to be particularly appropriate, because the animal exhibits circannual variations of its thermoregulatory system, such as thermogenic capacity and thermal insulation (Heidmaier and Steinlechner, 1981a, b; Heldmaier et al., 1982). Furthermore, this animal shows seasonal variations in body weight and spontaneous daily torpor during winter (Heldmaier and Steinlechner, 1981b).

Gangliosides, giyco-sphingohpids containing different numbers of negatively charged sialic (--neuraminic) acids (NeuAc), are characteristic components of all vertebrate cell membranes. In comparison with other organs, gangliosides are enriched in the nervous system where they are especially abundant in the synaptic membranes (Morgan et al., 1973; Eichberg et al., 1974). Gangliosides are assumed to be involved in the process of synaptic transmission (Rahmann, 1983) and also to play an essential role in thermal adaptation of vertebrates--for reviews see Rahmann (1981) and Rahmann and Hilbig (1983). Several changes, both in composition and content, of brain gangliosides of true hibernators, which are characterized by relatively long periods (days and weeks) of torpor, have been shown already by Hilbig and Rahmann (1979), Geiser et al. (1981), Robert et al. (1982), Demediuk and Moscatelli (1983), Sonnino et aL (1984) and Miihleisen et al. (1984). These studies have revealed the general tendency that, in adaptation to hibernation, the polarity of neuronal gangliosides was increased either by means of polysialylation or by sole existence of N- instead of N,O-acetylation of sialic acids. These differences have been shown to be region specific. Based on these findings it was of special interest to look for any variation, especially in composition of brain gangliosides, in animals being not true hibernators but showing daily variations in their body

MATERIALS AND METHODS

*Present address: II. Physiological Institute, University of Heidelberg, Heidelberg, F.R.G. tTo whom all reprint requests should be addressed.

Twenty-five Djungarian hamsters (P. sungorus) were maintained under a natural photoperiod. Ten of these hamsters were kept under outdoor conditions and killed by decapitation in July (summer animals, T, = 22°C), whereas the other 15 hamsters were kept until the end of February (winter animals, T , = - 3 ° C ) . Immediately after decapitation the brain was removed quickly and dissected into eight main regions: olfactory bulb, cortex, cerebellum, mid-brain (corpora quadrigemina), brain stem (thalamus, hypothalamus), pons, spinal bulb (medulla oblongata) and spinal cord. Respective brain parts were pooled in each group, yielding quantities of 250 mg fresh wt. The pools of each group consisted of 3-5 animals depending upon the weight of the respective brain region. The tissue samples were homogenized in water; protein concentrations were determined according to Lowry et al. (1951). The homogenates were iyophilized and gangliosides were extracted and prepared according to Svennerholm and Fredman (1980). The concentration of glycoprotein-bound sialic acid was determined according to Svennerholm (1957). For the deter-

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M. MOHLEISENet al.

120

Table 1. Concentration of proteins, sialo-glycoproteins and gangliosides in different brain structures of Djungarian hamsters (P sungorus) during summer and winter mg protein/g fresh wt Brain structure Olfactory bulb Cortex Corpora quadrigemina Pons Brain stem Cerebellum Spinal bulb Spinal cord

Summer 122 132 116 144 104 131 132 109

Winter 149 98* 119 147 112 118 123 132"

,ug sialic acid/g fresh wt Glycoprotein Gangliosides " " -Summer Winter Summer Winter 259 259 237 232 246 224 215 171

250 248 183* 182" 163" 223 149" 125"

784 1202 1039 1069 1248 829 889 673

1235" 724* 1542* 1217 772* 847 865 563

The data include methodological errors less than 10%. *Differences greater than methodological errors.

mination of ganglioside composition an appropriate amount of total gangliosides were applied and separated on pre-coated HP-TLC silica gel plates (Merck, Darmstadt, F.R.G.). The following solvent system was used: chloroform-methanol-12 mM MgC12-33~o ammonia (60:36:8.2:0.08). The small amount of ammonia in the solvent system which is necessary for good separation of the more-polar gangliosides will not hydrolyse alkali-labile gangliosides (Rahmann and Hilbig, 1983). Spots were visualized with resorcinol reagent (Svennerholm, 1957) and identified according to their migration rates by known standards. Quantitative composition was then determined by densitometric scanning at 580nm (Zeiss KM3) and subsequent planimetric determination of the

peak areas (Kontron Mop AMO-2). The nomenclature of individual gangliosides was according to Svennerholm (1964). The significance of the difference (Student t-test) between summer and winter animals was calculated from the means of the pooled samples. The chemical structures of the ganglioside molecular species are given in the Appendix. RESULTS

I. Concentrations of proteins, sialo-glycoproteins and gangliosides in heterothermic (winter) and euthermic (summer) Djungarian hamsters For characterization of any seasonal variations in

O-

(B G G

G G G G O-A G

G

Fig. 1. Thin-layer chromatogram (HP-TLC) ofbrain gangliosides from: (A) cortex, cerebellum, olfactory bulb, spinal bulb and standard, (B) pons, corpora quadrigemina region, brain stem, spinal cord and standard of Djungarian hamsters (P. sungorus) adapted to summer and winter conditions. Solvent system: chloroform-methanol-I 2 mM MgCI2 (aq. sol.)-33~o ammonia (60: 36:8.2 : 0.08)

Brain gangliosides in the Djungarian hamster

Figures I(A) and I(B) show that there exist marked seasonal differences in the composition of the various brain regions. First of all it is obvious that in all regions of winter-adapted animals the N,O-Ac-GT,b, which is less polar in comparison to N-Ac-GTIb, is absent as compared to the euthermic hamsters. In addition, the compositon of the individual brain gangliosides showed distinct differences between the different brain structures of summer and winter animals (Fig. 2). In the ganglioside pattern of the olfactory bulb and cortex GTib and GD~, predominated, whereas in the corpora quadrigemina region and cerebellum GT,b was the major compound. In all other structures GTIb and GM! prevailed. Besides these regional differences the brains of winter-adapted hamsters generally were characterized by up to a five-fold higher proportion of the polar Gq~b (Fig. 2, left-hand columns). This type of polysialylation during winter was especially obvious in the pons, brain stem, cerebellum and spinal bulb where increases from 5 + (summer) to 20 + 5% (winter) of the relative amounts of ganglioside-bound NeuAc were registered. The relative portion of GT,~ rose less than that of GQ~ during winter in most brain regions

brain components of Djungarian hamsters the concentrations of the proteins were first determined. In comparison to euthermic hamsters, during winter remarkable increases occurred only in the spinal cord, whereas in the cortex a decrease was observed (Table 1). In five out of eight brain regions the concentration of the protein-bound sialic acid during the summer period was higher than in winter (Table 1). In ganglioside-bound sialic acid changes also occurred, but differed from one brain regiori to another (Table 1). An increase in ganglioside content was registered during the winter in the olfactory bulb and the corpora quadrigemina region, simultaneously with a decrease in the cortex and brain stem.

2. Ganglioside composition in heterothermic (winter) and normothermic (summer) Djungarian hamsters The ganglioside composition of eight different brain regions from summer and winter hamsters was compared. Of particular interest were those regions which are known to be especially thermosensitive such as the ports, brain stem, regio quadrigemina and spinal bulb (Boulant, 1980).

1%

OLFACTORYBULB

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I 30" 20. 10"

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SPINALBULB

(MedullaoblongoiQ) . i n,,

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SPINAL Qib ' o ~ ' r,b~lbol,

X~ ' Oi. ' d3 ' x'3 ' , 4 T~

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a~b 'o&cJ rlbTIbOIb

/.00 I ,oo

X'l ' 6° ' o'3 ' x'3 ' M'3 T,o

X2

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Fig. 2. Relative composition (%) and concentration (#g/g fresh wt) of neuronal ganglioside-bound sialic acid per single fraction of eight brain structures from summer- and winter-adapted Djungarian hamsters (P. sungorus). XI-X3 are unidentified gangliosides, which were found only in traces in some structures.

122

M. MldHLEISEN et al.

(olfactory bulb, corpora quadrigemina region, pons, brain stem, cerebellum and spinal cord). In addition to these relative changes in ganglioside composition it was of interest to calculate also the concentration of ganglioside-bound sialic acid (/~g/g fresh wt) of the single gangliosides in order to evaluate quantitatively the seasonal changes in the polarity of the neuronal membranes in the respective brain regions (Fig. 2, right-hand columns). During winter a significant increase in the polar Go~b was observed in the olfactory bulb ( + 6 0 / a g NeuAc/g fresh wt), corpora quadrigemina region (+218/~g), pons ( + 1 8 5 # g ) , brain stem (+105/~g), cerebellum ( +95 #g) and spinal bulb ( + 163/ag). In the corpora quadrigemina and pons regions, which are known to be highly thermosensitive. Grt~ concomitantly increased from 330 to 570/~g and from 305 to 396pg NeuAc/g fresh wt, respectively. In the cortex however significant decreases in these two main gangliosides (G-nh and Gola) occurred during winter. The gangliosides of the spinal cord on the other hand remained constant. In summary, the change from normothermic to heterothermic life in the brain of hamsters is correlated with drastic region-specific changes in the polarity of membranous gangliosides. These changes are achieved by two strategies: firstly, by a drastic degradation of the less-polar alkali-labile gangliosides (i.e. selective disappearance of N,O-acetylated compounds) and secondly by an increase in the polar Go~b fraction. DISCUSSION Recently it has been shown that, usually in the whole brain of true hibernators. Grlb, instead of GD1, of non-hibernating mammals, is the main ganglioside (Rahmann and Hilbig, 1983). This has been confirmed for seven out of eight brain regions of the Djungarian hamster, which is not a true hibernator, but exhibits daily torpor during winter (Heldmaier and Steinlechner, 1981b). Furthermore, it is well known from true hibernators [fat dormouse, Glis glis (Geiser et al., 1981, Miihleisen et al., 1984); golden hamster, Mesocricetus auratus (Hilbig and Rahmann, 1978: Demediuk and Moscatelli, 1983); European hamster. Cricetus cricetus (Robert et al., 1982)] that Go~~ increases during winter. These previously reported polysialylation phenomena of gangliosides yielding a higher polarity of the neuronal membrane occurred to smaller extent compared with those in P. sungorus as presented in this paper. While in true hibernators the increase of Go~ ~ from summer to winter is usually less than two-fold, in the Djungarian hamster, however, the rise is more than four-fold in the cerebellum, medulla oblongata and brain stem and occurs also in all other brain regions. The total absence of N,O-Ac-Gr~b, which is less polar than N-Ac-Gv~b, in P. sungorus during winter is in accordance with previous results in the fat dormouse (MiJhleisen et al., 1984) where this alkalilabile ganglioside is only present in summer animals also. Sonnino et al. (1984) using two-dimensional TLC with intermittent alkali treatment, which gives better separation of the individual gangliosides, have shown that there are up to 15 alkali-labile gangli-

osides in the brain of fat dormice. These alkali-labile compounds were almost entirely absent during winter. Alkali-labile gangliosides are less polar than their alkali-stable counterparts. From this it might be concluded that in P. sungorus an increase in membranous polarity is obtained both by a rise of GI-~ and a simultaneous decrease in the amount of alkalilabile gangliosides. In total these results are a further example for the phenomenon that changes in the molecular composition of brain gangliosides are correlated with thermal adaptation in vertebrates. In particular, polysialylation seems to be widely distributed among animals which show heterothermic phases (mammals and birds) or thermal adaptation (fish and amphibians). Polysialylation does not occur in homeothermic mammals (mice) under cold stress (Miihleisen et al., 1984). Therefore, polysialylation of gangliosides in nervous membranes in the cold is considered to be a general strategy of vertebrates for survival at low ambient temperatures (Rahmann, 1981; Rahmann and Hilbig, 1981). The correlations between the physico-chemical properties of gangliosides (Probst and Rahmann, 1980; Miihleisen et al., 1983, Probst et al., 1984) and the observed in vivo implications of gangliosides in temperature adaptation lead to speculations on possible biological functions of gangliosides. The differences in the molecular composition of gangliosides reported here for summer, and winteracclimated Djungarian hamsters can be considered in the light of the following physico-chemical measurements using ganglioside monolayers. These showed that the physical properties of gangliosides from summer-adapted animals were more sensitive to variations in the temperature than those from winter animals (Probst et al., 1984). In these physico-chemical studies the surface area requirement of different polar gangliosides and ganglioside mixtures was shown to be readily modulated by temperature and/or Ca -,+ addition, in contrast to some phospholipids (lecithin, phosphatidylserine). Similarly, in vivo, ganglioside cluster areas--possibly comparable to pure ganglioside monolayers (Bach and Sela, 1980: Maggio et al., 1980) and probably present in neuronal, especially synaptic membranes (Sharom and Grant, 1978: Bunow and Bunow, 1979; Delmelle et al., 1980: Bach and Sela, 1980)--might be modulated depending upon the functional state of the synapse. By this modulation the structure and function of integral channels or enzyme proteins of membranes may be altered and/or the fluidity, and as a consequence permeability, of microdomains which constitute such a structure and function may be changed. The last assumption is in agreement with the findings of Cossins (1977) showing adaptive changes in the viscosity of synaptosomal membranes following temperature alterations. From the present in vivo data it can be concluded that molecular changes of the gangliosides may also occur at the very local site(s) of synaptic contact, thus assuring that the process of quantal transmitter release during transmission is the same although the temperature has changed. This assumption is in agreement with the finding of Krnjevic (1974), according to which in cold-blooded lower vertebrates

Brain gangliosides in the Djungarian hamster four or five Ca 2+ ions are necessary for the release of one transmitter quantum, but in warm-blooded mammals only two or three Ca 2+ ions are necessary. F r o m the in vivo results, and the physieo-chemical data, it might be concluded that in warm-adapted animals the molecular organization of neuronal ganglioside mixtures is labile to temperature changes, whereas in cold-adapted animals it is thermally stable. This, of course, includes the possibility of modulation of the membrane permeability. In the case of hibernating mammals the thermoadaptability of the neuronal membrane is accompanied by regionspecific peculiarities: in the pons and oblongated medulla, where thermoregulatory centres are located (Reaves and Hayward, 1979) and which, therefore, remain active during the phase of hibernation, temperature-dependent changes in polarity were found (this study; Sonnino et aL, 1984). These changes are correlated with alterations in surface behaviour of the gangliosides as reported by Probst et al. (1984). On the other hand, in the cortex and cerebellum, both of which are almost inactive during hibernation, only minor alterations occurred. In conclusion, the physico-chemical and biochemical data complement each other and strengthen the evidence for the important role of gangliosides in the modulation of membrane-mediated processes, in particular of synaptic transmission, in order to enable an undisturbed transfer of information despite changes in the thermal environment. Acknowledgements--We thank Professor G. Heldmaier, University of Marburg, for the donation of some Djungarian hamsters which served as a basis for our own breeding. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ra 166/13-6). REFERENCES

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APPENDIX GM4 GM3 GM2 GMj

NeuAc~ 2-3 Gal-cer NeuAc~ 2-3 Galfl 1-4Glc-cer Gal NAcfl 1-4 [NeuAcc~ 2-3] Galfl 1-4 Glc-cer Galfl 1-3Gal NAcfl l-4[NeuAc~ 2-3]Galfl 1-4 Glc-cer GD3 NeuAcc~2-8 NeuAca 2-3 Galp l-4Glc-cer GD2 Ga[ Nacfl l-4[NeuAca 2-8 NeuAc~2-3] Galfl I-4 Glc-cer

Gol,, [NeuAca 2-3] Galfl 1-3 Gal NAch' 1-4 [NeuAc~2-3] Galfl 1-4 Glc-cer GD~b Galfl l-3Gal NAcfl l-4[NeuAc~ 2-8NeuAc:~ 2-3] Galfl I-4 GIc-cer Grl a NeuAc'~ 2-8NeuAc~ 2-4Gal~ 1 3Gal NAc/~ 1-4 [NeuAc~x 2-3] Gatfl 1-4 Glc-cer GT~b NeuAca 2-3 Galfl 1-3 Gal NAc/~'l-4(NeuAc:~ 2-8 NeuAca 2-3] Galfl l-4Glc-cer G-rl~ Galfl 1-3 Gal NAc/3 l-4[NeuAc~ 2-8 NeuAc~ 2-8 NeuAca 2-3] Galfl | - 4 Glc-cer Go~b NeuAc 2-8NeuAco~ 2-3Galfl 1-3Gal NAc/; 1-4 [NeuAca 2-8 NeuAca 2-3] Gal~ | - 4 Glc-cer