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Deprivation of paradoxical sleep and in vitro cerebral protein synthesis in the rat
Life Sciences Vol. 10, Part II, pp" 1349-1357, 1971 . Printed in Great Britain
Pergamon Press
pEPRIVATION OF PARADOXICAL SLEEP AND IN VITRO CEREBRAL PROTEIN SYNTHESIS IN THE RAT P . Bobillier, F.- Sakai, S . Sequin and M . Jouvet Department of Experimental Medicine School of Medicine, Lyon, France
(Received 23 September 1971; in final form 1 November 1971) Brain proteins are metabolically highly active and play a. role in cellular metabolism and its regulation . In recent years a considerable amount of work has been reported on the possible relationship between protein synthesis in the brain and its specialized functions (1) . Investigations of the chemical correlates of learning and memory, in particular, have provided an extensive literature
(2) . However, little is known about the
possible alterations of the cerebral proteins during sleep (3,4) . Paradoxical sleep (PS) deprivation is a very useful working instrument since this specific phenomenon is followed by a. sub-
aequent compensatory rebound of this state of sleep . On the other hand, recent sleep studies have shown the importance of serotonin and norepinephrine in the biochemical regulatory mechanisms associated with the neurophysiological changes which occur during and after PS deprivation (5) . Evidence has also accumulated to
indicate that brain stimulation in vitro will alter the synthesis of protein in the brain (6) . It was then of interest to study the possibility that an increased activity of the neurons (which is
reflected by the fast cortical activity occuring during PS) may be associated with variations in the protein synthesizing capacity of the brain tissue .
In this paper, we examined the protein synthesis in vitro by measuring the incorporation of labelled aminoacids into total proteins during incubation of brain slices . The brain slices were taken from rats which have been submitted to PS deprivation or to PS recuperation .
*This work has been suppozted by the Air Force Office of Scientific Research (USAF Contract No . F61052-70-C-003), by D .R .M .E . (Grant 70-036) and I .N .S .E .R .M .
1349
1350
Paradaaícal Sleep anti Protein Synthesis
Vol. 10, No. 23
Material's and Methods Selective PS deprivation of rats was carried out by a method described previously (7) : the animals being placed upon small
supports surrounded by water, with food and water ad lib . 24 adult male Sprague-Dawley rats weighing about 250 qr . were equally divided into three groups 1) control animals, 2) animals deprived of PS for 54 hr . and 3) animals deprived of PS for 48 hr and then allowed to recuperate for 6 hr . in sound-proof cages . All control animals were isolated in cages in the same room at + 25 °C, with water and food ad lib ., during the same length of time . At the end of the experimental procedure the animals were
killed by a strong blow to the body and then decapitated . The brains were removed, (cerebellum and pineals being discarded), washed in 0 .9 $ saline and dissected in a cold room at + 4 ° C into
Telencephalon (cortex, hippòcampus and nucleus caudatus) and brainstem . Incubation procedure
From each part of the brain, 0 .4 mm thick tissue slices were prepared at + 4 °C with a Mc ILLWAIN tissue chopper . The slices were put into a 25 ml Erlenmeyer flask containing 2 ml of standard
incubation medium (Krebs-Ringer bicarbonate, PH 7 .5) and preincubated for 5 min . in a shaker bath at 37 ° C, type Dubnoff with a
gas mixture consisting of 95 8 0 2 and 5 8 C0 2 . 1 .4~C1 of L- 3 H-Amino-acids mixture (NEN Chemicals GmBH) was then added to the medium, the flasks were flushed briefly with the gas mixture and incubated for 45 minutes . After incubation, the slices were rinsed in a funnel of filter paper with 5 ml of cold incubation medium and frozen until analysis . Preparation of tissues
The specimens were placed in a Potter-Elvejhem apparatus with 10 vol . of 0 .003 M CaC1 2 in 0 .32 M-Sucrose and homogenized for about 1 min . 30 sec . with a loose-fitting teflon pestle .
Preparation of subcellular fractions Portions of brainstem homogenates were separated (8) into crude subcellular fractions by differential centrifugation, in a S~rvall refrigerated centrifuge : a nuclear fraction at 800 q for 10 min . and a mitochondrial fraction, at 13,000 g for 10 min . The
voi, io, No . as
Paraaoadoal sleep ana ~o~ein s9~tneeie
issi
supernatant from the crude mitochondrial pellet was centrifuged at 100,000 g for 1 hr . in a Spinco ultracentrigue to provide the cell sap (or soluble fraction) . Analysis of tissue protein The proteins in tissue homogenates from Telencephalon and each of the brainstem subcellular fractions were precipitated in cold 5 ~ TCA . After centrifugation, the precipitate was washed once with 5 8 TCA and heated to 90 °C for 30 min, then once more
with cold 5 8 TCA, once with Ethanol-Ether (1 : l,v/v) and heated to 37 °C for 30 min ., and finally, once with cold Ethanol-Ether . The pellets were redissolved with 0 .1 N NaOH and the proteins
estimated by fluorlmetric assay (9) with ovalbumine as a standard . Another portion was taken for the determination of radioactivity, the labelled proteins being counted in a Packard TRI
CARB Spectrometer with Ineta-Gel solvent system (Packard Inetrtiunent) . The specific activity (SA) of the proteins was expressed as dpm/mg protein . In order to estimate the pool of free 3H amino-acids, the radioactivity of the TCA-soluble supernatant was determined again using liquid scintillation counting, and the "amino acid specific activity" expressed as DPM/mg of proteins . Quenching corrections
were made with the external standard and channels ratio method . Significance of differences was tested using the Student's t test: Results
The tissue/medium ratio between the SA of the TCA-soluble fraction in the slices and that of the incubation medium was
always >1 in both brain structures studied, thus indicating that
the uptake of H3 amino acids occured well against a concentration gradient . Our results are summarized in Tables I, II and III .
As shown in Table I, the SA of the TCA-soluble fraction in the brainstem of rats deprived of PS, was significantly increased as compared to control animals and to PS. deprived animals allowed
to recuperate . A less pronounced Increase above controls, though not significant, was also observed in the incorporation of 3 H aminoacide in the intracellular pool of brainstem of rats deprived of PS and allowed to recuperate . In the telencephalon the SA of the TCA-soluble fraction was found to be similar in the three groups of rats studied .
1352
Paradrndcal 31eep and Protein Synthesis
Vol. 10, No. 23
TABLE I The in vitro incorporation (duratión 45 minutes) of L- 3 H-aminoacid mixture into the brain TCA-soluble fraction of rats
The animals were divided in 3 groups : Control animals (I), PS-deprived animals during 54 hr . (II) and animals in recuperation during 6 hr . after 48 hr . of PS deprivation (III) . Values are the mean + S .E .M . of 8 animals in each group. Brain regions Telencephalon Brainstem
Specific activity : DPM/mg protein 21013+893
48972+4465
21716+1112 69561+3584
20494+1158 51380+3462
P values from Student's t test : idtie < .O1 when compared with animals of group I ;~ ~ s< .O1 when group III is compared with
group II .
Conversely, the results of the in vitro incorporation of 3H aminoacids into the proteins of brainstem and telencephalon, as shown in Table II, did not indicate any significant differences between the two experimental groups and only a alight increase above controls (not significant) . TABLE II
The in vitro incorporation (duration 45 minutes) of L- 3H-amino acids mixture into the brain proteins of rats The groups of animals are expressed in the same way as in
Table I .
Fractions examined
Specific activity : DPM/mq protein
Telencephalon Brainstem Total homogenate
I 561+28
II 608+45
639+58
1095+54
1378+150
1136+165
Mitochondrial Soluble
2438+88
3215+361
2539+274
Nuclear
1113+52 1362+98
1289+174 1464+238
III
1310+175 1465+236
Vol. 10, No. 23
Paradaaical Sleep and Protein Synthesis
1353
Experiments have been done to determine whether or not some alterations in protein synthesis could be more particularly localized in any particular or soluble subcellular fractions of brain tissue . As in the total homogenate, only a slight increase of the
SA of proteins in the crude subcellular fractions of the brainstem in both experimental groups of rats can be noticed , this change not being significant when compared to control rats .
Thus, the slight (though generally increased) SA of proteins which affected the brainstem of rats deprived of PS, or allowed to recuperate after PS deprivation did not reflect the marked
increase which occured in the uptake of 3 H aminoacids into the TCA-soluble fraction of experimental animals .
The pool size of free amino acids may vary and affect the
protein synthesis (10) . To ascertain whether the slight increase in the SA of proteins was due to variations of the aminoacide or
to variations in the incorporation of the precursors intò the proteins, we determined the relative specific activity . This ratio may be used as an index of protein synthesis . No significant variations in the relative specific activity were obtained either in the Telencephalon or in the brainstem of either experimental or control rats (Table III) . These data clearly suggest that the
brain protein synthesis unlike 3H aminoacid upta9ce was not affected by the PS deprivation or by the PS rebound of rats under our experimental conditions . TABLE III
The effects of the PS deprivation on the relative
specific activity of proteins in the brain of rats . The groups of animals are expressed in the same way as in Table I . Brain regions
Telencephalon Brainstem
Specific activity of proteins Specific activity of TCA-soluble fraction III
Relative specific activity, I .027+ .001
.024+ .003
II
.028_+ .002 .020+ .002
.031+_ .002 .021+ .002
1354
Paradaadcal 81eep and Protein 3y~hesis
Vvl . 10, No . 23
DISCUSSION In the present study an attempt has been made to correlate protein synthesis in the central nervous system of rats with conditions
of increased functional activity during sleep . No significant alter ation of the protein synthesis was seen in the Telencephalon or in the Brainstem areas nor in the Brainstem particular or soluble subcellular fractions in the two experimental groups when compared to control animals . In contrast, we observed rises in the uptake of the labelled precursors in the Brainstem of the two experimental groups, but the significant one occured only in the PS deprived group .
In previous studies by other authors it was suggested that sleep gave rise to an increased protein synthesis of the activated
neurons . Thus, Shapot (3) found that the 35 S methionine incorpora tion was greatly increased in the brain proteins of sleeping rats which had been previously excited, till exhaustion by phenamine . During sleep of 20-day-old rate Reich (4) reported also an
increase in the incorporation of inorganic orthophosphate -32 P into a phosphoprotein fraction of the brain . Actually, most works (11) indicate that some stimulation of the central nervous system usually tends to increase the protein synthesis of the activated nerve cells, though exceptions to this occur . Interpretations of these results are difficult for several reasons ; among which is the non specific induction of sleep due to drug administration . Also, an increased amount of labelled proteins may also come about through changes in the circulation or through the alterations in the S .A . of the precursor pool of
aminoacida at the site of protein synthesis . Finally, the animals are submitted to stressful situations . In our own experiment, it is also probable that the physiolo-
gical procedure utilized to obtain a selective PS deprivation maintains the animals under a generalized stress . The increase in uptake of aminoacids into rat brain cortical slices has recently been attributed to stress (12) . It is likely that stress might
induce alterations in cell permeability and thus explains the above reported increase in uptake of 3 H amino acids into the intracellular pool of brainstem slices in both experimental groups of rats . However, no difference was seen in the free aminoacid pool
Vol . 10, No. 22
Paradoxical 81eep and Protein Synthesis
1355
of the Telencephalon . This seems to indicate that PS deprivation does not affect the aminoacid metabolism in all structures uniform-
ly . Other similar variations in the levels of free amino acids have been shown to occur in the brains of rats (13) and cats (14) in response to PS deprivation . From these results it can be suggested that the alterations of the brain aminoacid metabolism are mainly due to stress through some common mechanisms which involves horm~nal pathways and could thus secondarily influence the protein synthesis in the central nervous system . It should be noticed too that in these kinds of experiments, the problem is to dissociate the biochemical changes arising from specific and non specific
components . Among the several explanations for the failure to find any
variation in the protein synthesis during PS rebound, the most
simple one is that our procedures of analysis of total brain proteins are not sensitive enough to reveal the presence of additional proteins synthetized in response to increased neuronal activity . Another explanation would be an initial stimulation of
protein synthesis during the first minutes of PS recuperation followed by a depression in protein synthesis or a return to
steady state metabolism when sleep lasts for more than 2 hours . A high intensity of stress as well as its a long duration may also account for the lack .of effects observed . It has been hypo-
thesized (15) that macromolecules may be produced in order to alter or facilitate the synaptic transmission in neuronal .networks, but there is no evidence in our data to suggest that we are observing any part of that process during sleep . One can notice that if there is an effect it may not necessarily be uniform . And so, after evaluating these possibilities involving specific
proteins, a new experimental procedure of PS deprivation in which stress can be minimised, is required . Further detailed qualitative analysis of the protein synthesis in brain, such as fractionation of proteins by disc electrophoresis on polyacrylamide gels, is also needed . SIIMMARY The in vitro incorporation of L- 3H-aminoacids mixture into the proteins of brain slices was performed in order to determine
1358
Parado~cal 31eep aid Protein Synthesis
vol., io,, xo. a3
whether the PS deprivation would affect the protein metabolism in the central nervous system of rats . A significant increase of
3H amino acid uptake into the TCA-soluble fraction has been observed in the brainstem of rats deprived of PS . There was, however, no alteration of the protein synthesis observed either in the Telencephalon or in the crude subcellular fractions of brainstem, after PS-deprivation or after the rebound of PS following deprivation . The significance of these results is discussed in relation with stress .
ACRNOWLEDGEMENT We gratefully acknowledge the technical assistance of Misa L . Léger in the electron microscopic observation of brain subcellular fractions . REFERENCES RICHTER, in Protein Metabolism of the Nervous System , ed . Lajtha, p . 241 . Plenum Press, New York, London (1970) . GLASSMAN, Ann . Rev . Biochem . 38, 605 (1969) . V .S . SHAPOT, in Meta bolism of the Nervous System, ed . D . Richter, p . 257 . Pergamon Press (1957) . 4.
P . REICH, J .K . DRIVER and M .L . KARNOVSKY, Science , 157, 336
5.
F . HERY, J .F . PUJOL, M . LOPEZ, J . MACON and J . GLOWINSKI,
6.
F . ORREGO and F . LIPMANN, J . Biol . Çhem ., 242, 665 (1967)
7.
J .F . PUJOL, J . MOURET, M . JOUVET and J . GLOWINSKI, Science ,
8.
E. de ROBERTIS, A . PELLEGRINO de IRALDI, G . RODRIGUEZ de LORES ARNAIZ and L. SALGANICOFF, J . Neurochem ., 9, 23 (1962) .
9.
S . UDENFRIEND, Molecular Biology , III, p . 191, Academic Press,
(1967) .
Brain Res ., 21, 391 (1970) .
159, 112 (1968) .
London (1962) .
A . LAJTHA, In tern . Rev. Neurobiol . 7, 1 (1964) . D .A . RAPPOPORT and H.F . DAGINAWALA, in Protein Metabolism of the Nervous System , ed . A . Lajtha, p . 459 . Plenum Press New York, London (1970) .
Vol. 10, No. 23
Paradoxical Sleep and Protein Synthesis
1357
12 . B . JAROUBEK, B . SEMIGINOVSKY, M . KRAUS and R . ERDOSSOVA, Life Sci ., 9, 1169 (1970) . 13 . Y . GODILA and P . MANDEL, J . Neurochem., 12, 455 (1965) . 14 . V . KARAKZIC, D . MICIR and L . RAKIC, Experientia , 27, 509 (1971) . 15 . J . GAITO, in Molecular Psychobiology, ed . C . Thomas p . 33, Springfield Illinois USA (1966) .
Pergamon Press
pEPRIVATION OF PARADOXICAL SLEEP AND IN VITRO CEREBRAL PROTEIN SYNTHESIS IN THE RAT P . Bobillier, F.- Sakai, S . Sequin and M . Jouvet Department of Experimental Medicine School of Medicine, Lyon, France
(Received 23 September 1971; in final form 1 November 1971) Brain proteins are metabolically highly active and play a. role in cellular metabolism and its regulation . In recent years a considerable amount of work has been reported on the possible relationship between protein synthesis in the brain and its specialized functions (1) . Investigations of the chemical correlates of learning and memory, in particular, have provided an extensive literature
(2) . However, little is known about the
possible alterations of the cerebral proteins during sleep (3,4) . Paradoxical sleep (PS) deprivation is a very useful working instrument since this specific phenomenon is followed by a. sub-
aequent compensatory rebound of this state of sleep . On the other hand, recent sleep studies have shown the importance of serotonin and norepinephrine in the biochemical regulatory mechanisms associated with the neurophysiological changes which occur during and after PS deprivation (5) . Evidence has also accumulated to
indicate that brain stimulation in vitro will alter the synthesis of protein in the brain (6) . It was then of interest to study the possibility that an increased activity of the neurons (which is
reflected by the fast cortical activity occuring during PS) may be associated with variations in the protein synthesizing capacity of the brain tissue .
In this paper, we examined the protein synthesis in vitro by measuring the incorporation of labelled aminoacids into total proteins during incubation of brain slices . The brain slices were taken from rats which have been submitted to PS deprivation or to PS recuperation .
*This work has been suppozted by the Air Force Office of Scientific Research (USAF Contract No . F61052-70-C-003), by D .R .M .E . (Grant 70-036) and I .N .S .E .R .M .
1349
1350
Paradaaícal Sleep anti Protein Synthesis
Vol. 10, No. 23
Material's and Methods Selective PS deprivation of rats was carried out by a method described previously (7) : the animals being placed upon small
supports surrounded by water, with food and water ad lib . 24 adult male Sprague-Dawley rats weighing about 250 qr . were equally divided into three groups 1) control animals, 2) animals deprived of PS for 54 hr . and 3) animals deprived of PS for 48 hr and then allowed to recuperate for 6 hr . in sound-proof cages . All control animals were isolated in cages in the same room at + 25 °C, with water and food ad lib ., during the same length of time . At the end of the experimental procedure the animals were
killed by a strong blow to the body and then decapitated . The brains were removed, (cerebellum and pineals being discarded), washed in 0 .9 $ saline and dissected in a cold room at + 4 ° C into
Telencephalon (cortex, hippòcampus and nucleus caudatus) and brainstem . Incubation procedure
From each part of the brain, 0 .4 mm thick tissue slices were prepared at + 4 °C with a Mc ILLWAIN tissue chopper . The slices were put into a 25 ml Erlenmeyer flask containing 2 ml of standard
incubation medium (Krebs-Ringer bicarbonate, PH 7 .5) and preincubated for 5 min . in a shaker bath at 37 ° C, type Dubnoff with a
gas mixture consisting of 95 8 0 2 and 5 8 C0 2 . 1 .4~C1 of L- 3 H-Amino-acids mixture (NEN Chemicals GmBH) was then added to the medium, the flasks were flushed briefly with the gas mixture and incubated for 45 minutes . After incubation, the slices were rinsed in a funnel of filter paper with 5 ml of cold incubation medium and frozen until analysis . Preparation of tissues
The specimens were placed in a Potter-Elvejhem apparatus with 10 vol . of 0 .003 M CaC1 2 in 0 .32 M-Sucrose and homogenized for about 1 min . 30 sec . with a loose-fitting teflon pestle .
Preparation of subcellular fractions Portions of brainstem homogenates were separated (8) into crude subcellular fractions by differential centrifugation, in a S~rvall refrigerated centrifuge : a nuclear fraction at 800 q for 10 min . and a mitochondrial fraction, at 13,000 g for 10 min . The
voi, io, No . as
Paraaoadoal sleep ana ~o~ein s9~tneeie
issi
supernatant from the crude mitochondrial pellet was centrifuged at 100,000 g for 1 hr . in a Spinco ultracentrigue to provide the cell sap (or soluble fraction) . Analysis of tissue protein The proteins in tissue homogenates from Telencephalon and each of the brainstem subcellular fractions were precipitated in cold 5 ~ TCA . After centrifugation, the precipitate was washed once with 5 8 TCA and heated to 90 °C for 30 min, then once more
with cold 5 8 TCA, once with Ethanol-Ether (1 : l,v/v) and heated to 37 °C for 30 min ., and finally, once with cold Ethanol-Ether . The pellets were redissolved with 0 .1 N NaOH and the proteins
estimated by fluorlmetric assay (9) with ovalbumine as a standard . Another portion was taken for the determination of radioactivity, the labelled proteins being counted in a Packard TRI
CARB Spectrometer with Ineta-Gel solvent system (Packard Inetrtiunent) . The specific activity (SA) of the proteins was expressed as dpm/mg protein . In order to estimate the pool of free 3H amino-acids, the radioactivity of the TCA-soluble supernatant was determined again using liquid scintillation counting, and the "amino acid specific activity" expressed as DPM/mg of proteins . Quenching corrections
were made with the external standard and channels ratio method . Significance of differences was tested using the Student's t test: Results
The tissue/medium ratio between the SA of the TCA-soluble fraction in the slices and that of the incubation medium was
always >1 in both brain structures studied, thus indicating that
the uptake of H3 amino acids occured well against a concentration gradient . Our results are summarized in Tables I, II and III .
As shown in Table I, the SA of the TCA-soluble fraction in the brainstem of rats deprived of PS, was significantly increased as compared to control animals and to PS. deprived animals allowed
to recuperate . A less pronounced Increase above controls, though not significant, was also observed in the incorporation of 3 H aminoacide in the intracellular pool of brainstem of rats deprived of PS and allowed to recuperate . In the telencephalon the SA of the TCA-soluble fraction was found to be similar in the three groups of rats studied .
1352
Paradrndcal 31eep and Protein Synthesis
Vol. 10, No. 23
TABLE I The in vitro incorporation (duratión 45 minutes) of L- 3 H-aminoacid mixture into the brain TCA-soluble fraction of rats
The animals were divided in 3 groups : Control animals (I), PS-deprived animals during 54 hr . (II) and animals in recuperation during 6 hr . after 48 hr . of PS deprivation (III) . Values are the mean + S .E .M . of 8 animals in each group. Brain regions Telencephalon Brainstem
Specific activity : DPM/mg protein 21013+893
48972+4465
21716+1112 69561+3584
20494+1158 51380+3462
P values from Student's t test : idtie < .O1 when compared with animals of group I ;~ ~ s< .O1 when group III is compared with
group II .
Conversely, the results of the in vitro incorporation of 3H aminoacids into the proteins of brainstem and telencephalon, as shown in Table II, did not indicate any significant differences between the two experimental groups and only a alight increase above controls (not significant) . TABLE II
The in vitro incorporation (duration 45 minutes) of L- 3H-amino acids mixture into the brain proteins of rats The groups of animals are expressed in the same way as in
Table I .
Fractions examined
Specific activity : DPM/mq protein
Telencephalon Brainstem Total homogenate
I 561+28
II 608+45
639+58
1095+54
1378+150
1136+165
Mitochondrial Soluble
2438+88
3215+361
2539+274
Nuclear
1113+52 1362+98
1289+174 1464+238
III
1310+175 1465+236
Vol. 10, No. 23
Paradaaical Sleep and Protein Synthesis
1353
Experiments have been done to determine whether or not some alterations in protein synthesis could be more particularly localized in any particular or soluble subcellular fractions of brain tissue . As in the total homogenate, only a slight increase of the
SA of proteins in the crude subcellular fractions of the brainstem in both experimental groups of rats can be noticed , this change not being significant when compared to control rats .
Thus, the slight (though generally increased) SA of proteins which affected the brainstem of rats deprived of PS, or allowed to recuperate after PS deprivation did not reflect the marked
increase which occured in the uptake of 3 H aminoacids into the TCA-soluble fraction of experimental animals .
The pool size of free amino acids may vary and affect the
protein synthesis (10) . To ascertain whether the slight increase in the SA of proteins was due to variations of the aminoacide or
to variations in the incorporation of the precursors intò the proteins, we determined the relative specific activity . This ratio may be used as an index of protein synthesis . No significant variations in the relative specific activity were obtained either in the Telencephalon or in the brainstem of either experimental or control rats (Table III) . These data clearly suggest that the
brain protein synthesis unlike 3H aminoacid upta9ce was not affected by the PS deprivation or by the PS rebound of rats under our experimental conditions . TABLE III
The effects of the PS deprivation on the relative
specific activity of proteins in the brain of rats . The groups of animals are expressed in the same way as in Table I . Brain regions
Telencephalon Brainstem
Specific activity of proteins Specific activity of TCA-soluble fraction III
Relative specific activity, I .027+ .001
.024+ .003
II
.028_+ .002 .020+ .002
.031+_ .002 .021+ .002
1354
Paradaadcal 81eep and Protein 3y~hesis
Vvl . 10, No . 23
DISCUSSION In the present study an attempt has been made to correlate protein synthesis in the central nervous system of rats with conditions
of increased functional activity during sleep . No significant alter ation of the protein synthesis was seen in the Telencephalon or in the Brainstem areas nor in the Brainstem particular or soluble subcellular fractions in the two experimental groups when compared to control animals . In contrast, we observed rises in the uptake of the labelled precursors in the Brainstem of the two experimental groups, but the significant one occured only in the PS deprived group .
In previous studies by other authors it was suggested that sleep gave rise to an increased protein synthesis of the activated
neurons . Thus, Shapot (3) found that the 35 S methionine incorpora tion was greatly increased in the brain proteins of sleeping rats which had been previously excited, till exhaustion by phenamine . During sleep of 20-day-old rate Reich (4) reported also an
increase in the incorporation of inorganic orthophosphate -32 P into a phosphoprotein fraction of the brain . Actually, most works (11) indicate that some stimulation of the central nervous system usually tends to increase the protein synthesis of the activated nerve cells, though exceptions to this occur . Interpretations of these results are difficult for several reasons ; among which is the non specific induction of sleep due to drug administration . Also, an increased amount of labelled proteins may also come about through changes in the circulation or through the alterations in the S .A . of the precursor pool of
aminoacida at the site of protein synthesis . Finally, the animals are submitted to stressful situations . In our own experiment, it is also probable that the physiolo-
gical procedure utilized to obtain a selective PS deprivation maintains the animals under a generalized stress . The increase in uptake of aminoacids into rat brain cortical slices has recently been attributed to stress (12) . It is likely that stress might
induce alterations in cell permeability and thus explains the above reported increase in uptake of 3 H amino acids into the intracellular pool of brainstem slices in both experimental groups of rats . However, no difference was seen in the free aminoacid pool
Vol . 10, No. 22
Paradoxical 81eep and Protein Synthesis
1355
of the Telencephalon . This seems to indicate that PS deprivation does not affect the aminoacid metabolism in all structures uniform-
ly . Other similar variations in the levels of free amino acids have been shown to occur in the brains of rats (13) and cats (14) in response to PS deprivation . From these results it can be suggested that the alterations of the brain aminoacid metabolism are mainly due to stress through some common mechanisms which involves horm~nal pathways and could thus secondarily influence the protein synthesis in the central nervous system . It should be noticed too that in these kinds of experiments, the problem is to dissociate the biochemical changes arising from specific and non specific
components . Among the several explanations for the failure to find any
variation in the protein synthesis during PS rebound, the most
simple one is that our procedures of analysis of total brain proteins are not sensitive enough to reveal the presence of additional proteins synthetized in response to increased neuronal activity . Another explanation would be an initial stimulation of
protein synthesis during the first minutes of PS recuperation followed by a depression in protein synthesis or a return to
steady state metabolism when sleep lasts for more than 2 hours . A high intensity of stress as well as its a long duration may also account for the lack .of effects observed . It has been hypo-
thesized (15) that macromolecules may be produced in order to alter or facilitate the synaptic transmission in neuronal .networks, but there is no evidence in our data to suggest that we are observing any part of that process during sleep . One can notice that if there is an effect it may not necessarily be uniform . And so, after evaluating these possibilities involving specific
proteins, a new experimental procedure of PS deprivation in which stress can be minimised, is required . Further detailed qualitative analysis of the protein synthesis in brain, such as fractionation of proteins by disc electrophoresis on polyacrylamide gels, is also needed . SIIMMARY The in vitro incorporation of L- 3H-aminoacids mixture into the proteins of brain slices was performed in order to determine
1358
Parado~cal 31eep aid Protein Synthesis
vol., io,, xo. a3
whether the PS deprivation would affect the protein metabolism in the central nervous system of rats . A significant increase of
3H amino acid uptake into the TCA-soluble fraction has been observed in the brainstem of rats deprived of PS . There was, however, no alteration of the protein synthesis observed either in the Telencephalon or in the crude subcellular fractions of brainstem, after PS-deprivation or after the rebound of PS following deprivation . The significance of these results is discussed in relation with stress .
ACRNOWLEDGEMENT We gratefully acknowledge the technical assistance of Misa L . Léger in the electron microscopic observation of brain subcellular fractions . REFERENCES RICHTER, in Protein Metabolism of the Nervous System , ed . Lajtha, p . 241 . Plenum Press, New York, London (1970) . GLASSMAN, Ann . Rev . Biochem . 38, 605 (1969) . V .S . SHAPOT, in Meta bolism of the Nervous System, ed . D . Richter, p . 257 . Pergamon Press (1957) . 4.
P . REICH, J .K . DRIVER and M .L . KARNOVSKY, Science , 157, 336
5.
F . HERY, J .F . PUJOL, M . LOPEZ, J . MACON and J . GLOWINSKI,
6.
F . ORREGO and F . LIPMANN, J . Biol . Çhem ., 242, 665 (1967)
7.
J .F . PUJOL, J . MOURET, M . JOUVET and J . GLOWINSKI, Science ,
8.
E. de ROBERTIS, A . PELLEGRINO de IRALDI, G . RODRIGUEZ de LORES ARNAIZ and L. SALGANICOFF, J . Neurochem ., 9, 23 (1962) .
9.
S . UDENFRIEND, Molecular Biology , III, p . 191, Academic Press,
(1967) .
Brain Res ., 21, 391 (1970) .
159, 112 (1968) .
London (1962) .
A . LAJTHA, In tern . Rev. Neurobiol . 7, 1 (1964) . D .A . RAPPOPORT and H.F . DAGINAWALA, in Protein Metabolism of the Nervous System , ed . A . Lajtha, p . 459 . Plenum Press New York, London (1970) .
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