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Soluble protein pattern of different brain regions of old rats when they acquire a new behavior
BEHAVIORAL AND NEURAL BIOLOGY 27, 294--301
(1979)
Soluble Protein Pattern of Different Brain Regions of Old Rats When They Acquire a New Behavior t H . HYDI~N AND L . RONNBXCK
Institute of Neurobiology, University of Gi~teborg, GiSteborg, Sweden Three-year-old rats could easily establish a new behavior consisting of reversal of handedness in an instrumental learning test. These old animals acquired the new behavior at the same rate as young adult rats, but did not perform as well. Incorporation of [14C]valine into soluble protein of different parts of the limbic system and cortex cerehri was measured, as well as dye uptake into protein bands on polyacrylamide gel electrophoresis. In the hippocampus, entorhinal cortex, septum, and sensory-motor cortex, and one specific protein fraction increased in synthesis. We also report upon some other specific responses due to the acquisition of the new behavior of the very old rats.
This study presents results of brain protein analysis during a behavioral test in old rats (3 years old!). Increased incorporation of protein precursors and quantitative increase of specific proteins in localized parts of the brain, viz., the hippocampus and related areas of the limbic system, have previously been published by several laboratories, including our own (Matthies, Pohle, Popov, LSssner, Riitrich, Jork, & Ott, 1975; Shashoua, 1976; Hydrn & RSnnb~ick, 1979). Such studies require analysis at the microlevel. Previous studies have been performed on rats aged 2.0-2.5 months. An important question then is whether changes in behavior also induce a short-lasting alteration in the specific protein pattern of brain cells in animals of old age. We have therefore chosen rats aged 3 years which is considered to be very old. The following points have been considered. First, to see whether the brain cells of very old animals showed similar changes in soluble protein pattern which have been found in young adult rats on training. Second, to correlate behavior and the brain cell protein activity. Third, to compare the capacity of old and young animals to acquire the new behavior. A number of studies referred to above implicate that brain protein 1 This work was supported by a grant from the Swedish Medical Research Council No. B79-12X-00086-14C. The skillful technical assistance of Mrs. Inger Augustsson and Mrs. Mona Grthman is gratefully appreciated. 294 0163-1047/79/110294-08502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS
295
synthesis during or shortly after training is required for the formation of long-term memory of discrimination training, active and passive avoidance learning (Agranoff, 1970; Mark & Watts, 1971; Squire and Barondes, 1972), and habituation, a phylogenetically primitive form of long-lasting behavioral plasticity (Squire & B e c k e r , 1975). Using another approach, Flexner, Flexner, and Stellar (1963) and Bennett, Diamond, Krech, and Rosenzweig (1979) demonstrated an impairment in later performance of learned tasks by administration of hexemides, or anisomycin, to interfere with the RNA-dependent synthesis of proteins. During training to reverse handedness in rats, a wave of protein activity pervades the brain. As measured by incorporation of labeled amino acid into soluble protein, Hyd6n and Lange (1968, 1970a, 1970b, 1970c, 1972) have shown an initial increase in the hippocampus followed by an increased incorporation in different cortical areas with a temporal phase shift. Many lines of evidence suggest the decisive role of the hippocampus at an early stage of memory formation. After a brief training period (2 x 25 min transfer of handedness), at least one soluble protein with subunits (MW 15,000, 30,000, and 65,000) was identified in the hippocampus (R6nnbgtck and Hyd6n, 1979). This brain region is thought to control the processes of sensory inputs and their integration with motivational influences into complex cortical functions during the development of an acquired behavior (Adey, 1967; Beach, Emmens, Kimble, & Lickey, 1969; Douglas, 1967; Meissner, 1967). MATERIALS AND METHODS
Animals. Thirty-six-month-old rats, four experimental and four active controls of the Wistar strain, were used. They had been living in cages 100 x 50 × 35 cm 3 with 12 hr light, 12 hr dark, and were fed pellets and water ad libitum. Weight at the end of the experiment was around 380 g. Behavioral test. The behavioral test was reversal of handedness with training twice daily for 25 min during 4 days (Hyd6n and Lange, 1970c). Trained rats were compared with active controls, i.e., rats which used the preferred paw to retrieve food pills one by one for the same time and obtained the same amount of food as did the trained rats. Thus, the background of environmental and motor nontraining factors was equal for the two groups of animals. Experimental procedure. Immediately prior to the last training session of 25 min, the experimental animals and active controls got intraperitoneal injections of 700 p.Ci [1-'4C]valine (40-50 mCi/mmole Merck AG, Darmstadt, Germany) and were sacrificed 40 min thereafter. The brains were quickly taken out--hippocampus, anterior dorsal part of the thalamus, septum, entorhinal cortex, sensory-motor cortex, and visual
296
HYDfiN A N D R O N N B A C K TRAINED ACTIVE CONTROL
a NUMBER
OF CORRECT
REACHES 5O
40
A
30
10
I
I DAY 1
I
I DAY 2
I
I DAY 3
I
I DAY 4
TRAINING
PERIOD
FIG. l(a) Performance curve (number of correct reaches) as a function of training of the very old rats studied. All animals were trained for 4 days, 25 min twice daily, in the behavioral test described in the text. (b) Performance curve as a function of training of young adult rats and active controls (body weight around 150 g) under similar conditions as in Fig. la. Trained r a t s , - - ; active c o n t r o l s , - - - .
cortex (area 17) according to Craigie (1963). The samples were stored in -80°C until used. Extraction of protein. The brain samples were homogenized in 1.0 mM Tris-HC1 buffer, pH 7.2, containing 0.03 M NaC1 and 0.05 M sucrose on ice, centrifuged at 30,000g for 120 min. Protein was determined according to Lowry, Rosebrough, Farr, and Randall (1951). Electrophoresis of the supernatants was performed on 10% polyacrylamide gels (10 mm i.d.) according to Davis, (1964) and Ornstein (1964). In all cases, 750/xg of protein was applied on each gel. The gels were stained for 10 hr with a 0.2% solution of Coomassie brilliant blue (Imperial Chemical Industries Ltd., England) in a mixture of acetic acid, methanol, and water (1:5:10). Destaining was performed for 72 hr in the latter solution. The stained gels
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS b
297
NUMBER OF CORRECT REACHES
180
160
140
20
100
• \
80
/
\ \
I/
"~
6o
4o
20
I
I
DAY 1
i
I
DAY 2
I
I
DAY 3
I
I
DAY 4 TRAINING PERIOD
FIG. l(b)
were scanned in a microphotometer equipped with a Vitatron linear log recorder (Hyd~n & Lange, 1971). Each gel was sliced into 52 pieces, which were treated with Soluene 100 (Packard, Zfirich, Switzerland) for 48 hr and l0 ml of scintillation fluid (5.5 g Permablend III per liter toluene) was added. The radioactivity measurement was performed in a Packard liquid scintillation spectrometer (Model 3380, 544). The magnitude of the specific activity values of the protein fractions varied from 1600 to 17,000 cpm/mg protein. For every rat and area to be studied, the dentisometric and incorporation values from one gel was done in duplicate. Mean values of incorporation from trained animals and the corresponding mean values of active controls for each gel slice were divided into each other, and the quotients were expressed as percentage of active control values (_ SEM) (n --- 4, in duplicate). RESULTS Behavioral Test
All control old rats used in this study quickly took food pills out of the tube with their preferred paw. The learning old animals also rather quickly
298
HYDI~N AND RONNBACK
acquired their new tasks, taking food pills with their nonpreferred paw (Fig. la). Yet there were some differences between young and these very old animals in acquiring the new behavior (Fig. lb). The total number of reaches of the old rats was just one-third of that of the young ones. However, the old rats were tired, one having a quick, faint respiration, and one having symptoms of an early bronchitis.
Precursor Incorporation The incorporation of the trained rats was higher by a factor of 1.1-1.3 compared to the data of controls. In control vs control data such an analysis gives a value of 1.0. In trained vs control samples the calculations were therefore carried out using a factor which normalized a maximum number of data points. Deviation from the relative label ratio thus could be converted directly to a percentage change at each position along the gel. (See Shashoua, 1976). A straight line along the whole gel was obtained with the exception of some peaks corresponding to specific protein fractions with significantly higher incorporation values of trained rats compared to active controls. In Fig. 2 densitometric values of electrophoretic bands from different brain areas are shown together with differences in incorporation values of [14C]valine-trained animals/active controls of 2-mm gel slices expressed as percentage ___ SEM (indicated by bars). An increased incorporation was found in one protein fraction (Rs .31.34, p < .001) in hippocampus, septum, entorhinalis cortex, and sensory-motor cortex of trained old rats as compared to active controls. Another two more anodally migrating fractions (Re 1.0 and .86, p < .001) increased in incorporation in hippocampus, while sensory-motor cortex and visual cortex responded with a decreased incorporation in trained animals (Rs .53-.55, p < .05). No significant changes were found in thalamus. The dye uptake in different fractions (bands) are shown in Fig. 2. It can be seen that those fractions, showing increase in [14C]valine incorporation of trained rats to controls, also have an increased dye uptake, suggesting an increase in synthesis of these protein fractions. However, the differences in staining are smaller than those of radioactivity incorporation, probably due to the fact that dye uptake is calculated on whole bands, while [14C]valine incorporation values are based upon single gel slices of 2 mm each.
DISCUSSION It can be stressed that the rats used in this study are remarkably old, 3 years. Intracranial injections in such old animals could be hazardous, so we found it advisable to administer the precursors as intraperitoneal injections. The incorporation of [14C]valine into soluble protein was calculated for
299
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS % DPM
TR/C
50 40
HIPPOCAMPUS
30 20 10 0.34 16~7
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FIG. 2. Activity of [14C]valine (% dpm) incorporated into soluble proteins of hippocampus, entorhinal cortex, septum, sensory-motor cortex, visual cortex, and thalamus. Electrophoresis (10% polyacrylamide) was performed as described in the text; incorporation values of 2-mm sections from trained animals were divided by corresponding values from active controls. Densitometric, i.e., dye uptake values of individual bands expressed as percentage trained animals/active controls (% TR/C) and relative mobility (R f) values are given. One gel section (Re .31-.34) of hippocampus, entorhinal cortex, septum, and sensory-motor cortex responds with an increase in [14C]valine incorporation Co < .001) as well as a relative increase in dye uptake (11-18%), indicating an increase in synthesis. Two other fractions in hippocampus (Rs .86 and 1.0) react in a similar way (p < .001) while one gel area in sensory-motor cortex and visual cortex (Rs .53-.55) responds with a decrease in both relative incorporation of [14C]valine (p < .05) and dye uptake (-9-12%). (Anode to the right).
300
HYDEN AND RONNBACK
each protein fraction as quotients between trained rats and active controls. The rational for the determination is the following. The whole of the separation pattern was used and divided in 2-mm sections. Since the [14C]valine pool of each gel separation is the same for each protein band, the values of the ratio pooltrained/poolcontrol are constant. Therefore, a difference in the incorporation of [14C]valine at the same position in two different gel separations show up as a deviation from 1.0. Striking is an significant increase in incorporation values of a protein fraction, R s = .31-.34 in the three limbic areas, the hippocampus, entorhinal cortex, and septum. The activity of the hippocampal protein was also significantly increased at Rs = .86 and 1.0. An increase of labeling of three to four protein fractions as a function of training has previously been observed in the hippocampus, among them the brain-specific proteins, S 100 and 14.3.2 (Hyd6n & Lange, 1968, 1970a, 1970b, 1970c, 1971, 1972; Matthies et al., 1975; Hyd6n & RSnnb~ick, 1979). In contrast to the limbic areas, the protein changes of the sensory-motor and visual cortex after training are characterized by negative activity peaks. This is probably due to a temporal phase shift in response as has been shown to occur in younger animals' (Hyd6n & Lange, 1971) activity peaks. Summarizing the biochemical results, it seems that the reversal of handedness test provokes the same type of response in the pattern of brain-soluble proteins of the very old rats as it does in young ones. The trained old rats are compared with their active controls of the same age which perform the same task with the preferred paw and do not acquire a new behavior. As to the behavior of trained and controls, it is clear that the former begin at a lower level of performance, but their rate of acquisition improves considerably with training and surpasses that of the controls after half the training time. Averaged over training time, the number of successful reaches is similar for trained and control rats. Since the animal gets and eats one protein pill for each successful reaching, both trained and controls thus get a similar amount of food. The level of successful reaches per training session of the old rats is less than half of the capacity of the young ones. On observing the behavior of the old rats it was clear that they became tired sooner than the young ones. On the other hand, their energy and rate of acquisition was impressive. REFERENCES Adey, W. R. (1967). Hippocampal states and functional reactions with cortico subcortical system in attention and learning. In W. R. Adey and T. Tokizane (Eds.), Structure and Function o f the Limbic System, Progress in Brain Research, Vol. 27, Elsevier, Amsterdam, pp. 228-245. Agranoff, B. W. (1970). Recent studies on the stages of memory formation in the goldfish. In W. L. Byrne (Ed.) Molecular Approaches to Learning and Memory, pp. 35-39. New York: Academic Press.
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301
Beach, G., Emmens, M., Kimble, D. P., & Lickey, M. (1969). Autoradiographic demonstration of biochemical changes in the limbic system during avoidance training. Proceedings of the National Academy of Sciences, Washington, 62, 692-696. Bennett, E. K., Diamond, M. C., Krech, D., & Rosenzweig, M. R. (1964). Chemical and anatomical plasticity of the brain. Science, 146, 610-619. Craigie, E. H. (1963). Craigie's Neuroanatomy of the Rat, rev. ed. New York: Academic Press. Davis, B. J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences, 121, 404-427. Douglas, R. J. (1967). The hippocampus and behavior. Psychology Bulletin, 67, 416-442. Flexner, J. B., Flexner, L. B., & Stellar, E. (1963). Memory in mice as affected by intracerebral puromycin. Science, 141, 57-59. Hyd6n, H., & Lange, P. W. (1968). Protein synthesis in the hippocampal pyramidal cells of rats during a behavioral test. Science, 159, 1370-1373. Hyd~n, H., & Lange, P. W. (1970). Protein synthesis in limbic structures during change in behavior. Brain Research, 22, 423. (a) Hyd6n, H., & Lange, P. W. (1970). S 100 brain protein: Correlation with behavior. Proceedings of the National Academy of Sciences USA, 67, 1959. (b) Hyd6n, H., & Lange, P. W. (1970). Brain-cell protein synthesis specifically related to learning. Proceedings of the National Academy of Sciences USA, 65, 898. (c) Hyd6n, H., & Lange, P. W. (1971). Microelectrophoretic determination of protein and protein synthesis in the 10-7 to 10-9 gram range. In A. Niederwieser and G. Pataki (Eds.), New Techniques in Amino Acid Peptide and Protein Analysis, p. 271. Ann Arbor, Mich.: Ann Arbor Science Pub. Hyd6n, H., & Lange, P. W. (1972). Protein changes in different brain areas as a function of intermittent training. Proceeding of the National Academy of Sciences USA, 69, 1980. Hyd6n, H., & R6nnb~ick, L. (1979). Proteins S 100 and 14-3-2 in nerve cells of rats raised in enriched and impoverished environment. Distribution, quantification and cell separation by surface antigens. Behavioral and Neural Biology, 25, 371-379. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265. Mark, R. F., & Watts, M. E. (1971). Drug inhibition of memory formation in chickens. I. Long-term memory. Proceeding of the Royal Society of Britain, 178, 439-454. Matthies, H., Pohle, W., Popov, N., L6ssner, B., Riitrich, H. L., Jork, R., & Ott, T. (1975). Biochemical mechanisms correlated with learning and memory formation facts hypothesis. In K. Liss~k (Ed.), Neural and Neurohumoral, Organization of Motivated Behaviour, pp. 85-105. Proe. IVth Conf. Interbrain, P~cz, Hungary, May 19-23. Meissner, W. W. (1967). Hippocampus and learning. International Journal of Neuropsychiatry, 3, 298-310. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Annals of the New York Academy of Sciences, 121, 321-349. R6nnb~ick, L., & Hyd6n, H. (1979) Stimulation of a soluble protein fraction in hippocampus after a brief training period. (in press). Shashoua, V. E. (1976). Brain metabolism and the acquisition of new behaviors. I. Evidence for specific changes in the pattern of protein synthesis. Brain Research, 111,347-364. Squire, L. R., & Barondes, S. H. (1972). Inhibitors of RNA or protein synthesis and memory. In J. Gaito (Ed.), Macromolecules and Behavior, 2nd ed., pp. 61-82. New York: Appleton-Century-Crofts. Squire, L. R., & Becker, C. K. (1975). Inhibition of cerebral protein synthesis impairs long-term habituation. Brain Research, 97, 367-372.
(1979)
Soluble Protein Pattern of Different Brain Regions of Old Rats When They Acquire a New Behavior t H . HYDI~N AND L . RONNBXCK
Institute of Neurobiology, University of Gi~teborg, GiSteborg, Sweden Three-year-old rats could easily establish a new behavior consisting of reversal of handedness in an instrumental learning test. These old animals acquired the new behavior at the same rate as young adult rats, but did not perform as well. Incorporation of [14C]valine into soluble protein of different parts of the limbic system and cortex cerehri was measured, as well as dye uptake into protein bands on polyacrylamide gel electrophoresis. In the hippocampus, entorhinal cortex, septum, and sensory-motor cortex, and one specific protein fraction increased in synthesis. We also report upon some other specific responses due to the acquisition of the new behavior of the very old rats.
This study presents results of brain protein analysis during a behavioral test in old rats (3 years old!). Increased incorporation of protein precursors and quantitative increase of specific proteins in localized parts of the brain, viz., the hippocampus and related areas of the limbic system, have previously been published by several laboratories, including our own (Matthies, Pohle, Popov, LSssner, Riitrich, Jork, & Ott, 1975; Shashoua, 1976; Hydrn & RSnnb~ick, 1979). Such studies require analysis at the microlevel. Previous studies have been performed on rats aged 2.0-2.5 months. An important question then is whether changes in behavior also induce a short-lasting alteration in the specific protein pattern of brain cells in animals of old age. We have therefore chosen rats aged 3 years which is considered to be very old. The following points have been considered. First, to see whether the brain cells of very old animals showed similar changes in soluble protein pattern which have been found in young adult rats on training. Second, to correlate behavior and the brain cell protein activity. Third, to compare the capacity of old and young animals to acquire the new behavior. A number of studies referred to above implicate that brain protein 1 This work was supported by a grant from the Swedish Medical Research Council No. B79-12X-00086-14C. The skillful technical assistance of Mrs. Inger Augustsson and Mrs. Mona Grthman is gratefully appreciated. 294 0163-1047/79/110294-08502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS
295
synthesis during or shortly after training is required for the formation of long-term memory of discrimination training, active and passive avoidance learning (Agranoff, 1970; Mark & Watts, 1971; Squire and Barondes, 1972), and habituation, a phylogenetically primitive form of long-lasting behavioral plasticity (Squire & B e c k e r , 1975). Using another approach, Flexner, Flexner, and Stellar (1963) and Bennett, Diamond, Krech, and Rosenzweig (1979) demonstrated an impairment in later performance of learned tasks by administration of hexemides, or anisomycin, to interfere with the RNA-dependent synthesis of proteins. During training to reverse handedness in rats, a wave of protein activity pervades the brain. As measured by incorporation of labeled amino acid into soluble protein, Hyd6n and Lange (1968, 1970a, 1970b, 1970c, 1972) have shown an initial increase in the hippocampus followed by an increased incorporation in different cortical areas with a temporal phase shift. Many lines of evidence suggest the decisive role of the hippocampus at an early stage of memory formation. After a brief training period (2 x 25 min transfer of handedness), at least one soluble protein with subunits (MW 15,000, 30,000, and 65,000) was identified in the hippocampus (R6nnbgtck and Hyd6n, 1979). This brain region is thought to control the processes of sensory inputs and their integration with motivational influences into complex cortical functions during the development of an acquired behavior (Adey, 1967; Beach, Emmens, Kimble, & Lickey, 1969; Douglas, 1967; Meissner, 1967). MATERIALS AND METHODS
Animals. Thirty-six-month-old rats, four experimental and four active controls of the Wistar strain, were used. They had been living in cages 100 x 50 × 35 cm 3 with 12 hr light, 12 hr dark, and were fed pellets and water ad libitum. Weight at the end of the experiment was around 380 g. Behavioral test. The behavioral test was reversal of handedness with training twice daily for 25 min during 4 days (Hyd6n and Lange, 1970c). Trained rats were compared with active controls, i.e., rats which used the preferred paw to retrieve food pills one by one for the same time and obtained the same amount of food as did the trained rats. Thus, the background of environmental and motor nontraining factors was equal for the two groups of animals. Experimental procedure. Immediately prior to the last training session of 25 min, the experimental animals and active controls got intraperitoneal injections of 700 p.Ci [1-'4C]valine (40-50 mCi/mmole Merck AG, Darmstadt, Germany) and were sacrificed 40 min thereafter. The brains were quickly taken out--hippocampus, anterior dorsal part of the thalamus, septum, entorhinal cortex, sensory-motor cortex, and visual
296
HYDfiN A N D R O N N B A C K TRAINED ACTIVE CONTROL
a NUMBER
OF CORRECT
REACHES 5O
40
A
30
10
I
I DAY 1
I
I DAY 2
I
I DAY 3
I
I DAY 4
TRAINING
PERIOD
FIG. l(a) Performance curve (number of correct reaches) as a function of training of the very old rats studied. All animals were trained for 4 days, 25 min twice daily, in the behavioral test described in the text. (b) Performance curve as a function of training of young adult rats and active controls (body weight around 150 g) under similar conditions as in Fig. la. Trained r a t s , - - ; active c o n t r o l s , - - - .
cortex (area 17) according to Craigie (1963). The samples were stored in -80°C until used. Extraction of protein. The brain samples were homogenized in 1.0 mM Tris-HC1 buffer, pH 7.2, containing 0.03 M NaC1 and 0.05 M sucrose on ice, centrifuged at 30,000g for 120 min. Protein was determined according to Lowry, Rosebrough, Farr, and Randall (1951). Electrophoresis of the supernatants was performed on 10% polyacrylamide gels (10 mm i.d.) according to Davis, (1964) and Ornstein (1964). In all cases, 750/xg of protein was applied on each gel. The gels were stained for 10 hr with a 0.2% solution of Coomassie brilliant blue (Imperial Chemical Industries Ltd., England) in a mixture of acetic acid, methanol, and water (1:5:10). Destaining was performed for 72 hr in the latter solution. The stained gels
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS b
297
NUMBER OF CORRECT REACHES
180
160
140
20
100
• \
80
/
\ \
I/
"~
6o
4o
20
I
I
DAY 1
i
I
DAY 2
I
I
DAY 3
I
I
DAY 4 TRAINING PERIOD
FIG. l(b)
were scanned in a microphotometer equipped with a Vitatron linear log recorder (Hyd~n & Lange, 1971). Each gel was sliced into 52 pieces, which were treated with Soluene 100 (Packard, Zfirich, Switzerland) for 48 hr and l0 ml of scintillation fluid (5.5 g Permablend III per liter toluene) was added. The radioactivity measurement was performed in a Packard liquid scintillation spectrometer (Model 3380, 544). The magnitude of the specific activity values of the protein fractions varied from 1600 to 17,000 cpm/mg protein. For every rat and area to be studied, the dentisometric and incorporation values from one gel was done in duplicate. Mean values of incorporation from trained animals and the corresponding mean values of active controls for each gel slice were divided into each other, and the quotients were expressed as percentage of active control values (_ SEM) (n --- 4, in duplicate). RESULTS Behavioral Test
All control old rats used in this study quickly took food pills out of the tube with their preferred paw. The learning old animals also rather quickly
298
HYDI~N AND RONNBACK
acquired their new tasks, taking food pills with their nonpreferred paw (Fig. la). Yet there were some differences between young and these very old animals in acquiring the new behavior (Fig. lb). The total number of reaches of the old rats was just one-third of that of the young ones. However, the old rats were tired, one having a quick, faint respiration, and one having symptoms of an early bronchitis.
Precursor Incorporation The incorporation of the trained rats was higher by a factor of 1.1-1.3 compared to the data of controls. In control vs control data such an analysis gives a value of 1.0. In trained vs control samples the calculations were therefore carried out using a factor which normalized a maximum number of data points. Deviation from the relative label ratio thus could be converted directly to a percentage change at each position along the gel. (See Shashoua, 1976). A straight line along the whole gel was obtained with the exception of some peaks corresponding to specific protein fractions with significantly higher incorporation values of trained rats compared to active controls. In Fig. 2 densitometric values of electrophoretic bands from different brain areas are shown together with differences in incorporation values of [14C]valine-trained animals/active controls of 2-mm gel slices expressed as percentage ___ SEM (indicated by bars). An increased incorporation was found in one protein fraction (Rs .31.34, p < .001) in hippocampus, septum, entorhinalis cortex, and sensory-motor cortex of trained old rats as compared to active controls. Another two more anodally migrating fractions (Re 1.0 and .86, p < .001) increased in incorporation in hippocampus, while sensory-motor cortex and visual cortex responded with a decreased incorporation in trained animals (Rs .53-.55, p < .05). No significant changes were found in thalamus. The dye uptake in different fractions (bands) are shown in Fig. 2. It can be seen that those fractions, showing increase in [14C]valine incorporation of trained rats to controls, also have an increased dye uptake, suggesting an increase in synthesis of these protein fractions. However, the differences in staining are smaller than those of radioactivity incorporation, probably due to the fact that dye uptake is calculated on whole bands, while [14C]valine incorporation values are based upon single gel slices of 2 mm each.
DISCUSSION It can be stressed that the rats used in this study are remarkably old, 3 years. Intracranial injections in such old animals could be hazardous, so we found it advisable to administer the precursors as intraperitoneal injections. The incorporation of [14C]valine into soluble protein was calculated for
299
SOLUBLE PROTEIN PATTERN OF BRAIN REGIONS % DPM
TR/C
50 40
HIPPOCAMPUS
30 20 10 0.34 16~7
-10 -20
0.36 16t9 ENTORHINALIS
1.0 17~9
RF
DENStTOMETRY % TR/C
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0.53 -12 .'t 5 THALAMUS
-10 0.95 0
FIG. 2. Activity of [14C]valine (% dpm) incorporated into soluble proteins of hippocampus, entorhinal cortex, septum, sensory-motor cortex, visual cortex, and thalamus. Electrophoresis (10% polyacrylamide) was performed as described in the text; incorporation values of 2-mm sections from trained animals were divided by corresponding values from active controls. Densitometric, i.e., dye uptake values of individual bands expressed as percentage trained animals/active controls (% TR/C) and relative mobility (R f) values are given. One gel section (Re .31-.34) of hippocampus, entorhinal cortex, septum, and sensory-motor cortex responds with an increase in [14C]valine incorporation Co < .001) as well as a relative increase in dye uptake (11-18%), indicating an increase in synthesis. Two other fractions in hippocampus (Rs .86 and 1.0) react in a similar way (p < .001) while one gel area in sensory-motor cortex and visual cortex (Rs .53-.55) responds with a decrease in both relative incorporation of [14C]valine (p < .05) and dye uptake (-9-12%). (Anode to the right).
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each protein fraction as quotients between trained rats and active controls. The rational for the determination is the following. The whole of the separation pattern was used and divided in 2-mm sections. Since the [14C]valine pool of each gel separation is the same for each protein band, the values of the ratio pooltrained/poolcontrol are constant. Therefore, a difference in the incorporation of [14C]valine at the same position in two different gel separations show up as a deviation from 1.0. Striking is an significant increase in incorporation values of a protein fraction, R s = .31-.34 in the three limbic areas, the hippocampus, entorhinal cortex, and septum. The activity of the hippocampal protein was also significantly increased at Rs = .86 and 1.0. An increase of labeling of three to four protein fractions as a function of training has previously been observed in the hippocampus, among them the brain-specific proteins, S 100 and 14.3.2 (Hyd6n & Lange, 1968, 1970a, 1970b, 1970c, 1971, 1972; Matthies et al., 1975; Hyd6n & RSnnb~ick, 1979). In contrast to the limbic areas, the protein changes of the sensory-motor and visual cortex after training are characterized by negative activity peaks. This is probably due to a temporal phase shift in response as has been shown to occur in younger animals' (Hyd6n & Lange, 1971) activity peaks. Summarizing the biochemical results, it seems that the reversal of handedness test provokes the same type of response in the pattern of brain-soluble proteins of the very old rats as it does in young ones. The trained old rats are compared with their active controls of the same age which perform the same task with the preferred paw and do not acquire a new behavior. As to the behavior of trained and controls, it is clear that the former begin at a lower level of performance, but their rate of acquisition improves considerably with training and surpasses that of the controls after half the training time. Averaged over training time, the number of successful reaches is similar for trained and control rats. Since the animal gets and eats one protein pill for each successful reaching, both trained and controls thus get a similar amount of food. The level of successful reaches per training session of the old rats is less than half of the capacity of the young ones. On observing the behavior of the old rats it was clear that they became tired sooner than the young ones. On the other hand, their energy and rate of acquisition was impressive. REFERENCES Adey, W. R. (1967). Hippocampal states and functional reactions with cortico subcortical system in attention and learning. In W. R. Adey and T. Tokizane (Eds.), Structure and Function o f the Limbic System, Progress in Brain Research, Vol. 27, Elsevier, Amsterdam, pp. 228-245. Agranoff, B. W. (1970). Recent studies on the stages of memory formation in the goldfish. In W. L. Byrne (Ed.) Molecular Approaches to Learning and Memory, pp. 35-39. New York: Academic Press.
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