- Email: [email protected]
Alterations in synaptosomal calcium concentrations after rapid eye movement sleep deprivation in rats
~pergamon
PH:
Neuroscience Vol. 75, No.3, pp. 729-736, 1996 Copyright © 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain S0306-4522(96)OOl77-7 0306-4522/96 $15.00+0.00
ALTERATIONS IN SYNAPTOSOMAL CALCIUM CONCENTRATIONS AFTER RAPID EYE MOVEMENT SLEEP DEPRIVATION IN RATS B. N. MALLICK* and S. GULYANI School of Life Sciences, lawaharlal Nehru University, New Delhi, 110067, India Abstract-Rapid eye movement sleep deprivation alters behavioral and physiological, as well as cellular functioning and responsiveness. Since intracellular calcium concentration plays an important role in regulating cellular functions, it was hypothesized that such deprivation might induce changes in intracellular calcium concentration. Therefore, in this study, rats were deprived of rapid eye movement sleep by the flower-pot technique, and total, bound and free calcium concentrations were estimated in synaptosomal preparations from the cerebrum, cerebellum, brainstem, midbrain, pons and medulla. Rapid eye movement sleep deprivation was continued for two or four days and suitable control experiments were conducted to rule out the effects of non-specific factors. Total calcium concentration increased in the brainstem but showed a decrease in the cerebellum and cerebrum. After four days deprivation, the free calcium concentration always decreased; however, the bound calcium concentration decreased in the cerebrum and cerebellum but increased in the brainstem. After two days' deprivation, the medulla was the only region where the bound calcium increased while the free form decreased; only the free form decreased in the pons, while the midbrain was never affected. The results suggest that there was a net efflux of calcium in the cerebellum and cerebrum, but a net influx in the brainstem. The findings support our hypothesis and help to explain earlier observations. Since it is known that calcium plays an important role in cellular functioning, these changes in calcium concentration may be the underlying mechanism for rapid eye movement sleep deprivation-induced cellular expressions and behavior of neurons. Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Key words: bound-free-total calcium, deprivation, REM sleep, synaptosomes.
Rapid eye movement (REM) sleep is an integral part of sleep-wakefulness physiology. Although its precise mechanism of generation, functions and mechanism of action are unknown, evidence suggests that it is an essential physiological phenomenon to the extent that prolonged REM sleep deprivation may even be fatal. Deprivation studies have been used extensively to explore the physiological significance of REM sleep. Although the effects of varying periods of REM sleep deprivation on behavioral, t,47 psychologicaV physiological,4,6,36, neurotransmitter level 33 and biochemicaI9,10,15,21,42-45 processes have been investigated in some detail, studies on its effect on cellular physiology are relatively recent. Single neuronal responses were studied before, during and after REM sleep deprivation. The deprivation altered individual neuronal responsiveness to the stimulation to which they were responsive. After REM sleep deprivation, the REM-on and REM-off *To whom correspondence should be addressed. Abbreviations: DMSO, dimethylsulfoxide; EGTA, ethyl-
eneglycolbis(aminoethyl ether)tetra-acetate; Fura-2AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2(2'-amino-5'-methylphenoxy)-ethane- N,N,N,N-tetraacetic acid pentaacetoxymethyl ester; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; REM, rapid eye movement. 729
neurons increased and decreased, respectively, their firing rates.2° Similarly, neurons responsive to auditory stimulation showed a loss of inhibitory responses 18 when tested with auditory stimulation after the deprivation (compared to pre-deprivation response). REM sleep and its deprivation altered membrane bound ATPases,9,19 membrane fluidity,21 immediate early gene expression,31,39 mRNA levels,33 receptor sensitivity, densities 26 ,45 and hormonal secretion. 30 Alterations in each of these is potentially capable of inducing changes in cellular physiology and responses. Almost all the above mentioned cellular properties and responses are known to be affected by at least one common factor, the calcium ion concentration. The calcium concentration in a cell has been reported to be an important index and a regulatory factor in influencing membrane bound ATPase activities,50 gene expression, 7 neuronal firing rate,28 membrane fluidity, 5,16 receptor sensitivity and densities,28 and hormonal and neurotransmitter secretion.2 8 ,35 Hence, it was hypothesized that REM sleep deprivation may alter intracellular calcium levels which may induce changes in cellular physiology. Since synaptosomes provide a suitable system for studying the intracellular calcium concentration,27 total, free and bound synaptosomal calcium concentrations were estimated in the
B. N. Mallick and S. Gulyani
730
cerebrum, cerebellum, brainstem, midbrain, pons and medulla of non-REM sleep-deprived, REM sleep-deprived and control rats.
synaptosomes were resusupended (1 mg protein/ml) in HEPES buffer and stored on ice until the fluorescence was measured. Fluorescence measurement
EXPERIMENTAL PROCEDURES
Male Wistar inbred rats (235-275 g), obtained from the university animal facility were used in this study. The rats were deprived of REM sleep for 48 or 96 h by the standard flower-pot method using small platforms (diameter 6.5 cm) surrounded by a pool of water. To rule out the possibilities of non-specific effects, a group of rats was maintained on larger platforms (diameter 13.5 cm) projecting above a pool of water for periods identical to those of the small platform rats. In another group, REM sleep-deprived animals were allowed to recover from deprivation by maintaining them individually in dry rat cages for three days. At the end of the experiment, the REM sleep-deprived and control rats were killed by decapitation. The brains were rapidly removed and washed with chilled homogenizing buffer WC) before dissecting into the cerebrum, cerebellum, brainstern, midbrain, pons and medulla. 43 Isolation of synaptosomes
The tissue was homogenized in 0.32 M sucrose buffer (containing 5 mM HEPES, pH 7.4) and the synaptosomes were isolated following a standard method. s The homogenate was centrifuged at 3000 r.p.m. (1000 g) for 5 min. The supernatant was recentrifuged at 10,000 r.p.m. (12,000 g) for 20 min and the pellet thus obtained was layered on top of a discontinuous Ficoll (12%17.5%) gradient. This gradient was then centrifuged at 24,000 r.p.m. (68,000 g) for I h using a swing-out rotor. The synaptosomes were recovered at the 12%/7.5% interface. Following isolation, synaptosomes were washed in 20 volumes of ice-cold HEPES buffer containing 128 mM NaCI, 2.4 mM KCI, 1.2 mM MgS04 , 1.2 mM KH 2 P04 , 25 mM HEPES and 10 mM D-glucose. The synaptosomes were divided into two parts; one was used for free and the other for total calcium estimation. Estimation of total calcium concentration
The portion of synaptosomes used for total calcium estimation was resuspended in 1.5 ml of deionized water and freeze-thawed three times. Freeze-thawing was done by dipping this synaptosomal suspension in liquid nitrogen for I min followed by dipping into a water bath (60OC) for 10 min. The suspension was then centrifuged at 15,000 r.p.m. (20,000 g) for 15 min and an aliquot from the supernatant was assayed for total calcium by atomic absorption spectroscopy using a Perkin-Elmer model atomic absorption spectrometer. 38 The samples were assayed in a standard air-acetylene flame. The absorption wavelength was adjusted to the calcium resonance line at 4227 A (211.4 nm). Calcium standards were prepared by using calcium carbonate. Estimation offree calcium concentration
Washed synaptosomes were resuspended (5 mg protein/ ml) in HEPES buffer for loading with Fura-2AM (Sigma) dissolved in dimethylsulfoxide.(DMSO; 20% stock). Synaptosome and Fura-2AM suspensions were mixed so that the final loading concentrations were 2.5 mg protein/ml, 5 11M Fura-2AM and 0.2'Yo DMSO. 49 This mixture was incubated at 30°C for 30 min in a temperature-controlled shaking water bath. The loading was terminated by addition of 20 volumes of ice-cold HEPES buffer and centrifuged at 10,000 r.p.m. (15,000 g) for 15 min. Fura-2AM-Ioaded
The Fura-2AM ester is accumulated by the synaptosomes and subsequently enzymatically hydrolysed. The product of hydrolysis forms a complex with intracellular calcium to give a characteristic fluorescent signal. The Fura-2AM bound to the calcium ion is excited by light at 333 nm and Fura-2AM not bound to the calcium ion is excited at 375 nm. Thus, the fluorescence spectrum of Fura-2AM shifts depending on whether it is bound to calcium. Hence, the estimate of fluorescence is proportional to bound and free calcium levels. The ratio of fluorescence at the two wavelengths will be directly related to the ratio of the two forms of Fura-2AM and can therefore be used to calculate calcium concentrations. The advantage of this ratio approach is that the Fura-2AM concentration terms, path length and instrument sensitivity parameters cancel out at the time of measurement. The background value of the relative fluorescence ratio (R min ) due to free Fura-2AM and/or that associated with extracellular structures was corrected by estimating the Fura-2AM fluorescence signal in the presence of excess EGTA (2 mM prepared in Tris, pH 8.2). The maximum fluorescence ratio (R max ) was determined when Fura-2AM was maximally bound to calcium following lysis of synaptosomes with 0.1% Triton X-100 and addition of a saturating concentration of calcium (5 mM). This calibration was done with each batch of Fura-2AMloaded synaptosomes. In addition, pure Fura-2AM (unloaded with calcium) was incubated with DMSO for 30 min to obtain the value of autofluorescence. Correction of signals at 333 and 375 nm for autofluorescence preceded determination of the 333/375 nm fluorescence ratio. The free calcium concentration was estimated49 using 224 nm at the K d for Fura-2AM and the following equation: 2+
Free (Ca )i = K d X
(R - Rmin ) S}2 ) X -, (R max - R Sb2
where R is the fluorescence ratio of Fura-2AM (333/ 375 nm), Sf2 is the fluorescence of Fura-2AM at zero Ca 2 + (375 nm) and Sb2 is the fluorescence of Fura-2AM at Ca 2 + saturation (375 nm). All the measurements were done using an RF-540 spectrofluorimeter (Shimadzu), with excitation wavelengths set at 333 and 375 nm (5 mm slit width), and emission was monitored at 485 nm (5 mm slit width). Synaptosomes were incubated at room temperature for 2 min in a quartz cuvette in a sample chamber of the fluorimeter to allow equilibration to the appropriate temperature before monitoring the calcium concentrations. Estimation of bound calcium concentration
The difference between the total and free calcium concentrations was taken as the bound calcium concentration. The protein concentration in the brain tissue sample was estimated using the method of Lowry et al. 1? Data collected from six to 12 rats in each group were statistically analysed using ANOVA. The significance levels were determined by applying Scheffe's test. The means of calcium concentrations of the REM sleep-deprived group of rats were compared with those in free-moving control, large-platform control and recovery groups of rats. The large-platform and recovery values were also compared with those of the free-moving controls. RESULTS
The experimental rats on the small platform could not assume the relaxed posture necessary for REM
REM sleep deprivation and synaptosomal Ca 2 + concentrations Table I. Effect of rapid eye movement sleep deprivation on total synaptosomal calcium levels in different brain areas Cerebellum
Brainstem FM E4 E2 LP4 LP2 R4
0.358 0.739 0.475 0.397 0.373 0.351
± ± ± ± ± ±
0.031(8; 0.063** (6) 0.057***(6) 0.049(8) 0.097(4) 0.049(6)
0.643 0.532 0.572 0.614 0.670 0.617
± ± ± ± ± ±
0.021(8) 0.040***(6) 0.112(6) 0.014(8) 0.112(4) 0.021(6)
731 (~mol/mg
protein ± S.E.M.)
Cerebrum 0.533 0.331 0.597 0.495 0.472 0.514
± ± ± ± ± ±
0.096(8) 0.040***(6) 0.091(6) 0.074(8) 0.106(4) 0.045(6)
***p < 0.001; significant in comparison to free-moving control.
FM, free-moving control; other abbreviations as in Fig. 1. Numbers in parentheses are the number of observations in the respective group. Table 2. Effect of rapid eye movement sleep deprivation on total synaptosomal calcium levels in brainstem areas
FM E2 LP
(~mol/mg
protein ± S.E.M.)
Midbrain
Pons
Medulla
0.170 ± 0.027(6) 0.175 ± 0.072(5) 0.208 ± 0.097(4)
0.280 ± 0.057(6) 0.250 ± 0.031(5) 0.327 ± 0.086(5)
0.402 ± 0.100(6) 0.586 ± 0.089***(6) 0.472 ± 0.072(5)
***p < 0.001; significant in comparison to free-moving control. Abbreviations: as in Fig. I and Table 1. Numbers in parentheses are the number of observations in the respective group.
sleep (due to muscle atonia) without falling into the surrounding water, and were deprived of REM sleep. Rarely, the rats on the small platform were seen falling into the water after the first day of deprivation. However, during deprivation, any rat seen falling into the water during the second day onwards was not considered in this study. Some of the rats on the larger platforms were seen sleeping on the edges of the large platforms and waking up with a jerk, presumably at the onset of REM sleep. Those animals were also not used for large-platform control experiments. Although the experimental rats did not show any significant loss in body weight or lesion or ulceration on the skin or foot pad, they showed other signs of REM sleep deprivation, namely irritability, reduced grooming and extra sensitivity to external stimuli. Although no quantitative test was performed, the experimental rats fought with each other if left together and reacted aggressively to touch. However, these symptoms were absent in the other control and recovered rats.
Effects of rapid eye movement sleep deprivation on total calcium concentration in different brain areas The brainstem showed a significant increase (F1• 13 = 278; P < 0.001) in total calcium levels after four days of REM sleep deprivation, while the cerebellum (F1 ,1O = 91.81; P < 0,001) and cerebrum (F 1 ,1O = 122; P < 0,001) showed a significant decrease when compared to free-moving controls (Table I), Total calcium concentrations in the large-platform control group in all the brain regions studied were comparable to free-moving controls, Altered calcium
levels returned to normal after recovery, Two days' REM sleep deprivation also increased the total calcium level in the brainstem (F1 ,13 = 61.38; P < 0,001), although the cerebellum and the cerebrum remained unaffected (Table I). Changes in total calcium concentration in the brainstem areas
Since two days' REM sleep deprivation increased total calcium concentration in the brainstem only, it was further estimated in the midbrain, pons and medulla, This was attempted since pontomedullary regions, within the brainstem, are known to be responsible for REM sleep generation and also since other cellular parameters are reported to be affected first in those regions after REM sleep deprivation, It was found that the total calcium level increased significantly in the medulla only (F1,10 = 131.67; P < 0,001), while the midbrain and pons remained unaffected (Table 2), The total calcium concentrations in the midbrain, pons and medulla of the large-platform control rats also remained unaffected (Table 2), Effects ofrapid eye movement sleep deprivation on free calcium concentration in different brain areas The free calcium concentration decreased in the brainstem (Fig. I), cerebellum (Fig, 2) and cerebrum (Fig, 3) after two and four days' REM sleep deprivation compared to respective free-moving control groups of rats, The alteration after four days' deprivation was always higher than that at two days' deprivation. The significance levels after four days'
B. N. Mallick and S. Gulyani
732 Brain stem 120
Cerebellum
•••
10 0
50
...
l>{)
cco
...
0 -10
l>{)
~
5~
... i=l-.
-25
13
13
5~
Bound calcium
... i=l-.
10 0
Bound calcium 10
0
-50
_
L..-~'-"-
-25 l-E4
E2
LP4
LP2
•••
_
R4
Free calcium Fig. 1. Percentage changes (± S.E.M.) in free and bound synaptosomal calcium concentrations in the brainstem of REM sleep-deprived and control rats, as compared to free-moving control, taken as 100%. Abbreviations: E4 and E2, four and two days' REM sleep deprivation, respectively; LP4 and LP2, four and two days on large platform; R4, four days' recovery. Significance levels are: •P < 0.05; "p < 0.01; "'P < 0.001.
E4
E2
LP4
LP2
R4
Free calcium Fig. 2. Percentage changes (± S.E.M.) in free and bound synaptosomal Ca 2 + in REM sleep-deprived and control rat brain cerebellum compared to free-moving control, taken as baseline (100%). Abbreviations and significance levels are as in Fig. I.
Effects of rapid eye movement sleep deprivation on bound calcium concentration in different brain areas
deprivation were: cerebrum, F I ,IO = 605.5, P < 0.001; cerebellum, FI,ll = 79.33, P < 0.001; brainstem, F I 12 =264, P < 0.001. After two days' deprivation they were: cerebrum, F I 8 = 51.69, P < 0.01; cerebellum, F I ,IO = 12.44, P < 0.01; brainstem, F I 10 =21.26, P < 0.01. In the large-platform control gr~up of animals, free calcium levels remained unaffected in all the regions, except the cerebellum (FI 10 = 31.83; P < 0.01). All the altered calcium lev~ls returned to normal after recovery.
The bound calcium concentration increased in the brainstem (Fig. I; F I ,13 = 278; P < 0.001), but decreased in the cerebellum (Fig. 2; Fl,lo =91.81; P < 0.001) and the cerebrum (Fig. 3; FI,lO = 122; P < 0.001) after four days of REM sleep deprivation. After two days' deprivation it increased in the brainstem only (Fig. I), while it remained unaffected in the cerebellum and the cerebrum. The altered levels returned to normal after recovery. The bound calcium levels were not affected in the large-platform control group of rats.
Changes in free calcium concentration in the brainstem areas
Changes in bound calcium concentration in brainstem areas
Free calcium concentration was estimated in freemoving control, large-platform control and two-day REM sleep-deprived rats. The latter group of rats showed a significant decrease in free calcium level in the medulla (Fig. 4; F I ,5 = 412.24; P < 0.001) and the pons (Fig. 4; F I ,5 = 933.62; P < 0.001) as compared to that of the respective free-moving controls. The midbrain (Fig. 4) did not show any significant change. Since the altered free calcium levels returned to normal in the recovery group after four days' REM sleep deprivation, a recovery study after shorter REM sleep deprivation (two days) was not undertaken.
The bound calcium concentration increased in the medulla (Fig. 4; Fl,lo = 131.67; P < 0.001). However, it remained unaffected in the pons (Fig. 4) and the midbrain (Fig. 4). It was not 'significantly affected in the large-platform control group of animals either. Ratio of changes in bound to free calcium concentration
The ratio of changes in bound to free calcium levels was close to unity in the cerebellum and cerebrum in all control and experimental groups of rats (Fig. 5). However, in the case of the brainstem
733
REM sleep deprivation and synaptosomal Ca2+ concentrations Cerebrum
150
•••
Medulla
20
100
o SO
0
"O§ll
-SO
'ti
'~5
"
Pons
150
"c:
Oll
BOWld calcium
100
20
..c:u'" ~ I:l..
SO
0
= "
I:l..
"
0
Midbrain
150
-so
L..--'!.l!.il!....
E4
_
E2
LP4
LP2
R4
100
Free calcium Fig. 3. Percentage changes (± S.E.M.) in free and bound synaptosomal Ca 2 + in rat brain cerebrum in REM sleepdeprived and control rats as compared to free-moving control, taken as 100%. Abbreviations and significance levels are as in Fig. l.
SO
0
BOWld calcium
after four days' REM sleep deprivation, there were disproportionate changes in bound to free calcium levels. The bound form increased, while the free form decreased. DISCUSSION
Behaviorally, the experimental rats in this study expressed signs of REM sleep deprivation. I ,47 The total, free and bound calcium levels were estimated in synaptosomal preparations from anatomically identified brain regions. After REM sleep deprivation, there was a decrease in all forms of calcium concentration in the cerebrum and the cerebellum. However, for the entire brainstem and medulla, although there were decreases in the free form, the total and bound calcium concentrations increased. The flower-pot method was chosen in this study for REM sleep deprivation because it has been most widely used II for investigating the effects of REM sleep deprivation on psychological, physiological, biochemical and behavioral parameters. This method has been shown to induce maximum REM sleep deprivation without significantly affecting non-REM sleep. The platform sizes were selected based on an earlier report,48 Almost total loss of REM sleep by this method in cats 20 and in rats 23 ,46 has been reported. However, there is a non-significant loss in non-REM sleep, especially within 24 h of deprivation. Since the large-platform control animals also
Free calcium
Fig. 4. Changes in bound and free calcium concentrations in the medulla, pons and midbrain in two-day REM sleepdeprived and control groups of rats. The values were compared to free-moving control (FM), taken as baseline. Abbreviations and significance levels are as in Fig. 1.
experienced a loss in non-REM sleep similar to that of small-platform animals,23 and the effect of deprivation was observed in the small-platform group of rats only (except in the case of free calcium concentration in the cerebellum), it is reasonable to accept that the effects observed in the small-platform rats were due to REM sleep deprivation and not to a small loss in non-REM sleep. Further, it was also found that the REM sleep deprivation-induced alterations in calcium concentrations returned to normal levels in the recovery group of rats. Rapid eye movement sleep deprivation and changes in synaptosomal calcium levels
In the normal situation, more than 99% of total intracellular calcium exists in bound form,2 supporting the observations in this study. A significant decrease in total, free and bound calcium in the cerebrum and the cerebellum suggests a net efflux of calcium after REM sleep deprivation. Although it is difficult to comment on the precise cellular mechanism of this efflux, the following possibility, via stimulation of a highly sensitive calcium pump, fits in and
B. N. Mallick and S. Gulyani
734
8
°u'"
3
" u
~I '" ~ ~ u.. '"
e---e Brain stem
e'\.
Cerebellum _ _ Cerebmm
6---{;
'\.
2
bJJ
e
cOJ e
•--.---.• ~ .•
o
0'-:-:-:-------::,.,------,---------
-=u''""
15-
i
e<:
~
~
~
~
M
Fig. 5. The ratio of percentage change in bound synaptosomal concentration to that in free synaptosomal concentration in different brain regions in control and experimental rats. Abbreviations are as in Fig. I. helps to explain the situation. The calcium concentration in a cell is known to be regulated by an Na-Ca exchange pump which, though it is less sensitive than the Ca pump, has a greater capacity for exchanging calcium. 34 This exchange protein functions in coordination with the Na-K pump. The Na+ gradient maintained by Na-K ATPase is used by the Na-Ca exchange pump to release Ca 2+.J4,35 It was reported earlier that REM sleep deprivation increased Na-K ATPase activity.9 This increased Na-K ATPase activity will induce a steep concentration gradient, resulting in a net influx of Na+ and consequently efflux of Ca 2+, This would thus cause a net decrease in intracellular Ca 2+. However, this explanation does not hold true in the brainstem where, unlike the cerebrum and the cerebellum, although there was a decrease in free form, the bound form increased. This study does not allow us to comment as to whether Ca 2+ moves from one compartment to the other. There was a decrease in total, free and bound calcium concentrations in the cerebellum and the cerebrum. In these areas, since there was a proportionate decrease in the free and bound forms of calcium (Fig. 5), it is likely that there would be a net efflux of Ca2+ from the neuron. Consequently, it is unlikely that there would be conversion of Ca 2+ from one form to the other in the cerebellum and cerebrum. However, in the brainstem the bound calcium, which forms the major pool in the neuron,2 increased while the free form, which is effectively responsible for neuronal functions, decreased after deprivation. The ratio of percentage changes in bound to free calcium concentrations was maintained at a fairly constant level in the cerebellum and the cerebrum, but not in the brainstem (Fig. 5). This proportionate decrease in total, bound and free calcium in the cerebellum and cerebrum may be explained by the fact that the ratio of intracellular bound to free calcium is very strictly regulated. 2.35 Nevertheless, in the brainstem the bound form increased while the free form decreased. Thus, the results suggest that the homeostatic mechanism which maintains the ratio of intracellular bound to
free calcium was not affected in the cerebrum and the cerebellum, though it failed in the brainstem, after REM sleep deprivation. The relative changes of calcium concentrations were considered and not the absolute values, since the former is more important for cell functions and, unlike the latter, remains virtually unaffected by the size of neurons and mass of area studied. The homeostatic mechanism failed first in the brainstem possibly because this area is involved in REM sleep generation, as discussed below. It was shown previously that other factors were also affected first in the brainstem after REM sleep deprivation. 9,42,43 The pons and the medulla were the first sites to be affected, while the midbrain remained unaffected. The bound calcium concentration increased only in the medulla, while free calcium decreased both in the medulla and in the pons, These differential responses of calcium concentrations in the brainstern regions assume significance since the pontomedullary regions are responsible for REM sleep generation. 40 It is important to note that after REM sleep deprivation Na-K ATPase,9 acetylcholinesterase 42 and monoamine oxidase43 were also affected first in the pontomedullary regions. While extensive studies are needed for conclusive evidence, the possible role of norepinephrine as suggested earlier for REM sleep deprivation-induced increase in Na-K ATPase 10 activity also holds true in this case. The locus coeruleus norepinephrinergic REM-off neurons cease firing during REM sleep l3 and have monosynaptic projections to the medulla. 37 During the normal situation, although these REM-off neurons cease firing during REM sleep, they keep firing continuously during REM sleep deprivation. 20 Hence, during REM sleep deprivation there would be a continuous supply and a relative increase in norepinephrine locally in the projected area, the medulla in this instance. This may also be supported by the recent report that mild stimulation of the locus coeruleus, which is likely to increase norepinephrine in the projected site, induced an effect similar to that of REM sleep deprivation. 41 This local increase in the level of norepinephrine is likely to enhance calcium influx. 24 However, a possible argument against this explanation would be that locus coeruleus neurons also project to the cortex, but similar changes were not observed in the cerebrum. This is possibly because the cortex alone has not been studied; instead, the entire cerebrum was taken, where the effect is likely to be diluted or reduced (as obtained). It would also depend on the proportion of projections. This is further substantiated by the observation that the effects were opposite in the pons, where the locus coeruleus neurons were located, and due to constant firing by REM-off neurons the norepinephrine is expected to be depleted. In addition, this increase could also be due to selective local presence of neurohormone and/or sleep-inducing peptides 12 in the medulla.
REM sleep deprivation and synaptosomal Ca 2 + concentrations
Physiological significance
An alteration in calcium level would modulate the basic physiology and functioning of a neuron. 32 ,35 Modulation of neuronal excitability, firing rate and nerve impulse conduction due to changes in Ca2+ are well known. 28 ,35 It has been reported that intracellular calcium plays an important role in memory processing, 2 gene expression, 7 receptor sensitivities, receptor-mediated cellular functioning and enzyme activities,35 and membrane fluidity. 5, 16 Independent and isolated studies have shown that all these parameters, including calcium concentration in this study, are affected after REM sleep deprivation. Thus, pending further direct evidence, based on the indirect evidence available it is tempting to suggest that changes in intracellular calcium concentration could be at least one of the primary underlying
735
mechanisms for REM sleep deprivation-induced alterations in overall physiological responses of a neuron, and ultimately the functioning of the nervous system. Finally, the role of calcium and membrane fluidity in maintaining cellular integrity, functioning and longevity has been reported;5,22,29,35 these results, along with the results of this study, would certainly go a long way to help explain the underlying basic molecular mechanism of the role of REM sleep in cellular growth and maturation. 14 ,25 Acknowledgements-The financial support received from the Indian Council of Medical Research is duly acknowledged. We are grateful to Prof. V. Subramaniam, School of Environmental Sciences, Jawaharlal Nehru University for kindly allowing us to use the atomic absorption spectrometer.
REFERENCES
Albert l. B. (1975) REM sleep deprivation. Bio!. Psychiat. 10, 341~351. 2. Borle A. B. (1981) Control, modulation and regulation of cell calcium. Rev. Physiol. Biochem. Pharmac. 90, 13-153. 3. Clemes S. and Dement W. C. (1967) Effect of REM sleep deprivation on psychological functioning. J. nerv. ment. Dis. 144, 485--489. 4. Cohen H. B. and Dement W. C. (1965) Sleep: changes in threshold to electroconvulsive shQck in rats after deprivation of "paradoxical" phase. Science 150, 1318~1319. 5. Daniell L. C. and Harris R. A. (1988) Neuronal intracellular calcium concentrations are altered by anesthetics: relationship to membrane fluidization. J. Pharmac. expo Ther. 245, 1~7. 6. Fishbein W. and Gutwein B. M. (1977) Paradoxical sleep and memory storage processes. Behav. Bioi. 19,425--464. 7. Ginty D. J., Bading H. and Greenberg M. E. (1992) Trans-synaptic regulation of gene expression. Curro Opin. Neurobiol. 3,312-316. 8. Gordon-Weeks P. R. (1987) Isolation of synaptosomes, growth cones and their subcellular components. In Neurochemistry: A Practical Approach (eds Turner J. A. and Bachlard S. H.), pp. 1~25. IRL Press, Oxford. 9. Gulyani S. and Mallick B. N. (1993) Effect of rapid eye movement sleep deprivation on rat brain Na-K ATPase activity. J. Sleep Res. 2, 45~50. 10. Gulyani S. and Mallick B. N. (1995) Possible mechanism of rapid eye movement sleep deprivation induced increase in Na-K ATPase activity. Neuroscience 64, 255-260. II. Hicks R. A., Okuda A. and Thomsen D. (1977) Depriving rats of REM sleep: the identification of a methodological problem. Am. J. Psychol. 90, 95~102. 12. Inoue S. (1989) Biology of Sleep Substances. CRC Press, Boca Raton, FL. 13. Jacobs B. L. (1986) Single unit activity of locus coeruleus neurons in behaving animals. Prog. Neurobio!. 27, 183-194. 14. Krueger J. M. and Obal F. Jr (1993) A neuronal group theory of sleep function. J. Sleep Res. 2,63-69. 15. Kushida A. c., Bergmann B. M. and Rechtschaffen A. (1989) Sleep deprivation in rat: IV. Paradoxical sleep deprivation. Sleep 12, 22-30. 16. Lebel C. P. and Schatz R. A. (1990) Altered synaptosomal phospholipid metabolism after toluene: possible relationship with membrane fluidity, Na-K adenosine triphosphatase and phospholipid methylation. J. Pharmac. expo Ther. 253, 1189~1l97. 17. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with Folin phenol reagent. J. bioi. Chem. 193, 265~275. 18. Mallick B. N., Fahringer H., Wu M. F. and Siegel J. M. (1991) REM sleep deprivation reduces auditory evoked inhibition of dorsolateral pontine neurons. Brain Res. 552, 333-337. 19. Mallick B. N. and Gulyani S. (1993) Rapid eye movement sleep deprivation increases chloride-sensitive Mg-ATPase activity in the rat brain. Pharmac. Biochem. Behav. 45, 359-362. 20. Mallick B. N., Siegel J. M. and Fahringer H. (1989) Changes in pontine unit activity with REM sleep deprivation. Brain Res. 515, 94-98. 21. Mallick B. N., Thakkar M. and Gangabhagirathi R. (1995) Rapid eye movement sleep deprivation decreases membrane fluidity in the rat brain. Neurosci. Res. 22, 117-122. 22. McConkey D. J., Hartzell P., Nicoterra P. and Orrenius S. (1989) Calcium activated DNA fragmentation kills immature thymocytes. Fedn Proc. Fedn Am. Socs expo Bioi. 3, 1843-1848. 23. Mendelson W. B. (1974) The flower pot technique of rapid eye movement (REM) sleep deprivation. Pharmac. Biochem. Behav. 2, 553-556. 24. Minneman K. P. (1981) Adrenergic receptor molecules. In Neurotransmitter Receptors (eds Yamamura l. H. and Enna J. S.), pp. 187-257. Chapman & Hall, London. 25. Mirmiran M. and Van Someren E. (1993) The importance of REM sleep for brain maturation. J. Sleep Res. 2, I.
188~192.
26.
Mogilnicka E., Przewlocka B., Van-Iuijtelaar E. L. J. M., Klimek V. and Coenen M. L. (1986) Effects of REM sleep deprivation on central alpha-I and beta-adrenoceptors in rat brain. Pharmac. Biochem. Behav. 25,329-332.
736 27. 28. 29. 30. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
B. N. Mallick and S. Gulyani Nicholls D. G., Sihra T. S. and Sanchez-Prieto J. (1987) The role of plasma membrane and intracellular organelles in synaptosomal calcium regulation. In Cell Calcium and the Control ofMembrane Transport (eds Mandel J. L. and Eaton C. D.), pp. 32-42. Rockefeller University Press, New York. Nicoll R. A. (1988) Neurotransmitters and ion channels. Science 241, 545-551. Nicotera P., McConkey D., Svensson S. A., Bellomo G. and Orrenius S. (1988) Correlation between cytosolic Ca 2 + concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology 52, 55-63. Obal F. Jr, Fang J., Payne L. C. and Krueger J. M. (1995) Growth-horrnone-releasing hormone mediates the sleep promoting activity of interleukin-I in rats. Neuroendocrinology 61, 559-565. O'Hara B. F., Young K. A., Watson F. L., Heller H. C. and KilduffT. S. (1993) Immediate early gene expression in brain during sleep deprivation: preliminary observations. Sleep 16, 1-7. Penner R., Fasolato C. and Hoth M. (1993) Calcium influx and its control by calcium release. Curro Opin. Neurobiol. 3,368-374. Porkka-Heiskanen T., Smith S. E., Taira T., Urban J. H., Levine J. E., Turek F. W. and Stenberg D. (1995) Noradrenergic activity in rat brain during rapid eye movement sleep deprivation and rebound sleep. Am. J. Physiol. 268, RI456-1463. Rahamimoff H. (1990) Na+-Ca 2 + exchanger: the elusive protein. Curro Topics Cell Regul. 31,241-271. Rasmussen H. and Rasmussen J. E. (1990) Calcium as intracellular messenger: from simplicity to complexity. Curro Topics Cell Regul. 31, 1-109. Rechtschaffen A., Bergmann B. M., Everson C. A., Kushida C. A. and Gilliland M. A. (1989) Sleep deprivation in the rat: integration and discussion of the findings. Sleep 12, 68-87. Sakai K. (1985) Anatomical and physiological basis of paradoxical sleep. In Brain Mechanisms of Sleep (eds McGinty D. J., Drucker-Colin R., Morrison A. and Parmeggiani P. L.), pp. 111-138. Raven Press, New York. Schmidt W. K. and Way E. L. (1979) Direct assay for calcium in brain homogenates and synaptosomal pellets. J. Neurochem. 32, 1095-1098. Shiromani P. J., KilduffT. S., Bloom F. E. and McCarley R. W. (1992) Cholinergically induced REM sleep triggers Fos-likeimmunoreactivity in dorsolateral pontine regions associated with REM sleep. Brain Res. 580, 351-357. Siegel J. M. (1985) Pontomedullary interactions in the generation of REM sleep. In Brain Mechanisms of Sleep (eds McGinty D. J., Drucker-Colin R., Morrison A. and Parrneggiani P. L.), pp. 157-174. Raven Press, New York. Singh S. and Mallick B. N. (1996) Mild electrical stimulation of pontine tegmentum around locus coeruleus reduces rapid eye movement sleep in rats. Neurosci. Res. 24, 227-235. Thakkar M. and Mallick B. N. (1991) Effect of REM sleep deprivation on rat brain acetylcholinesterase. Pharmac. Biochem. Behav. 39,211-214. Thakkar M. and Mallick B. N. (1993) Effect of rapid eye movement sleep deprivation on rat brain monoamine oxidases. Neuroscience 55, 677-683. Thakkar M. and Mallick B. N. (1993) Rapid eye movement sleep deprivation induced changes in glucose metabolic enzymes in rat brain. Sleep 16, 691-694. Tsai L. L., Mergmann B. M., Perry B. D. and Rechtschaffen A. (1994) Effects of chronic sleep deprivation on central cholinergic receptors in rat brain. Brain Res. 642, 95-103. Van Luijtelaar E. L. J. M. and Coenen A. M. L. (1986) Electrophysiological evaluation of three paradoxical sleep deprivation techniques in rats. Physiol. Behav. 36, 603-609. Vogel G. W. (1975) A review of REM sleep deprivation. Archs gen. Psychiat. 32,749-761. Yanik G. and Radulovacki M. (1987) REM sleep deprivation up-regulates adenosine Al receptors. Brain Res. 402, 362-364. Yates S. L., Fluhler E. N. and Lippiello P. M. (1992) Advances in the use of the fluoresecent probe fura-2 for the estimation of intrasynaptosomal calcium. J. Neurosci. Res. 32, 255-260. Yingst D. R. (1988) Modulation of Na,K ATPase by Ca and intracellular proteins. A. Rev. Physiol. 50,291-303. (Accepted 20 March 1996)
PH:
Neuroscience Vol. 75, No.3, pp. 729-736, 1996 Copyright © 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain S0306-4522(96)OOl77-7 0306-4522/96 $15.00+0.00
ALTERATIONS IN SYNAPTOSOMAL CALCIUM CONCENTRATIONS AFTER RAPID EYE MOVEMENT SLEEP DEPRIVATION IN RATS B. N. MALLICK* and S. GULYANI School of Life Sciences, lawaharlal Nehru University, New Delhi, 110067, India Abstract-Rapid eye movement sleep deprivation alters behavioral and physiological, as well as cellular functioning and responsiveness. Since intracellular calcium concentration plays an important role in regulating cellular functions, it was hypothesized that such deprivation might induce changes in intracellular calcium concentration. Therefore, in this study, rats were deprived of rapid eye movement sleep by the flower-pot technique, and total, bound and free calcium concentrations were estimated in synaptosomal preparations from the cerebrum, cerebellum, brainstem, midbrain, pons and medulla. Rapid eye movement sleep deprivation was continued for two or four days and suitable control experiments were conducted to rule out the effects of non-specific factors. Total calcium concentration increased in the brainstem but showed a decrease in the cerebellum and cerebrum. After four days deprivation, the free calcium concentration always decreased; however, the bound calcium concentration decreased in the cerebrum and cerebellum but increased in the brainstem. After two days' deprivation, the medulla was the only region where the bound calcium increased while the free form decreased; only the free form decreased in the pons, while the midbrain was never affected. The results suggest that there was a net efflux of calcium in the cerebellum and cerebrum, but a net influx in the brainstem. The findings support our hypothesis and help to explain earlier observations. Since it is known that calcium plays an important role in cellular functioning, these changes in calcium concentration may be the underlying mechanism for rapid eye movement sleep deprivation-induced cellular expressions and behavior of neurons. Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Key words: bound-free-total calcium, deprivation, REM sleep, synaptosomes.
Rapid eye movement (REM) sleep is an integral part of sleep-wakefulness physiology. Although its precise mechanism of generation, functions and mechanism of action are unknown, evidence suggests that it is an essential physiological phenomenon to the extent that prolonged REM sleep deprivation may even be fatal. Deprivation studies have been used extensively to explore the physiological significance of REM sleep. Although the effects of varying periods of REM sleep deprivation on behavioral, t,47 psychologicaV physiological,4,6,36, neurotransmitter level 33 and biochemicaI9,10,15,21,42-45 processes have been investigated in some detail, studies on its effect on cellular physiology are relatively recent. Single neuronal responses were studied before, during and after REM sleep deprivation. The deprivation altered individual neuronal responsiveness to the stimulation to which they were responsive. After REM sleep deprivation, the REM-on and REM-off *To whom correspondence should be addressed. Abbreviations: DMSO, dimethylsulfoxide; EGTA, ethyl-
eneglycolbis(aminoethyl ether)tetra-acetate; Fura-2AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2(2'-amino-5'-methylphenoxy)-ethane- N,N,N,N-tetraacetic acid pentaacetoxymethyl ester; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; REM, rapid eye movement. 729
neurons increased and decreased, respectively, their firing rates.2° Similarly, neurons responsive to auditory stimulation showed a loss of inhibitory responses 18 when tested with auditory stimulation after the deprivation (compared to pre-deprivation response). REM sleep and its deprivation altered membrane bound ATPases,9,19 membrane fluidity,21 immediate early gene expression,31,39 mRNA levels,33 receptor sensitivity, densities 26 ,45 and hormonal secretion. 30 Alterations in each of these is potentially capable of inducing changes in cellular physiology and responses. Almost all the above mentioned cellular properties and responses are known to be affected by at least one common factor, the calcium ion concentration. The calcium concentration in a cell has been reported to be an important index and a regulatory factor in influencing membrane bound ATPase activities,50 gene expression, 7 neuronal firing rate,28 membrane fluidity, 5,16 receptor sensitivity and densities,28 and hormonal and neurotransmitter secretion.2 8 ,35 Hence, it was hypothesized that REM sleep deprivation may alter intracellular calcium levels which may induce changes in cellular physiology. Since synaptosomes provide a suitable system for studying the intracellular calcium concentration,27 total, free and bound synaptosomal calcium concentrations were estimated in the
B. N. Mallick and S. Gulyani
730
cerebrum, cerebellum, brainstem, midbrain, pons and medulla of non-REM sleep-deprived, REM sleep-deprived and control rats.
synaptosomes were resusupended (1 mg protein/ml) in HEPES buffer and stored on ice until the fluorescence was measured. Fluorescence measurement
EXPERIMENTAL PROCEDURES
Male Wistar inbred rats (235-275 g), obtained from the university animal facility were used in this study. The rats were deprived of REM sleep for 48 or 96 h by the standard flower-pot method using small platforms (diameter 6.5 cm) surrounded by a pool of water. To rule out the possibilities of non-specific effects, a group of rats was maintained on larger platforms (diameter 13.5 cm) projecting above a pool of water for periods identical to those of the small platform rats. In another group, REM sleep-deprived animals were allowed to recover from deprivation by maintaining them individually in dry rat cages for three days. At the end of the experiment, the REM sleep-deprived and control rats were killed by decapitation. The brains were rapidly removed and washed with chilled homogenizing buffer WC) before dissecting into the cerebrum, cerebellum, brainstern, midbrain, pons and medulla. 43 Isolation of synaptosomes
The tissue was homogenized in 0.32 M sucrose buffer (containing 5 mM HEPES, pH 7.4) and the synaptosomes were isolated following a standard method. s The homogenate was centrifuged at 3000 r.p.m. (1000 g) for 5 min. The supernatant was recentrifuged at 10,000 r.p.m. (12,000 g) for 20 min and the pellet thus obtained was layered on top of a discontinuous Ficoll (12%17.5%) gradient. This gradient was then centrifuged at 24,000 r.p.m. (68,000 g) for I h using a swing-out rotor. The synaptosomes were recovered at the 12%/7.5% interface. Following isolation, synaptosomes were washed in 20 volumes of ice-cold HEPES buffer containing 128 mM NaCI, 2.4 mM KCI, 1.2 mM MgS04 , 1.2 mM KH 2 P04 , 25 mM HEPES and 10 mM D-glucose. The synaptosomes were divided into two parts; one was used for free and the other for total calcium estimation. Estimation of total calcium concentration
The portion of synaptosomes used for total calcium estimation was resuspended in 1.5 ml of deionized water and freeze-thawed three times. Freeze-thawing was done by dipping this synaptosomal suspension in liquid nitrogen for I min followed by dipping into a water bath (60OC) for 10 min. The suspension was then centrifuged at 15,000 r.p.m. (20,000 g) for 15 min and an aliquot from the supernatant was assayed for total calcium by atomic absorption spectroscopy using a Perkin-Elmer model atomic absorption spectrometer. 38 The samples were assayed in a standard air-acetylene flame. The absorption wavelength was adjusted to the calcium resonance line at 4227 A (211.4 nm). Calcium standards were prepared by using calcium carbonate. Estimation offree calcium concentration
Washed synaptosomes were resuspended (5 mg protein/ ml) in HEPES buffer for loading with Fura-2AM (Sigma) dissolved in dimethylsulfoxide.(DMSO; 20% stock). Synaptosome and Fura-2AM suspensions were mixed so that the final loading concentrations were 2.5 mg protein/ml, 5 11M Fura-2AM and 0.2'Yo DMSO. 49 This mixture was incubated at 30°C for 30 min in a temperature-controlled shaking water bath. The loading was terminated by addition of 20 volumes of ice-cold HEPES buffer and centrifuged at 10,000 r.p.m. (15,000 g) for 15 min. Fura-2AM-Ioaded
The Fura-2AM ester is accumulated by the synaptosomes and subsequently enzymatically hydrolysed. The product of hydrolysis forms a complex with intracellular calcium to give a characteristic fluorescent signal. The Fura-2AM bound to the calcium ion is excited by light at 333 nm and Fura-2AM not bound to the calcium ion is excited at 375 nm. Thus, the fluorescence spectrum of Fura-2AM shifts depending on whether it is bound to calcium. Hence, the estimate of fluorescence is proportional to bound and free calcium levels. The ratio of fluorescence at the two wavelengths will be directly related to the ratio of the two forms of Fura-2AM and can therefore be used to calculate calcium concentrations. The advantage of this ratio approach is that the Fura-2AM concentration terms, path length and instrument sensitivity parameters cancel out at the time of measurement. The background value of the relative fluorescence ratio (R min ) due to free Fura-2AM and/or that associated with extracellular structures was corrected by estimating the Fura-2AM fluorescence signal in the presence of excess EGTA (2 mM prepared in Tris, pH 8.2). The maximum fluorescence ratio (R max ) was determined when Fura-2AM was maximally bound to calcium following lysis of synaptosomes with 0.1% Triton X-100 and addition of a saturating concentration of calcium (5 mM). This calibration was done with each batch of Fura-2AMloaded synaptosomes. In addition, pure Fura-2AM (unloaded with calcium) was incubated with DMSO for 30 min to obtain the value of autofluorescence. Correction of signals at 333 and 375 nm for autofluorescence preceded determination of the 333/375 nm fluorescence ratio. The free calcium concentration was estimated49 using 224 nm at the K d for Fura-2AM and the following equation: 2+
Free (Ca )i = K d X
(R - Rmin ) S}2 ) X -, (R max - R Sb2
where R is the fluorescence ratio of Fura-2AM (333/ 375 nm), Sf2 is the fluorescence of Fura-2AM at zero Ca 2 + (375 nm) and Sb2 is the fluorescence of Fura-2AM at Ca 2 + saturation (375 nm). All the measurements were done using an RF-540 spectrofluorimeter (Shimadzu), with excitation wavelengths set at 333 and 375 nm (5 mm slit width), and emission was monitored at 485 nm (5 mm slit width). Synaptosomes were incubated at room temperature for 2 min in a quartz cuvette in a sample chamber of the fluorimeter to allow equilibration to the appropriate temperature before monitoring the calcium concentrations. Estimation of bound calcium concentration
The difference between the total and free calcium concentrations was taken as the bound calcium concentration. The protein concentration in the brain tissue sample was estimated using the method of Lowry et al. 1? Data collected from six to 12 rats in each group were statistically analysed using ANOVA. The significance levels were determined by applying Scheffe's test. The means of calcium concentrations of the REM sleep-deprived group of rats were compared with those in free-moving control, large-platform control and recovery groups of rats. The large-platform and recovery values were also compared with those of the free-moving controls. RESULTS
The experimental rats on the small platform could not assume the relaxed posture necessary for REM
REM sleep deprivation and synaptosomal Ca 2 + concentrations Table I. Effect of rapid eye movement sleep deprivation on total synaptosomal calcium levels in different brain areas Cerebellum
Brainstem FM E4 E2 LP4 LP2 R4
0.358 0.739 0.475 0.397 0.373 0.351
± ± ± ± ± ±
0.031(8; 0.063** (6) 0.057***(6) 0.049(8) 0.097(4) 0.049(6)
0.643 0.532 0.572 0.614 0.670 0.617
± ± ± ± ± ±
0.021(8) 0.040***(6) 0.112(6) 0.014(8) 0.112(4) 0.021(6)
731 (~mol/mg
protein ± S.E.M.)
Cerebrum 0.533 0.331 0.597 0.495 0.472 0.514
± ± ± ± ± ±
0.096(8) 0.040***(6) 0.091(6) 0.074(8) 0.106(4) 0.045(6)
***p < 0.001; significant in comparison to free-moving control.
FM, free-moving control; other abbreviations as in Fig. 1. Numbers in parentheses are the number of observations in the respective group. Table 2. Effect of rapid eye movement sleep deprivation on total synaptosomal calcium levels in brainstem areas
FM E2 LP
(~mol/mg
protein ± S.E.M.)
Midbrain
Pons
Medulla
0.170 ± 0.027(6) 0.175 ± 0.072(5) 0.208 ± 0.097(4)
0.280 ± 0.057(6) 0.250 ± 0.031(5) 0.327 ± 0.086(5)
0.402 ± 0.100(6) 0.586 ± 0.089***(6) 0.472 ± 0.072(5)
***p < 0.001; significant in comparison to free-moving control. Abbreviations: as in Fig. I and Table 1. Numbers in parentheses are the number of observations in the respective group.
sleep (due to muscle atonia) without falling into the surrounding water, and were deprived of REM sleep. Rarely, the rats on the small platform were seen falling into the water after the first day of deprivation. However, during deprivation, any rat seen falling into the water during the second day onwards was not considered in this study. Some of the rats on the larger platforms were seen sleeping on the edges of the large platforms and waking up with a jerk, presumably at the onset of REM sleep. Those animals were also not used for large-platform control experiments. Although the experimental rats did not show any significant loss in body weight or lesion or ulceration on the skin or foot pad, they showed other signs of REM sleep deprivation, namely irritability, reduced grooming and extra sensitivity to external stimuli. Although no quantitative test was performed, the experimental rats fought with each other if left together and reacted aggressively to touch. However, these symptoms were absent in the other control and recovered rats.
Effects of rapid eye movement sleep deprivation on total calcium concentration in different brain areas The brainstem showed a significant increase (F1• 13 = 278; P < 0.001) in total calcium levels after four days of REM sleep deprivation, while the cerebellum (F1 ,1O = 91.81; P < 0,001) and cerebrum (F 1 ,1O = 122; P < 0,001) showed a significant decrease when compared to free-moving controls (Table I), Total calcium concentrations in the large-platform control group in all the brain regions studied were comparable to free-moving controls, Altered calcium
levels returned to normal after recovery, Two days' REM sleep deprivation also increased the total calcium level in the brainstem (F1 ,13 = 61.38; P < 0,001), although the cerebellum and the cerebrum remained unaffected (Table I). Changes in total calcium concentration in the brainstem areas
Since two days' REM sleep deprivation increased total calcium concentration in the brainstem only, it was further estimated in the midbrain, pons and medulla, This was attempted since pontomedullary regions, within the brainstem, are known to be responsible for REM sleep generation and also since other cellular parameters are reported to be affected first in those regions after REM sleep deprivation, It was found that the total calcium level increased significantly in the medulla only (F1,10 = 131.67; P < 0,001), while the midbrain and pons remained unaffected (Table 2), The total calcium concentrations in the midbrain, pons and medulla of the large-platform control rats also remained unaffected (Table 2), Effects ofrapid eye movement sleep deprivation on free calcium concentration in different brain areas The free calcium concentration decreased in the brainstem (Fig. I), cerebellum (Fig, 2) and cerebrum (Fig, 3) after two and four days' REM sleep deprivation compared to respective free-moving control groups of rats, The alteration after four days' deprivation was always higher than that at two days' deprivation. The significance levels after four days'
B. N. Mallick and S. Gulyani
732 Brain stem 120
Cerebellum
•••
10 0
50
...
l>{)
cco
...
0 -10
l>{)
~
5~
... i=l-.
-25
13
13
5~
Bound calcium
... i=l-.
10 0
Bound calcium 10
0
-50
_
L..-~'-"-
-25 l-E4
E2
LP4
LP2
•••
_
R4
Free calcium Fig. 1. Percentage changes (± S.E.M.) in free and bound synaptosomal calcium concentrations in the brainstem of REM sleep-deprived and control rats, as compared to free-moving control, taken as 100%. Abbreviations: E4 and E2, four and two days' REM sleep deprivation, respectively; LP4 and LP2, four and two days on large platform; R4, four days' recovery. Significance levels are: •P < 0.05; "p < 0.01; "'P < 0.001.
E4
E2
LP4
LP2
R4
Free calcium Fig. 2. Percentage changes (± S.E.M.) in free and bound synaptosomal Ca 2 + in REM sleep-deprived and control rat brain cerebellum compared to free-moving control, taken as baseline (100%). Abbreviations and significance levels are as in Fig. I.
Effects of rapid eye movement sleep deprivation on bound calcium concentration in different brain areas
deprivation were: cerebrum, F I ,IO = 605.5, P < 0.001; cerebellum, FI,ll = 79.33, P < 0.001; brainstem, F I 12 =264, P < 0.001. After two days' deprivation they were: cerebrum, F I 8 = 51.69, P < 0.01; cerebellum, F I ,IO = 12.44, P < 0.01; brainstem, F I 10 =21.26, P < 0.01. In the large-platform control gr~up of animals, free calcium levels remained unaffected in all the regions, except the cerebellum (FI 10 = 31.83; P < 0.01). All the altered calcium lev~ls returned to normal after recovery.
The bound calcium concentration increased in the brainstem (Fig. I; F I ,13 = 278; P < 0.001), but decreased in the cerebellum (Fig. 2; Fl,lo =91.81; P < 0.001) and the cerebrum (Fig. 3; FI,lO = 122; P < 0.001) after four days of REM sleep deprivation. After two days' deprivation it increased in the brainstem only (Fig. I), while it remained unaffected in the cerebellum and the cerebrum. The altered levels returned to normal after recovery. The bound calcium levels were not affected in the large-platform control group of rats.
Changes in free calcium concentration in the brainstem areas
Changes in bound calcium concentration in brainstem areas
Free calcium concentration was estimated in freemoving control, large-platform control and two-day REM sleep-deprived rats. The latter group of rats showed a significant decrease in free calcium level in the medulla (Fig. 4; F I ,5 = 412.24; P < 0.001) and the pons (Fig. 4; F I ,5 = 933.62; P < 0.001) as compared to that of the respective free-moving controls. The midbrain (Fig. 4) did not show any significant change. Since the altered free calcium levels returned to normal in the recovery group after four days' REM sleep deprivation, a recovery study after shorter REM sleep deprivation (two days) was not undertaken.
The bound calcium concentration increased in the medulla (Fig. 4; Fl,lo = 131.67; P < 0.001). However, it remained unaffected in the pons (Fig. 4) and the midbrain (Fig. 4). It was not 'significantly affected in the large-platform control group of animals either. Ratio of changes in bound to free calcium concentration
The ratio of changes in bound to free calcium levels was close to unity in the cerebellum and cerebrum in all control and experimental groups of rats (Fig. 5). However, in the case of the brainstem
733
REM sleep deprivation and synaptosomal Ca2+ concentrations Cerebrum
150
•••
Medulla
20
100
o SO
0
"O§ll
-SO
'ti
'~5
"
Pons
150
"c:
Oll
BOWld calcium
100
20
..c:u'" ~ I:l..
SO
0
= "
I:l..
"
0
Midbrain
150
-so
L..--'!.l!.il!....
E4
_
E2
LP4
LP2
R4
100
Free calcium Fig. 3. Percentage changes (± S.E.M.) in free and bound synaptosomal Ca 2 + in rat brain cerebrum in REM sleepdeprived and control rats as compared to free-moving control, taken as 100%. Abbreviations and significance levels are as in Fig. l.
SO
0
BOWld calcium
after four days' REM sleep deprivation, there were disproportionate changes in bound to free calcium levels. The bound form increased, while the free form decreased. DISCUSSION
Behaviorally, the experimental rats in this study expressed signs of REM sleep deprivation. I ,47 The total, free and bound calcium levels were estimated in synaptosomal preparations from anatomically identified brain regions. After REM sleep deprivation, there was a decrease in all forms of calcium concentration in the cerebrum and the cerebellum. However, for the entire brainstem and medulla, although there were decreases in the free form, the total and bound calcium concentrations increased. The flower-pot method was chosen in this study for REM sleep deprivation because it has been most widely used II for investigating the effects of REM sleep deprivation on psychological, physiological, biochemical and behavioral parameters. This method has been shown to induce maximum REM sleep deprivation without significantly affecting non-REM sleep. The platform sizes were selected based on an earlier report,48 Almost total loss of REM sleep by this method in cats 20 and in rats 23 ,46 has been reported. However, there is a non-significant loss in non-REM sleep, especially within 24 h of deprivation. Since the large-platform control animals also
Free calcium
Fig. 4. Changes in bound and free calcium concentrations in the medulla, pons and midbrain in two-day REM sleepdeprived and control groups of rats. The values were compared to free-moving control (FM), taken as baseline. Abbreviations and significance levels are as in Fig. 1.
experienced a loss in non-REM sleep similar to that of small-platform animals,23 and the effect of deprivation was observed in the small-platform group of rats only (except in the case of free calcium concentration in the cerebellum), it is reasonable to accept that the effects observed in the small-platform rats were due to REM sleep deprivation and not to a small loss in non-REM sleep. Further, it was also found that the REM sleep deprivation-induced alterations in calcium concentrations returned to normal levels in the recovery group of rats. Rapid eye movement sleep deprivation and changes in synaptosomal calcium levels
In the normal situation, more than 99% of total intracellular calcium exists in bound form,2 supporting the observations in this study. A significant decrease in total, free and bound calcium in the cerebrum and the cerebellum suggests a net efflux of calcium after REM sleep deprivation. Although it is difficult to comment on the precise cellular mechanism of this efflux, the following possibility, via stimulation of a highly sensitive calcium pump, fits in and
B. N. Mallick and S. Gulyani
734
8
°u'"
3
" u
~I '" ~ ~ u.. '"
e---e Brain stem
e'\.
Cerebellum _ _ Cerebmm
6---{;
'\.
2
bJJ
e
cOJ e
•--.---.• ~ .•
o
0'-:-:-:-------::,.,------,---------
-=u''""
15-
i
e<:
~
~
~
~
M
Fig. 5. The ratio of percentage change in bound synaptosomal concentration to that in free synaptosomal concentration in different brain regions in control and experimental rats. Abbreviations are as in Fig. I. helps to explain the situation. The calcium concentration in a cell is known to be regulated by an Na-Ca exchange pump which, though it is less sensitive than the Ca pump, has a greater capacity for exchanging calcium. 34 This exchange protein functions in coordination with the Na-K pump. The Na+ gradient maintained by Na-K ATPase is used by the Na-Ca exchange pump to release Ca 2+.J4,35 It was reported earlier that REM sleep deprivation increased Na-K ATPase activity.9 This increased Na-K ATPase activity will induce a steep concentration gradient, resulting in a net influx of Na+ and consequently efflux of Ca 2+, This would thus cause a net decrease in intracellular Ca 2+. However, this explanation does not hold true in the brainstem where, unlike the cerebrum and the cerebellum, although there was a decrease in free form, the bound form increased. This study does not allow us to comment as to whether Ca 2+ moves from one compartment to the other. There was a decrease in total, free and bound calcium concentrations in the cerebellum and the cerebrum. In these areas, since there was a proportionate decrease in the free and bound forms of calcium (Fig. 5), it is likely that there would be a net efflux of Ca2+ from the neuron. Consequently, it is unlikely that there would be conversion of Ca 2+ from one form to the other in the cerebellum and cerebrum. However, in the brainstem the bound calcium, which forms the major pool in the neuron,2 increased while the free form, which is effectively responsible for neuronal functions, decreased after deprivation. The ratio of percentage changes in bound to free calcium concentrations was maintained at a fairly constant level in the cerebellum and the cerebrum, but not in the brainstem (Fig. 5). This proportionate decrease in total, bound and free calcium in the cerebellum and cerebrum may be explained by the fact that the ratio of intracellular bound to free calcium is very strictly regulated. 2.35 Nevertheless, in the brainstem the bound form increased while the free form decreased. Thus, the results suggest that the homeostatic mechanism which maintains the ratio of intracellular bound to
free calcium was not affected in the cerebrum and the cerebellum, though it failed in the brainstem, after REM sleep deprivation. The relative changes of calcium concentrations were considered and not the absolute values, since the former is more important for cell functions and, unlike the latter, remains virtually unaffected by the size of neurons and mass of area studied. The homeostatic mechanism failed first in the brainstem possibly because this area is involved in REM sleep generation, as discussed below. It was shown previously that other factors were also affected first in the brainstem after REM sleep deprivation. 9,42,43 The pons and the medulla were the first sites to be affected, while the midbrain remained unaffected. The bound calcium concentration increased only in the medulla, while free calcium decreased both in the medulla and in the pons, These differential responses of calcium concentrations in the brainstern regions assume significance since the pontomedullary regions are responsible for REM sleep generation. 40 It is important to note that after REM sleep deprivation Na-K ATPase,9 acetylcholinesterase 42 and monoamine oxidase43 were also affected first in the pontomedullary regions. While extensive studies are needed for conclusive evidence, the possible role of norepinephrine as suggested earlier for REM sleep deprivation-induced increase in Na-K ATPase 10 activity also holds true in this case. The locus coeruleus norepinephrinergic REM-off neurons cease firing during REM sleep l3 and have monosynaptic projections to the medulla. 37 During the normal situation, although these REM-off neurons cease firing during REM sleep, they keep firing continuously during REM sleep deprivation. 20 Hence, during REM sleep deprivation there would be a continuous supply and a relative increase in norepinephrine locally in the projected area, the medulla in this instance. This may also be supported by the recent report that mild stimulation of the locus coeruleus, which is likely to increase norepinephrine in the projected site, induced an effect similar to that of REM sleep deprivation. 41 This local increase in the level of norepinephrine is likely to enhance calcium influx. 24 However, a possible argument against this explanation would be that locus coeruleus neurons also project to the cortex, but similar changes were not observed in the cerebrum. This is possibly because the cortex alone has not been studied; instead, the entire cerebrum was taken, where the effect is likely to be diluted or reduced (as obtained). It would also depend on the proportion of projections. This is further substantiated by the observation that the effects were opposite in the pons, where the locus coeruleus neurons were located, and due to constant firing by REM-off neurons the norepinephrine is expected to be depleted. In addition, this increase could also be due to selective local presence of neurohormone and/or sleep-inducing peptides 12 in the medulla.
REM sleep deprivation and synaptosomal Ca 2 + concentrations
Physiological significance
An alteration in calcium level would modulate the basic physiology and functioning of a neuron. 32 ,35 Modulation of neuronal excitability, firing rate and nerve impulse conduction due to changes in Ca2+ are well known. 28 ,35 It has been reported that intracellular calcium plays an important role in memory processing, 2 gene expression, 7 receptor sensitivities, receptor-mediated cellular functioning and enzyme activities,35 and membrane fluidity. 5, 16 Independent and isolated studies have shown that all these parameters, including calcium concentration in this study, are affected after REM sleep deprivation. Thus, pending further direct evidence, based on the indirect evidence available it is tempting to suggest that changes in intracellular calcium concentration could be at least one of the primary underlying
735
mechanisms for REM sleep deprivation-induced alterations in overall physiological responses of a neuron, and ultimately the functioning of the nervous system. Finally, the role of calcium and membrane fluidity in maintaining cellular integrity, functioning and longevity has been reported;5,22,29,35 these results, along with the results of this study, would certainly go a long way to help explain the underlying basic molecular mechanism of the role of REM sleep in cellular growth and maturation. 14 ,25 Acknowledgements-The financial support received from the Indian Council of Medical Research is duly acknowledged. We are grateful to Prof. V. Subramaniam, School of Environmental Sciences, Jawaharlal Nehru University for kindly allowing us to use the atomic absorption spectrometer.
REFERENCES
Albert l. B. (1975) REM sleep deprivation. Bio!. Psychiat. 10, 341~351. 2. Borle A. B. (1981) Control, modulation and regulation of cell calcium. Rev. Physiol. Biochem. Pharmac. 90, 13-153. 3. Clemes S. and Dement W. C. (1967) Effect of REM sleep deprivation on psychological functioning. J. nerv. ment. Dis. 144, 485--489. 4. Cohen H. B. and Dement W. C. (1965) Sleep: changes in threshold to electroconvulsive shQck in rats after deprivation of "paradoxical" phase. Science 150, 1318~1319. 5. Daniell L. C. and Harris R. A. (1988) Neuronal intracellular calcium concentrations are altered by anesthetics: relationship to membrane fluidization. J. Pharmac. expo Ther. 245, 1~7. 6. Fishbein W. and Gutwein B. M. (1977) Paradoxical sleep and memory storage processes. Behav. Bioi. 19,425--464. 7. Ginty D. J., Bading H. and Greenberg M. E. (1992) Trans-synaptic regulation of gene expression. Curro Opin. Neurobiol. 3,312-316. 8. Gordon-Weeks P. R. (1987) Isolation of synaptosomes, growth cones and their subcellular components. In Neurochemistry: A Practical Approach (eds Turner J. A. and Bachlard S. H.), pp. 1~25. IRL Press, Oxford. 9. Gulyani S. and Mallick B. N. (1993) Effect of rapid eye movement sleep deprivation on rat brain Na-K ATPase activity. J. Sleep Res. 2, 45~50. 10. Gulyani S. and Mallick B. N. (1995) Possible mechanism of rapid eye movement sleep deprivation induced increase in Na-K ATPase activity. Neuroscience 64, 255-260. II. Hicks R. A., Okuda A. and Thomsen D. (1977) Depriving rats of REM sleep: the identification of a methodological problem. Am. J. Psychol. 90, 95~102. 12. Inoue S. (1989) Biology of Sleep Substances. CRC Press, Boca Raton, FL. 13. Jacobs B. L. (1986) Single unit activity of locus coeruleus neurons in behaving animals. Prog. Neurobio!. 27, 183-194. 14. Krueger J. M. and Obal F. Jr (1993) A neuronal group theory of sleep function. J. Sleep Res. 2,63-69. 15. Kushida A. c., Bergmann B. M. and Rechtschaffen A. (1989) Sleep deprivation in rat: IV. Paradoxical sleep deprivation. Sleep 12, 22-30. 16. Lebel C. P. and Schatz R. A. (1990) Altered synaptosomal phospholipid metabolism after toluene: possible relationship with membrane fluidity, Na-K adenosine triphosphatase and phospholipid methylation. J. Pharmac. expo Ther. 253, 1189~1l97. 17. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with Folin phenol reagent. J. bioi. Chem. 193, 265~275. 18. Mallick B. N., Fahringer H., Wu M. F. and Siegel J. M. (1991) REM sleep deprivation reduces auditory evoked inhibition of dorsolateral pontine neurons. Brain Res. 552, 333-337. 19. Mallick B. N. and Gulyani S. (1993) Rapid eye movement sleep deprivation increases chloride-sensitive Mg-ATPase activity in the rat brain. Pharmac. Biochem. Behav. 45, 359-362. 20. Mallick B. N., Siegel J. M. and Fahringer H. (1989) Changes in pontine unit activity with REM sleep deprivation. Brain Res. 515, 94-98. 21. Mallick B. N., Thakkar M. and Gangabhagirathi R. (1995) Rapid eye movement sleep deprivation decreases membrane fluidity in the rat brain. Neurosci. Res. 22, 117-122. 22. McConkey D. J., Hartzell P., Nicoterra P. and Orrenius S. (1989) Calcium activated DNA fragmentation kills immature thymocytes. Fedn Proc. Fedn Am. Socs expo Bioi. 3, 1843-1848. 23. Mendelson W. B. (1974) The flower pot technique of rapid eye movement (REM) sleep deprivation. Pharmac. Biochem. Behav. 2, 553-556. 24. Minneman K. P. (1981) Adrenergic receptor molecules. In Neurotransmitter Receptors (eds Yamamura l. H. and Enna J. S.), pp. 187-257. Chapman & Hall, London. 25. Mirmiran M. and Van Someren E. (1993) The importance of REM sleep for brain maturation. J. Sleep Res. 2, I.
188~192.
26.
Mogilnicka E., Przewlocka B., Van-Iuijtelaar E. L. J. M., Klimek V. and Coenen M. L. (1986) Effects of REM sleep deprivation on central alpha-I and beta-adrenoceptors in rat brain. Pharmac. Biochem. Behav. 25,329-332.
736 27. 28. 29. 30. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
B. N. Mallick and S. Gulyani Nicholls D. G., Sihra T. S. and Sanchez-Prieto J. (1987) The role of plasma membrane and intracellular organelles in synaptosomal calcium regulation. In Cell Calcium and the Control ofMembrane Transport (eds Mandel J. L. and Eaton C. D.), pp. 32-42. Rockefeller University Press, New York. Nicoll R. A. (1988) Neurotransmitters and ion channels. Science 241, 545-551. Nicotera P., McConkey D., Svensson S. A., Bellomo G. and Orrenius S. (1988) Correlation between cytosolic Ca 2 + concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology 52, 55-63. Obal F. Jr, Fang J., Payne L. C. and Krueger J. M. (1995) Growth-horrnone-releasing hormone mediates the sleep promoting activity of interleukin-I in rats. Neuroendocrinology 61, 559-565. O'Hara B. F., Young K. A., Watson F. L., Heller H. C. and KilduffT. S. (1993) Immediate early gene expression in brain during sleep deprivation: preliminary observations. Sleep 16, 1-7. Penner R., Fasolato C. and Hoth M. (1993) Calcium influx and its control by calcium release. Curro Opin. Neurobiol. 3,368-374. Porkka-Heiskanen T., Smith S. E., Taira T., Urban J. H., Levine J. E., Turek F. W. and Stenberg D. (1995) Noradrenergic activity in rat brain during rapid eye movement sleep deprivation and rebound sleep. Am. J. Physiol. 268, RI456-1463. Rahamimoff H. (1990) Na+-Ca 2 + exchanger: the elusive protein. Curro Topics Cell Regul. 31,241-271. Rasmussen H. and Rasmussen J. E. (1990) Calcium as intracellular messenger: from simplicity to complexity. Curro Topics Cell Regul. 31, 1-109. Rechtschaffen A., Bergmann B. M., Everson C. A., Kushida C. A. and Gilliland M. A. (1989) Sleep deprivation in the rat: integration and discussion of the findings. Sleep 12, 68-87. Sakai K. (1985) Anatomical and physiological basis of paradoxical sleep. In Brain Mechanisms of Sleep (eds McGinty D. J., Drucker-Colin R., Morrison A. and Parmeggiani P. L.), pp. 111-138. Raven Press, New York. Schmidt W. K. and Way E. L. (1979) Direct assay for calcium in brain homogenates and synaptosomal pellets. J. Neurochem. 32, 1095-1098. Shiromani P. J., KilduffT. S., Bloom F. E. and McCarley R. W. (1992) Cholinergically induced REM sleep triggers Fos-likeimmunoreactivity in dorsolateral pontine regions associated with REM sleep. Brain Res. 580, 351-357. Siegel J. M. (1985) Pontomedullary interactions in the generation of REM sleep. In Brain Mechanisms of Sleep (eds McGinty D. J., Drucker-Colin R., Morrison A. and Parrneggiani P. L.), pp. 157-174. Raven Press, New York. Singh S. and Mallick B. N. (1996) Mild electrical stimulation of pontine tegmentum around locus coeruleus reduces rapid eye movement sleep in rats. Neurosci. Res. 24, 227-235. Thakkar M. and Mallick B. N. (1991) Effect of REM sleep deprivation on rat brain acetylcholinesterase. Pharmac. Biochem. Behav. 39,211-214. Thakkar M. and Mallick B. N. (1993) Effect of rapid eye movement sleep deprivation on rat brain monoamine oxidases. Neuroscience 55, 677-683. Thakkar M. and Mallick B. N. (1993) Rapid eye movement sleep deprivation induced changes in glucose metabolic enzymes in rat brain. Sleep 16, 691-694. Tsai L. L., Mergmann B. M., Perry B. D. and Rechtschaffen A. (1994) Effects of chronic sleep deprivation on central cholinergic receptors in rat brain. Brain Res. 642, 95-103. Van Luijtelaar E. L. J. M. and Coenen A. M. L. (1986) Electrophysiological evaluation of three paradoxical sleep deprivation techniques in rats. Physiol. Behav. 36, 603-609. Vogel G. W. (1975) A review of REM sleep deprivation. Archs gen. Psychiat. 32,749-761. Yanik G. and Radulovacki M. (1987) REM sleep deprivation up-regulates adenosine Al receptors. Brain Res. 402, 362-364. Yates S. L., Fluhler E. N. and Lippiello P. M. (1992) Advances in the use of the fluoresecent probe fura-2 for the estimation of intrasynaptosomal calcium. J. Neurosci. Res. 32, 255-260. Yingst D. R. (1988) Modulation of Na,K ATPase by Ca and intracellular proteins. A. Rev. Physiol. 50,291-303. (Accepted 20 March 1996)