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Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain
Neuroscience Letters 520 (2012) 62–66
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain Sudhuman Singh, Megha Amar, Birendra N. Mallick ∗ School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
h i g h l i g h t s
REMSD is reported to elevate neurotransmitter, including noradrenalin, in the brain. Phosphorylation of synapsinI holds key to neurotransmitter release. REMSD increased synapsinI, total and its phospho-form, synapsinI-phosphoSer603. Findings explain intracellular mechanism of REMSD associated increased neurotransmitter release.
a r t i c l e
i n f o
Article history: Received 3 March 2012 Received in revised form 29 April 2012 Accepted 8 May 2012 Keywords: Na-K ATPase Noradrenalin REMSD SynapsinI-phosphoSer603 Synaptic vesicle Total synapsinI
a b s t r a c t Rapid eye movement sleep (REMS) deprivation (REMSD) has been reported to elevate neurotransmitter level in the brain; however, intracellular mechanism of its increased release was not studied. Phosphorylation of synapsinI, a synaptic vesicle-associated protein, is involved in the regulation of neurotransmitter release. In this study, rats were REMS deprived by classical flowerpot method; free moving control (FMC), large platform control (LPC) and recovery control (REC) was carried out. In another set REMS deprived rats were intraperitoneally (i.p.) injected with ␣1-adrenoceptor antagonist, prazosin (PRZ). Effects of REMSD on Na-K ATPase activity and on the total synapsinI as well as phosphorylated synapsinI levels were estimated in synaptosomes prepared from whole brain. It was observed that REMSD significantly increased synaptosomal Na-K ATPase activity, which was prevented by PRZ. Western blotting of the same samples by anti-synapsinI and anti-synapsinI-phosphoSer603 showed that REMSD increased both the total as well as phospho-form of synapsinI as compared to respective levels in FMC and LPC samples. These findings suggest a functional link between REMSD and synaptic vesicular mobilization at the presynaptic terminal, a process that is essential for neurotransmitter release. The findings help explaining the intracellular mechanism of elevated neurotransmitter release associated to REMSD. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction REMS is an autonomically regulated instinct behavior, expressed by animals higher in evolution, including mammals, studied so far. Its loss affects several basic physiological processes [16] including brain excitability leading to the proposition that REMS serves as house keeping function of the brain [23]. Fundamentally, the functions of neurons and the brain are mediated by the release of neurotransmitters. Many studies reported alterations in the levels of neurotransmitters in relation to REMS and REMSD [17,26,31,36]. Neurotransmitter mediated modulation of REMS [21] and REMSD induced changes in neurotransmitter levels [5,28,30] have been reported, however, the cellular basis of REMSD-associated increased release of neurotransmitter was
∗ Corresponding author. Tel.: +91 11 26704522; fax: +91 11 26742558. E-mail address: [email protected] (B.N. Mallick). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.031
unknown. The effective levels of neurotransmitters in the brain and at the synaptic cleft depend on their relative synthesis, release and degradation. Several studies have looked into the changes in biomolecules in relation to REMS and REMSD [16]. Isolated studies have reported modulation of gene expressions [8,34] and changes in the levels of some synaptic proteins [11] in the brain in relation to sleep–wake like states. However, very little is known on the intracellular step(s) for neurotransmitter release especially in relation to REMS and REMSD, which is necessary for detailed understanding of normal and patho-physiology of REMS, REMSD and associated disorders. Neurotransmitters are stored in synaptic vesicles (SVs), which remain bound to the presynaptic cytoskeleton preventing its mobility and release. The release of neurotransmitter at the synaptic cleft is a complex process comprising of a series of steps, starting with detachment of SVs from cytoskeleton followed by its mobilization, targeting and docking at the active zone for exocytosis. SynapsinI is a neuron specific SV-associated protein, which is
S. Singh et al. / Neuroscience Letters 520 (2012) 62–66
localized exclusively at the presynaptic terminal that keeps the SVs attached to the actin based cytoskeleton. It has two subunits each having a globular head and a tail with multiple phosphorylation sites [35]; Ser603 residue is one of the pivotal sites for neurotransmitter release [14]. Phosphorylation of synapsinI has been shown to induce conformational changes leading to dissociation of SVs and allowing the latter to move from the reserve-pool to the releasablepool and finally to the active zone for release of neurotransmitter by exocytosis. Thus, essentially phosphorylation of synapsinI is the key and rate limiting step regulating the efficiency of neurotransmitter release [6,7,14]. Therefore, as a first step towards advancing our understanding on the intracellular mechanism of neurotransmitter release in response to REMSD, we examined total synapsinI and synapsinI-phosphoSer603 in the synaptosomes prepared from whole brain of REMS deprived and control rats. We have reported that REMSD elevates synaptosomal Na-K ATPase activity and that it is mediated by increased noradrenalin (NA) in the brain [20,23]. Na-K ATPase is responsible for ionic exchange through the membrane and plays an important role in the maintenance of resting potential and propagation of neuronal impulse leading to neurotransmitter release. Hence, as a reference for achieving REMSD as well as to correlate with changes in synapsinI levels, we also estimated the Na-K ATPase activity from the same sample.
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2.3. Western blotting
2. Materials and methods
The band densities of total and phosphorylated forms of synapsinI in synaptosomes prepared from control and experimental animals were estimated by western blotting and compared statistically. Equal amounts of total protein (30 g) were separated on 8%-polyacrylamide gel and transferred onto 0.45 m nitrocellulose membrane (MDI, India) using a semi-dry transfer apparatus (Bio-Rad, Australia). The membranes were blocked in 3%-BSA in Tris-buffered saline (TBS) for 2 h at room-temperature and then incubated overnight at 4 ◦ C with primary anti-synapsinI (polyclonal rabbit antibody, AB1543; Millipore, USA, 1:1000) and anti-synapsinI-phosphoSer603 (polyclonal rabbit antibody, AB5883; Millipore, USA, 1:1000); for loading control anti-GAPDH (polyclonal chicken antibody, AB2302, Millipore, USA, 1:1000) was used. Following primary antibody incubation, the membranes were washed four times with TBS containing 0.1% Tween-20 (TBS-T) and probed with species appropriate horseradish peroxidaseconjugated secondary antibody (goat anti-rabbit, sc-3837, Santa Cruz Biotechnology, USA; rabbit anti-chicken, A9046, Sigma, USA, 1:10,000) for 1.5 h at room-temperature. The membranes were then washed 3-times with TBS-T followed by with TBS and subsequently developed following enhanced chemiluminescence method. The intensities of the desired bands were densitometrically estimated using alpha image software (Alpha Innotech, USA).
2.1. Experimental animals
2.4. Estimation of Na-K ATPase activity
The experiments were conducted on male wistar rats (220–250 g) housed under standard laboratory conditions at controlled temperature, 12/12-h light/dark cycle and food and water available ad libitum. Experimental protocols were approved by the Institutional Animal Ethics Committee and conformed to NIH guidelines. All efforts were made to use minimum number of animals and to minimize sufferings to the animals during the period of the study. Normal rats maintained in dry cages were taken as FMC. Experimental rats were REMS deprived for 4 days by the classical flowerpot method [15] described earlier in detail [12]. In brief, for REMSD rats were kept on 6.5 cm diameter platform projected above a pool of water. To rule out non-specific effects, control rats were kept on larger (LPC) platform (13 cm diameter) projected above a pool of water and maintained for the same period in the same room adjacent to the experimental animals. In another REC group, rats were REMS deprived and then transferred to dry cages and allowed 3 days of recovery sleep. To check whether the REMSD induced effect was mediated due to elevated level of NA, in another set, rats were treated with ␣1-adrenoceptor antagonist, PRZ (4 mg/kg, i.p.) 48 h REMSD onwards for 2 days. Thus, in each set of experiment, there was one rat each of FMC, LPC, REMSD, REC, PRZ and 5 such sets were carried out.
As it has been consistently shown that REMSD stimulates NaK ATPase activity and it is mediated by induced NA [23], in this study we estimated Na-K ATPase activity from the same samples used to estimate synapsinI and syapsinI-phosphoSer603 for two reasons. One, as a support that REMSD was achieved and two, whether the effects on Na-K ATPase, synapsinI and synapsinIphosphoSer603 were all mediated by elevated NA level in the brain. The enzyme activity was estimated as reported earlier [12] following the method of Akagawa and Tsukada [2]. The reaction mixture contained 100 mM NaCl, 20 mM KCl, 5 mM MgCl2 , 3 mM ATP and 50 mM Tris (pH 7.4). An aliquot (30–40 g protein) of the synaptosomes was incubated with the reaction mixture at 37 ◦ C for 20 min and ATP was used as the substrate while ouabain was used as a specific blocker of Na-K ATPase. The reaction was stopped by 10% ice-cold tri-chloroacetic acid, the mixture was centrifuged at 1000 × g (3000 rpm) and the released inorganic phosphate (Pi) in the supernatant was estimated [10] using a PerkinElmer spectrophotometer. The ouabain-sensitive Na-K ATPase activity was estimated and expressed as mol Pi released/mg protein/h.
2.2. Synaptosome preparation At the end of experiment the rats were decapitated after cervical dislocation and brains were taken out in ice-cold buffer and processed for synaptosome preparation as reported earlier [4]. Briefly, each brain was homogenized in 10 ml ice-cold buffer containing 0.32 M sucrose, 12 mM Tris (pH 7.4) and 1 mM EDTA and centrifuged for 5 min at 3000 × g (6000 rpm). The supernatant was centrifuged for 20 min at 11,000 × g (12,000 rpm). The pellet was re-suspended in 1.0 ml of homogenizing buffer and layered onto a preformed sucrose density gradient of 1.2 M and 0.8 M and ultracentrifuged at 1,00,000 × g (25,000 rpm) for 2 h. The band obtained at the interface of 1.2 M and 0.8 M was re-suspended in 3-volumes of homogenizing buffer and used as synaptosomes. Protein concentration was estimated by Lowry’s method [18].
2.5. Statistical analysis Data was collected from 5 sets of experiments in each group and the results were expressed as mean ± SEM. The differences in the mean intensities of synapsinI and phosphorylated synapsinI bands and Na-K ATPase enzyme activities in samples from experimental and control rat brains were statistically compared with that of FMC using one-way analysis of variance (ANOVA). Significance levels were evaluated by applying Tukey’s test (Sigmaplot 10 software); at least P values < 0.05 were considered statistically significant. 3. Results 3.1. Effects of REMSD on total synapsinI Four days REMSD by the flowerpot method significantly increased total synapsinI to 64% as compared to FMC
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S. Singh et al. / Neuroscience Letters 520 (2012) 62–66
Fig. 1. (A) Western blot bands of total synapsinI in synaptosomes prepared from one representative set of experiment under FMC, LPC, REMSD, REC and PRZ-injected rat brain have been shown in the upper panel. Western blot of GAPDH bands in the same samples have been shown in the lower panel as loading control. (B) Percentage changes in total synapsinI Western blot band intensities (mean ± SEM) in synaptosomal samples prepared from 5 sets of experiments have been shown. ***P < 0.001, significant as compared to FMC. Abbreviations as in the text.
[F(4,21) = 36.257; P < 0.001]. The significant (P < 0.001) up-regulation of total synapsinI continued even after 3 days REC as well as in PRZ injected rats; i.e. synapsinI did not return to FMC level in REC or PRZ rats. The total synapsinI however, remained unaffected in LPC rats (Fig. 1). 3.2. Effects of REMSD on synapsinI-phosphoSer603 As phosphorylation of synapsinI is key for release of SVs from its attachment with cytoskeleton, we then asked whether REMSD affected the level of phosphorylated synapsinI. It was evaluated by western blotting with antibodies against the phosphorylated form of Ser603 residue of synapsinI and estimating the band intensities. The results showed that REMSD significantly increased the phosphorylated form of synapsinI to about 55% as compared to that of FMC samples [F(4,21) = 40.636; P < 0.001]. The elevated levels of synapsinI-phosphoSer603 continued in the samples prepared from REC and PRZ treated rats; however, it was not affected in the LPC rats (Fig. 2).
Fig. 2. (A) Western blot of synapsinI-phosphoSer603 bands in synaptosomes prepared from one representative set of experiment under FMC, LPC, REMSD, REC and PRZ injected rat brain have been shown in the upper panel. Western blot of GAPDH bands in the same samples have been shown in the lower panel as loading control. (B) Percentage changes in the band intensities (mean ± SEM) of synapsinIphosphoSer603 in synaptosomal samples prepared from 5 sets of experiments have been shown. ***P < 0.001 significant as compared to FMC. Abbreviations as in the text.
In contrast, although REMSD increased synapsinI and synapsinIphosphoSer603 levels in the rat brain, neither returned to baseline after recovery nor were they prevented by PRZ (Fig. 3). 4. Discussion An optimum level of neurotransmitter is necessary for normal functioning of the brain and expression of behaviors including REMS. Levels of neurotransmitters in specific region of the brain alter with modulation of spontaneous sleep-waking-REMS states [27,31,36] as well as after REMSD [5,17]. Therefore, we hypothesized that REMSD might affect factors responsible for neurotransmitter release. The level of neurotransmitter at the synaptic cleft and the brain as a whole would depend on its release, which in turn is modulated by phosphorylation of the SV-associated protein, synapsinI [14,33]. Involvement of synapsinI in regulating the reserve pool of SVs at the presynaptic terminal may be supported by the fact that decreased number of the SVs has been reported in the absence of synapsinI [29,33]. We observed in this study that total synapsinI was up-regulated in the brain after REMSD as compared to FMC and LPC rats and it continued at elevated level even after 3 days of
3.3. Effects of REMSD on synaptosomal Na-K ATPase activity Na-K ATPase activity was estimated in the same rat brain synaptosomes where total and phosphorylated forms of synapsinI were estimated. The enzyme activity (mean ± SEM) significantly [F(4,21) = 17.424; P < 0.001] increased by 47% in samples prepared from the REMSD rat brains as compared to samples from FMC. The enzyme activity in REMSD was 13.17 ± 1.84 mol Pi released/mg protein/h as compared to that of 9.02 ± 1.36 mol Pi released/mg protein/h in FMC samples. The enzyme activities in all other controls and PRZ treated samples were comparable. 3.4. Comparison of simultaneous effects on Na-K ATPase, synapsinI and synapsinI-phosphoSer603 REMSD increased Na-K ATPase activity, which returned to baseline level after 3 days recovery as well as by PRZ treatment.
Fig. 3. Relative percent changes in Na-K ATPase activity, synapsinI and synapsinIphosphoSer603 expressions under various conditions, as compared to respective FMC values taken as baseline, have been compared. N = 5, ***P < 0.001 significant as compared to FMC. Abbreviations as in the text.
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recovery sleep. Increased total synapsinI induced by REMSD supports increased release of NA in the rat brain after REMSD [30]. This is because phosphorylation of synapsinI releases SVs, from their attachment with the cytoskeleton, which then travels towards the synaptic terminal for release of neurotransmitter. The arguments given above may be supported by the fact that the level of synapsinI regulates availability of SVs for exocytosis in an activity dependent manner [6,7]. In resting condition of a neuron, synapsinI cross-links or tether SVs to one another and with actin-based cytoskeleton to form relatively immobile reserve pool of SVs away from the active zone. However, in active state of neurons, it gets phosphorylated (involving calcium/calmodulindependent protein kinaseII), which in turn reduces its affinity for SVs and actin filaments allowing their movements towards active zone for release of neurotransmitters [14]. Since phosphorylation of Ser603 residue is crucial for SVs mobilization [14], we estimated the level of synapsinI-phosphoSer603 and found REMSD increased phosphorylated form of synapsinI as compared to its level in the FMC and LPC rats. NA and serotonin are known to increase phosphorylation of synapsinI support our views [3,25]. Thus the results of this study support our contention that REMSD promotes release of neurotransmitter; it also helps understanding the intracellular mechanism for increased release of neurotransmitter after REMSD. It has been reported that NA acting on ␣1-adrenoceptor modulates several REMSD associated changes in the rat brain [23]. Under identical REMSD condition several enzyme activities [16,23], neuronal cytomorphology [19,32] and phosphorylation of Na-K ATPase [9] were altered. Several of those REMSD-associated effects returned to FMC level in REC rats and most of the effects were largely reduced by PRZ suggesting the effects were induced by NA and not due to non-specific factors. However, in this study we observed that levels of total synapsinI and its phosphorylated form were not affected by PRZ. This suggested that upon REMSD synapsinI and its phosphorylation at Ser603 residue were unlikely to be regulated by NA acting through ␣1-adrenoceptor. The roles of other receptor-subtypes and neurotransmitters, if any, have not been evaluated. Also, as REMSD affects several parameters in the whole brain, we studied synaptosomes prepared from whole brain, localized brain areas are likely to be affected but quantum of effects may vary and need further study. Classical flowerpot method for REMSD [13,24], if continued beyond 48 h, is known to induce reasonably selective REMS loss and the effects were not due to stress [30]. In this study REMSD was successfully achieved because Na-K ATPase activity increased in the experimental rats and it returned to baseline level after recovery as reported earlier [23]. Although synapsinI and its phosphorylated forms increased after REMSD, they were unaffected in LPC rats suggesting that the effects were specific to REMSD and unlikely due to non-specific factors. However their increased expressions did not return to baseline level after recovery or after PRZ injection (Fig. 3). These findings suggest that in this study the rats were REMS deprived and NA was increased because Na-K ATPase activity increased as reported earlier [12]. However, increase in expression of total synapsinI and its phosphorylated form was either not due to NA or the action of NA was not mediated through ␣1adrenoceptors. It is also evident that although 3 days recovery was sufficient for REMSD induced Na-K ATPase activity to return to normal level, it was not sufficient for recovery of synapsinI and its phosphorylated form. This may be because unlike allosteric modulation of NA induced stimulation of Na-K ATPase activity [1], the REMSD might stimulate expressions of synapsinI, possibly by stimulating its transcription. In conclusion, the findings of this study provide intracellular evidence for increased release of neurotransmitter upon REMSD. Increased phosphorylation of synapsinI supports continued enhanced release of neurotransmitter during REMSD. The
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observation compliments earlier reports that upon REMSD at least REM-OFF neurons continue firing [22] and level of NA is increased in the brain. Acknowledgements SS received fellowship from DBT. Research funding from DST, PURSE, Resource Networking, and JC Bose fellowship to BNM are acknowledged. References [1] H.V. Adya, B.N. Mallick, Uncompetitive stimulation of rat brain Na-K ATPase activity by rapid eye movement sleep deprivation, Neurochemistry International 36 (2000) 249–253. [2] K. Akagawa, Y. Tsukada, Presence and characteristics of catecholaminesensitive Na-K ATPase in rat striatum, Journal of Neurochemistry 32 (1979) 269–271. [3] A. Angers, D. Fioravante, J. Chin, L.J. Cleary, A.J. Bean, J.H. Byrne, Serotonin stimulates phosphorylation of Aplysia synapsin and alters its subcellular distribution in sensory neurons, Journal of Neuroscience 22 (2002) 5412–5422. [4] H.V. Anupama Adya, B.N. Mallick, Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions, Neurochemistry International 33 (1998) 283–286. [5] C.A. Blanco-Centurion, R.J. Salin-Pascual, Extracellular serotonin levels in the medullary reticular formation during normal sleep and after REM sleep deprivation, Brain Research 923 (2001) 128–136. [6] P. Chi, P. Greengard, T.A. Ryan, Synapsin dispersion and reclustering during synaptic activity, Nature Neuroscience 4 (2001) 1187–1193. [7] P. Chi, P. Greengard, T.A. Ryan, Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies, Neuron 38 (2003) 69–78. [8] C. Cirelli, G. Tononi, Differences in gene expression during sleep and wakefulness, Annals of Medicine 31 (1999) 117–124. [9] G. Das, A. Gopalakrishnan, M. Faisal, B.N. Mallick, Stimulatory role of calcium in rapid eye movement sleep deprivation-induced noradrenaline-mediated increase in Na-K-ATPase activity in rat brain, Neuroscience 155 (2008) 76–89. [10] C.H. Fiske, Y. Subbarow, The colorimetric determination of phosphorus, Journal of Biological Chemistry 66 (1925) 375–380. [11] G.F. Gilestro, G. Tononi, C. Cirelli, Widespread changes in synaptic markers as a function of sleep and wakefulness in Drosophila, Science 324 (2009) 109–112. [12] S. Gulyani, B.N. Mallick, Effect of rapid eye movement sleep deprivation on rat brain Na-K ATPase activity, Journal of Sleep Research 2 (1993) 45–50. [13] S. Gulyani, B.N. Mallick, Possible mechanism of rapid eye movement sleep deprivation induced increase in Na-K ATPase activity, Neuroscience 64 (1995) 255–260. [14] S. Hilfiker, V.A. Pieribone, A.J. Czernik, H.T. Kao, G.J. Augustine, P. Greengard, Synapsins as regulators of neurotransmitter release, Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences 354 (1999) 269–279. [15] D. Jouvet, P. Vimont, F. Delorme, Study of selective deprivation of the paradoxal phase of sleep in the cat, Journal de Physiologie 56 (1964) 381. [16] C.A. Kushida, Sleep Deprivation; Basic Science, Physiology and Behavior, 192, Marcel-Dekker, New York, 2005. [17] I. Lena, S. Parrot, O. Deschaux, S. Muffat-Joly, V. Sauvinet, B. Renaud, M.F. Suaud-Chagny, C. Gottesmann, Variations in extracellular levels of dopamine, noradrenaline, glutamate, and aspartate across the sleep–wake cycle in the medial prefrontal cortex and nucleus accumbens of freely moving rats, Journal of Neuroscience Research 81 (2005) 891–899. [18] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, Journal of Biological Chemistry 193 (1951) 265–275. [19] S. Majumdar, B.N. Mallick, Cytomorphometric changes in rat brain neurons after rapid eye movement sleep deprivation, Neuroscience 135 (2005) 679–690. [20] B.N. Mallick, H.V. Adya, S. Thankachan, REM sleep deprivation alters factors affecting neuronal excitability: role of norepinephrine and its possible mechanism of action, in: B.N. Mallick, S. Inoue (Eds.), Rapid Eye Movement Sleep, Narosa, 1999, pp. 338–354. [21] B.N. Mallick, S.R. Pandi-Permual, R.W. McCarley, A.R. Morrison (Eds.), Rapid Eye Movement Sleep—Regulation and Function, Cambridge University Press, Cambridge, 2011. [22] B.N. Mallick, J.M. Siegel, H. Fahringer, Changes in pontine unit activity with REM sleep deprivation, Brain Research 515 (1990) 94–98. [23] B.N. Mallick, A. Singh, REM sleep loss increases brain excitability: role of noradrenaline and its mechanism of action, Sleep Medicine Reviews 15 (2011) 165–178. [24] W.B. Mendelson, R.D. Guthrie, G. Frederick, R.J. Wyatt, The flower pot technique of rapid eye movement (REM) sleep deprivation, Pharmacology Biochemistry and Behavior 2 (1974) 553–556. [25] P. Mobley, P. Greengard, Evidence for widespread effects of noradrenaline on axon terminals in the rat frontal cortex, Proceedings of the National Academy of Sciences of the United States of America 82 (1985) 945–947.
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Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain Sudhuman Singh, Megha Amar, Birendra N. Mallick ∗ School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
h i g h l i g h t s
REMSD is reported to elevate neurotransmitter, including noradrenalin, in the brain. Phosphorylation of synapsinI holds key to neurotransmitter release. REMSD increased synapsinI, total and its phospho-form, synapsinI-phosphoSer603. Findings explain intracellular mechanism of REMSD associated increased neurotransmitter release.
a r t i c l e
i n f o
Article history: Received 3 March 2012 Received in revised form 29 April 2012 Accepted 8 May 2012 Keywords: Na-K ATPase Noradrenalin REMSD SynapsinI-phosphoSer603 Synaptic vesicle Total synapsinI
a b s t r a c t Rapid eye movement sleep (REMS) deprivation (REMSD) has been reported to elevate neurotransmitter level in the brain; however, intracellular mechanism of its increased release was not studied. Phosphorylation of synapsinI, a synaptic vesicle-associated protein, is involved in the regulation of neurotransmitter release. In this study, rats were REMS deprived by classical flowerpot method; free moving control (FMC), large platform control (LPC) and recovery control (REC) was carried out. In another set REMS deprived rats were intraperitoneally (i.p.) injected with ␣1-adrenoceptor antagonist, prazosin (PRZ). Effects of REMSD on Na-K ATPase activity and on the total synapsinI as well as phosphorylated synapsinI levels were estimated in synaptosomes prepared from whole brain. It was observed that REMSD significantly increased synaptosomal Na-K ATPase activity, which was prevented by PRZ. Western blotting of the same samples by anti-synapsinI and anti-synapsinI-phosphoSer603 showed that REMSD increased both the total as well as phospho-form of synapsinI as compared to respective levels in FMC and LPC samples. These findings suggest a functional link between REMSD and synaptic vesicular mobilization at the presynaptic terminal, a process that is essential for neurotransmitter release. The findings help explaining the intracellular mechanism of elevated neurotransmitter release associated to REMSD. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction REMS is an autonomically regulated instinct behavior, expressed by animals higher in evolution, including mammals, studied so far. Its loss affects several basic physiological processes [16] including brain excitability leading to the proposition that REMS serves as house keeping function of the brain [23]. Fundamentally, the functions of neurons and the brain are mediated by the release of neurotransmitters. Many studies reported alterations in the levels of neurotransmitters in relation to REMS and REMSD [17,26,31,36]. Neurotransmitter mediated modulation of REMS [21] and REMSD induced changes in neurotransmitter levels [5,28,30] have been reported, however, the cellular basis of REMSD-associated increased release of neurotransmitter was
∗ Corresponding author. Tel.: +91 11 26704522; fax: +91 11 26742558. E-mail address: [email protected] (B.N. Mallick). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.031
unknown. The effective levels of neurotransmitters in the brain and at the synaptic cleft depend on their relative synthesis, release and degradation. Several studies have looked into the changes in biomolecules in relation to REMS and REMSD [16]. Isolated studies have reported modulation of gene expressions [8,34] and changes in the levels of some synaptic proteins [11] in the brain in relation to sleep–wake like states. However, very little is known on the intracellular step(s) for neurotransmitter release especially in relation to REMS and REMSD, which is necessary for detailed understanding of normal and patho-physiology of REMS, REMSD and associated disorders. Neurotransmitters are stored in synaptic vesicles (SVs), which remain bound to the presynaptic cytoskeleton preventing its mobility and release. The release of neurotransmitter at the synaptic cleft is a complex process comprising of a series of steps, starting with detachment of SVs from cytoskeleton followed by its mobilization, targeting and docking at the active zone for exocytosis. SynapsinI is a neuron specific SV-associated protein, which is
S. Singh et al. / Neuroscience Letters 520 (2012) 62–66
localized exclusively at the presynaptic terminal that keeps the SVs attached to the actin based cytoskeleton. It has two subunits each having a globular head and a tail with multiple phosphorylation sites [35]; Ser603 residue is one of the pivotal sites for neurotransmitter release [14]. Phosphorylation of synapsinI has been shown to induce conformational changes leading to dissociation of SVs and allowing the latter to move from the reserve-pool to the releasablepool and finally to the active zone for release of neurotransmitter by exocytosis. Thus, essentially phosphorylation of synapsinI is the key and rate limiting step regulating the efficiency of neurotransmitter release [6,7,14]. Therefore, as a first step towards advancing our understanding on the intracellular mechanism of neurotransmitter release in response to REMSD, we examined total synapsinI and synapsinI-phosphoSer603 in the synaptosomes prepared from whole brain of REMS deprived and control rats. We have reported that REMSD elevates synaptosomal Na-K ATPase activity and that it is mediated by increased noradrenalin (NA) in the brain [20,23]. Na-K ATPase is responsible for ionic exchange through the membrane and plays an important role in the maintenance of resting potential and propagation of neuronal impulse leading to neurotransmitter release. Hence, as a reference for achieving REMSD as well as to correlate with changes in synapsinI levels, we also estimated the Na-K ATPase activity from the same sample.
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2.3. Western blotting
2. Materials and methods
The band densities of total and phosphorylated forms of synapsinI in synaptosomes prepared from control and experimental animals were estimated by western blotting and compared statistically. Equal amounts of total protein (30 g) were separated on 8%-polyacrylamide gel and transferred onto 0.45 m nitrocellulose membrane (MDI, India) using a semi-dry transfer apparatus (Bio-Rad, Australia). The membranes were blocked in 3%-BSA in Tris-buffered saline (TBS) for 2 h at room-temperature and then incubated overnight at 4 ◦ C with primary anti-synapsinI (polyclonal rabbit antibody, AB1543; Millipore, USA, 1:1000) and anti-synapsinI-phosphoSer603 (polyclonal rabbit antibody, AB5883; Millipore, USA, 1:1000); for loading control anti-GAPDH (polyclonal chicken antibody, AB2302, Millipore, USA, 1:1000) was used. Following primary antibody incubation, the membranes were washed four times with TBS containing 0.1% Tween-20 (TBS-T) and probed with species appropriate horseradish peroxidaseconjugated secondary antibody (goat anti-rabbit, sc-3837, Santa Cruz Biotechnology, USA; rabbit anti-chicken, A9046, Sigma, USA, 1:10,000) for 1.5 h at room-temperature. The membranes were then washed 3-times with TBS-T followed by with TBS and subsequently developed following enhanced chemiluminescence method. The intensities of the desired bands were densitometrically estimated using alpha image software (Alpha Innotech, USA).
2.1. Experimental animals
2.4. Estimation of Na-K ATPase activity
The experiments were conducted on male wistar rats (220–250 g) housed under standard laboratory conditions at controlled temperature, 12/12-h light/dark cycle and food and water available ad libitum. Experimental protocols were approved by the Institutional Animal Ethics Committee and conformed to NIH guidelines. All efforts were made to use minimum number of animals and to minimize sufferings to the animals during the period of the study. Normal rats maintained in dry cages were taken as FMC. Experimental rats were REMS deprived for 4 days by the classical flowerpot method [15] described earlier in detail [12]. In brief, for REMSD rats were kept on 6.5 cm diameter platform projected above a pool of water. To rule out non-specific effects, control rats were kept on larger (LPC) platform (13 cm diameter) projected above a pool of water and maintained for the same period in the same room adjacent to the experimental animals. In another REC group, rats were REMS deprived and then transferred to dry cages and allowed 3 days of recovery sleep. To check whether the REMSD induced effect was mediated due to elevated level of NA, in another set, rats were treated with ␣1-adrenoceptor antagonist, PRZ (4 mg/kg, i.p.) 48 h REMSD onwards for 2 days. Thus, in each set of experiment, there was one rat each of FMC, LPC, REMSD, REC, PRZ and 5 such sets were carried out.
As it has been consistently shown that REMSD stimulates NaK ATPase activity and it is mediated by induced NA [23], in this study we estimated Na-K ATPase activity from the same samples used to estimate synapsinI and syapsinI-phosphoSer603 for two reasons. One, as a support that REMSD was achieved and two, whether the effects on Na-K ATPase, synapsinI and synapsinIphosphoSer603 were all mediated by elevated NA level in the brain. The enzyme activity was estimated as reported earlier [12] following the method of Akagawa and Tsukada [2]. The reaction mixture contained 100 mM NaCl, 20 mM KCl, 5 mM MgCl2 , 3 mM ATP and 50 mM Tris (pH 7.4). An aliquot (30–40 g protein) of the synaptosomes was incubated with the reaction mixture at 37 ◦ C for 20 min and ATP was used as the substrate while ouabain was used as a specific blocker of Na-K ATPase. The reaction was stopped by 10% ice-cold tri-chloroacetic acid, the mixture was centrifuged at 1000 × g (3000 rpm) and the released inorganic phosphate (Pi) in the supernatant was estimated [10] using a PerkinElmer spectrophotometer. The ouabain-sensitive Na-K ATPase activity was estimated and expressed as mol Pi released/mg protein/h.
2.2. Synaptosome preparation At the end of experiment the rats were decapitated after cervical dislocation and brains were taken out in ice-cold buffer and processed for synaptosome preparation as reported earlier [4]. Briefly, each brain was homogenized in 10 ml ice-cold buffer containing 0.32 M sucrose, 12 mM Tris (pH 7.4) and 1 mM EDTA and centrifuged for 5 min at 3000 × g (6000 rpm). The supernatant was centrifuged for 20 min at 11,000 × g (12,000 rpm). The pellet was re-suspended in 1.0 ml of homogenizing buffer and layered onto a preformed sucrose density gradient of 1.2 M and 0.8 M and ultracentrifuged at 1,00,000 × g (25,000 rpm) for 2 h. The band obtained at the interface of 1.2 M and 0.8 M was re-suspended in 3-volumes of homogenizing buffer and used as synaptosomes. Protein concentration was estimated by Lowry’s method [18].
2.5. Statistical analysis Data was collected from 5 sets of experiments in each group and the results were expressed as mean ± SEM. The differences in the mean intensities of synapsinI and phosphorylated synapsinI bands and Na-K ATPase enzyme activities in samples from experimental and control rat brains were statistically compared with that of FMC using one-way analysis of variance (ANOVA). Significance levels were evaluated by applying Tukey’s test (Sigmaplot 10 software); at least P values < 0.05 were considered statistically significant. 3. Results 3.1. Effects of REMSD on total synapsinI Four days REMSD by the flowerpot method significantly increased total synapsinI to 64% as compared to FMC
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Fig. 1. (A) Western blot bands of total synapsinI in synaptosomes prepared from one representative set of experiment under FMC, LPC, REMSD, REC and PRZ-injected rat brain have been shown in the upper panel. Western blot of GAPDH bands in the same samples have been shown in the lower panel as loading control. (B) Percentage changes in total synapsinI Western blot band intensities (mean ± SEM) in synaptosomal samples prepared from 5 sets of experiments have been shown. ***P < 0.001, significant as compared to FMC. Abbreviations as in the text.
[F(4,21) = 36.257; P < 0.001]. The significant (P < 0.001) up-regulation of total synapsinI continued even after 3 days REC as well as in PRZ injected rats; i.e. synapsinI did not return to FMC level in REC or PRZ rats. The total synapsinI however, remained unaffected in LPC rats (Fig. 1). 3.2. Effects of REMSD on synapsinI-phosphoSer603 As phosphorylation of synapsinI is key for release of SVs from its attachment with cytoskeleton, we then asked whether REMSD affected the level of phosphorylated synapsinI. It was evaluated by western blotting with antibodies against the phosphorylated form of Ser603 residue of synapsinI and estimating the band intensities. The results showed that REMSD significantly increased the phosphorylated form of synapsinI to about 55% as compared to that of FMC samples [F(4,21) = 40.636; P < 0.001]. The elevated levels of synapsinI-phosphoSer603 continued in the samples prepared from REC and PRZ treated rats; however, it was not affected in the LPC rats (Fig. 2).
Fig. 2. (A) Western blot of synapsinI-phosphoSer603 bands in synaptosomes prepared from one representative set of experiment under FMC, LPC, REMSD, REC and PRZ injected rat brain have been shown in the upper panel. Western blot of GAPDH bands in the same samples have been shown in the lower panel as loading control. (B) Percentage changes in the band intensities (mean ± SEM) of synapsinIphosphoSer603 in synaptosomal samples prepared from 5 sets of experiments have been shown. ***P < 0.001 significant as compared to FMC. Abbreviations as in the text.
In contrast, although REMSD increased synapsinI and synapsinIphosphoSer603 levels in the rat brain, neither returned to baseline after recovery nor were they prevented by PRZ (Fig. 3). 4. Discussion An optimum level of neurotransmitter is necessary for normal functioning of the brain and expression of behaviors including REMS. Levels of neurotransmitters in specific region of the brain alter with modulation of spontaneous sleep-waking-REMS states [27,31,36] as well as after REMSD [5,17]. Therefore, we hypothesized that REMSD might affect factors responsible for neurotransmitter release. The level of neurotransmitter at the synaptic cleft and the brain as a whole would depend on its release, which in turn is modulated by phosphorylation of the SV-associated protein, synapsinI [14,33]. Involvement of synapsinI in regulating the reserve pool of SVs at the presynaptic terminal may be supported by the fact that decreased number of the SVs has been reported in the absence of synapsinI [29,33]. We observed in this study that total synapsinI was up-regulated in the brain after REMSD as compared to FMC and LPC rats and it continued at elevated level even after 3 days of
3.3. Effects of REMSD on synaptosomal Na-K ATPase activity Na-K ATPase activity was estimated in the same rat brain synaptosomes where total and phosphorylated forms of synapsinI were estimated. The enzyme activity (mean ± SEM) significantly [F(4,21) = 17.424; P < 0.001] increased by 47% in samples prepared from the REMSD rat brains as compared to samples from FMC. The enzyme activity in REMSD was 13.17 ± 1.84 mol Pi released/mg protein/h as compared to that of 9.02 ± 1.36 mol Pi released/mg protein/h in FMC samples. The enzyme activities in all other controls and PRZ treated samples were comparable. 3.4. Comparison of simultaneous effects on Na-K ATPase, synapsinI and synapsinI-phosphoSer603 REMSD increased Na-K ATPase activity, which returned to baseline level after 3 days recovery as well as by PRZ treatment.
Fig. 3. Relative percent changes in Na-K ATPase activity, synapsinI and synapsinIphosphoSer603 expressions under various conditions, as compared to respective FMC values taken as baseline, have been compared. N = 5, ***P < 0.001 significant as compared to FMC. Abbreviations as in the text.
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recovery sleep. Increased total synapsinI induced by REMSD supports increased release of NA in the rat brain after REMSD [30]. This is because phosphorylation of synapsinI releases SVs, from their attachment with the cytoskeleton, which then travels towards the synaptic terminal for release of neurotransmitter. The arguments given above may be supported by the fact that the level of synapsinI regulates availability of SVs for exocytosis in an activity dependent manner [6,7]. In resting condition of a neuron, synapsinI cross-links or tether SVs to one another and with actin-based cytoskeleton to form relatively immobile reserve pool of SVs away from the active zone. However, in active state of neurons, it gets phosphorylated (involving calcium/calmodulindependent protein kinaseII), which in turn reduces its affinity for SVs and actin filaments allowing their movements towards active zone for release of neurotransmitters [14]. Since phosphorylation of Ser603 residue is crucial for SVs mobilization [14], we estimated the level of synapsinI-phosphoSer603 and found REMSD increased phosphorylated form of synapsinI as compared to its level in the FMC and LPC rats. NA and serotonin are known to increase phosphorylation of synapsinI support our views [3,25]. Thus the results of this study support our contention that REMSD promotes release of neurotransmitter; it also helps understanding the intracellular mechanism for increased release of neurotransmitter after REMSD. It has been reported that NA acting on ␣1-adrenoceptor modulates several REMSD associated changes in the rat brain [23]. Under identical REMSD condition several enzyme activities [16,23], neuronal cytomorphology [19,32] and phosphorylation of Na-K ATPase [9] were altered. Several of those REMSD-associated effects returned to FMC level in REC rats and most of the effects were largely reduced by PRZ suggesting the effects were induced by NA and not due to non-specific factors. However, in this study we observed that levels of total synapsinI and its phosphorylated form were not affected by PRZ. This suggested that upon REMSD synapsinI and its phosphorylation at Ser603 residue were unlikely to be regulated by NA acting through ␣1-adrenoceptor. The roles of other receptor-subtypes and neurotransmitters, if any, have not been evaluated. Also, as REMSD affects several parameters in the whole brain, we studied synaptosomes prepared from whole brain, localized brain areas are likely to be affected but quantum of effects may vary and need further study. Classical flowerpot method for REMSD [13,24], if continued beyond 48 h, is known to induce reasonably selective REMS loss and the effects were not due to stress [30]. In this study REMSD was successfully achieved because Na-K ATPase activity increased in the experimental rats and it returned to baseline level after recovery as reported earlier [23]. Although synapsinI and its phosphorylated forms increased after REMSD, they were unaffected in LPC rats suggesting that the effects were specific to REMSD and unlikely due to non-specific factors. However their increased expressions did not return to baseline level after recovery or after PRZ injection (Fig. 3). These findings suggest that in this study the rats were REMS deprived and NA was increased because Na-K ATPase activity increased as reported earlier [12]. However, increase in expression of total synapsinI and its phosphorylated form was either not due to NA or the action of NA was not mediated through ␣1adrenoceptors. It is also evident that although 3 days recovery was sufficient for REMSD induced Na-K ATPase activity to return to normal level, it was not sufficient for recovery of synapsinI and its phosphorylated form. This may be because unlike allosteric modulation of NA induced stimulation of Na-K ATPase activity [1], the REMSD might stimulate expressions of synapsinI, possibly by stimulating its transcription. In conclusion, the findings of this study provide intracellular evidence for increased release of neurotransmitter upon REMSD. Increased phosphorylation of synapsinI supports continued enhanced release of neurotransmitter during REMSD. The
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observation compliments earlier reports that upon REMSD at least REM-OFF neurons continue firing [22] and level of NA is increased in the brain. Acknowledgements SS received fellowship from DBT. Research funding from DST, PURSE, Resource Networking, and JC Bose fellowship to BNM are acknowledged. References [1] H.V. Adya, B.N. Mallick, Uncompetitive stimulation of rat brain Na-K ATPase activity by rapid eye movement sleep deprivation, Neurochemistry International 36 (2000) 249–253. [2] K. Akagawa, Y. Tsukada, Presence and characteristics of catecholaminesensitive Na-K ATPase in rat striatum, Journal of Neurochemistry 32 (1979) 269–271. [3] A. Angers, D. Fioravante, J. Chin, L.J. Cleary, A.J. Bean, J.H. Byrne, Serotonin stimulates phosphorylation of Aplysia synapsin and alters its subcellular distribution in sensory neurons, Journal of Neuroscience 22 (2002) 5412–5422. [4] H.V. Anupama Adya, B.N. Mallick, Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions, Neurochemistry International 33 (1998) 283–286. [5] C.A. Blanco-Centurion, R.J. Salin-Pascual, Extracellular serotonin levels in the medullary reticular formation during normal sleep and after REM sleep deprivation, Brain Research 923 (2001) 128–136. [6] P. Chi, P. Greengard, T.A. Ryan, Synapsin dispersion and reclustering during synaptic activity, Nature Neuroscience 4 (2001) 1187–1193. [7] P. Chi, P. Greengard, T.A. Ryan, Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies, Neuron 38 (2003) 69–78. [8] C. Cirelli, G. Tononi, Differences in gene expression during sleep and wakefulness, Annals of Medicine 31 (1999) 117–124. [9] G. Das, A. Gopalakrishnan, M. Faisal, B.N. Mallick, Stimulatory role of calcium in rapid eye movement sleep deprivation-induced noradrenaline-mediated increase in Na-K-ATPase activity in rat brain, Neuroscience 155 (2008) 76–89. [10] C.H. Fiske, Y. Subbarow, The colorimetric determination of phosphorus, Journal of Biological Chemistry 66 (1925) 375–380. [11] G.F. Gilestro, G. Tononi, C. Cirelli, Widespread changes in synaptic markers as a function of sleep and wakefulness in Drosophila, Science 324 (2009) 109–112. [12] S. Gulyani, B.N. Mallick, Effect of rapid eye movement sleep deprivation on rat brain Na-K ATPase activity, Journal of Sleep Research 2 (1993) 45–50. [13] S. Gulyani, B.N. Mallick, Possible mechanism of rapid eye movement sleep deprivation induced increase in Na-K ATPase activity, Neuroscience 64 (1995) 255–260. [14] S. Hilfiker, V.A. Pieribone, A.J. Czernik, H.T. Kao, G.J. Augustine, P. Greengard, Synapsins as regulators of neurotransmitter release, Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences 354 (1999) 269–279. [15] D. Jouvet, P. Vimont, F. Delorme, Study of selective deprivation of the paradoxal phase of sleep in the cat, Journal de Physiologie 56 (1964) 381. [16] C.A. Kushida, Sleep Deprivation; Basic Science, Physiology and Behavior, 192, Marcel-Dekker, New York, 2005. [17] I. Lena, S. Parrot, O. Deschaux, S. Muffat-Joly, V. Sauvinet, B. Renaud, M.F. Suaud-Chagny, C. Gottesmann, Variations in extracellular levels of dopamine, noradrenaline, glutamate, and aspartate across the sleep–wake cycle in the medial prefrontal cortex and nucleus accumbens of freely moving rats, Journal of Neuroscience Research 81 (2005) 891–899. [18] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, Journal of Biological Chemistry 193 (1951) 265–275. [19] S. Majumdar, B.N. Mallick, Cytomorphometric changes in rat brain neurons after rapid eye movement sleep deprivation, Neuroscience 135 (2005) 679–690. [20] B.N. Mallick, H.V. Adya, S. Thankachan, REM sleep deprivation alters factors affecting neuronal excitability: role of norepinephrine and its possible mechanism of action, in: B.N. Mallick, S. Inoue (Eds.), Rapid Eye Movement Sleep, Narosa, 1999, pp. 338–354. [21] B.N. Mallick, S.R. Pandi-Permual, R.W. McCarley, A.R. Morrison (Eds.), Rapid Eye Movement Sleep—Regulation and Function, Cambridge University Press, Cambridge, 2011. [22] B.N. Mallick, J.M. Siegel, H. Fahringer, Changes in pontine unit activity with REM sleep deprivation, Brain Research 515 (1990) 94–98. [23] B.N. Mallick, A. Singh, REM sleep loss increases brain excitability: role of noradrenaline and its mechanism of action, Sleep Medicine Reviews 15 (2011) 165–178. [24] W.B. Mendelson, R.D. Guthrie, G. Frederick, R.J. Wyatt, The flower pot technique of rapid eye movement (REM) sleep deprivation, Pharmacology Biochemistry and Behavior 2 (1974) 553–556. [25] P. Mobley, P. Greengard, Evidence for widespread effects of noradrenaline on axon terminals in the rat frontal cortex, Proceedings of the National Academy of Sciences of the United States of America 82 (1985) 945–947.
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