The Effects of Nicotine on Spatial Learning and Memory and DREAM Protein Expression on REM Sleep Deprived Rat Hippocampus
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This study investigated the effects of nicotine treatment on spatial learning, memory, and DREAM protein expression in the hippocampus of rats deprived of REM sleep. Rats were divided into four groups: control, control treated with nicotine, REM sleep deprived, and REM sleep deprived treated with nicotine. REM sleep deprivation impaired spatial learning and memory as shown by decreased time in the target area during testing. Nicotine treatment prevented this impairment and improved performance of REM deprived rats. Preliminary results found DREAM protein was expressed in the hippocampus of all groups but differences between groups require further investigation to understand the protein's role. Future studies are needed to clarify the relationship between DREAM expression and cognitive performance.
The Effects of Nicotine on Spatial Learning and Memory and DREAM Protein Expression on REM Sleep Deprived Rat Hippocampus
- 1. THE EFFECTS OF NICOTINE TREATMENT ON SPATIAL LEARNING AND MEMORY AND DREAM PROTEIN EXPRESSION IN REM-SLEEP DEPRIVED RAT HIPPOCAMPUS MOHAMMAD JAMEL MOHD ALWAI SCHOOL OF HEALTH SCIENCES, UNIVERSITI SAINS MALAYSIA HEALTH CAMPUS
- 2. Introduction • Sleep is critical for the maintenance of health and support of life. • Two types of sleep; rapid eye movement (REM) and non-REM sleep (Campbell, 2009) • REM sleep is important for learning and memory formation in the hippocampus (Stickgold & Walker, 2007) • REM sleep deprivation can affect hippocampal synaptic plasticity – vital for learning acquisition and memory consolidation (Yang et al., 2008)
- 3. DREAM Protein • Downstream Regulatory Antagonistic Modulator (DREAM) protein involved in learning and memory processes. • Acts as negative regulator of the key memory factor cAMP- response element binding (CREB) protein in a Ca2+-dependent manner. • DREAM gene knockout facilitates CREB-dependent transcription and markedly enhances learning and synaptic plasticity with improved cognition. (Fontan-Lozano et al., 2009)
- 4. Nicotine (C10H14N2) • Derived from plants of family Solanaceae, from genus Nicotiana spp. (Chase et al., 2003) • An agonist for nicotinic acetylcholine receptor (Poorthuis et al., 2009). • Acute treatment of nicotine has been shown to prevent impairment of memory and synaptic plasticity of CA1 region of hippocampus due to REM sleep-deprivation (Aleisa et al., 2011) Figure 1: Chemical structure of nicotine
- 5. Rationale of Study To study how nicotine treatment can prevent impairment of learning and memory induced by REM sleep deprivation.
- 6. Research Question Whether administration of nicotine can prevent impairment of spatial learning and memory and synaptic plasticity through modulation of DREAM protein expression in REM sleep-deprived rat hippocampus.
- 7. General Objective To determine the effects of nicotine treatment on spatial learning and memory and DREAM protein expression in REM sleep- deprived rat hippocampus.
- 8. Specific Objectives • To determine the effects of nicotine (nicotinic acetylcholine receptor agonist) treatment on spatial learning and memory performance on REM sleep-deprived rats. • To determine the effects of nicotine (nicotinic acetylcholine receptor agonist) treatment on DREAM protein expression in REM sleep- deprived rat hippocampus.
- 9. Methodology
- 10. Drug Administration • Nicotine (1mg/kg) (Aleisa et al., 2006) was injected subcutaneously every 12h during 72h REM sleep-deprivation. • The dose given is known to produce nicotine blood levels similar to those of chronic smokers (Le Houezec et al., 1993)
- 11. REM sleep-deprivation • REM sleep deprivation used modified inverted flowerpot method (Siran et al., 2009). • Non-REMsd rats were placed in normal propylene cages.
- 12. Morris Water Maze • Circular pool filled within 20cm off the top of wall with water (20-22ºC). • Water were made opaque by skim milk powder • Platform (Ø 12cm, white Plexiglas).
- 13. Morris Water Maze • A camera was mounted on top of the maze and connected to a PC with MWM software. • Before performing the task, rats were placed on the platform for 10 s to familiarise it-self with the maze. • Rats were trained to find platform from 4 different directions around the edge of the pool for every day for 5 days. Figure 2: Morris Water Maze procedure
- 14. Morris Water Maze • The time (s) of escape latency were recorded to evaluate spatial learning and memory performance. • If rats failed to find platform in 60s, they were placed on the platform; latency were recorded as 60s. • Surrounding objects/visual objects were fixed to serve as cues for the rats to locate the platform. • Probe test was performed at day 6 without the platform to measure time rats spent in original platform position (target quadrant)
- 15. Immunohistochemistry analysis • After 24h MWM, rats were sacrificed via overdose injection of sodium pentobarbitone. • Brains were dissected out, cryoprotected in sucrose 20% overnight. • Brain sections were cut 40 µm thin using cryostat and hippocampus region collected as free-floating sections in PBS.
- 16. Immunohistochemistry analysis • Sections were rinsed with Tris-buffered saline (TBS) and incubated with primary antibodies for DREAM protein for 48h. • Then, the sections were incubated with biotinylated secondary antibody for 1h. • Sections were then reacted with Avidin-biotin complex (ABC) and stained with diaminobenzidine and hydrogen peroxide until brown colour is seen • Section then were mounted on slides, air-dried, dehydrated and put cover slip on it.
- 17. Body weight gain Comparison between groups in body weight gain during dry and wet condition. Dry condition Wet condition Body weight gain was reduced in both REMsd and REMsd+N groups during wet condition. RESULTS:
- 18. Food Intake Comparison in food intake during dry and wet condition for all groups. There was no significant difference in food intake during dry and wet condition for all groups.
- 19. Comparison in water intake during dry and wet condition for all groups.*, p < 0.01, compared between dry and wet conditions Water intake was reduced in REM sleep deprivation groups during wet condition. Water intake
- 20. Escape latency Escape latency time for all groups during MWM 5 days trial. Within days, escape latency time for all groups were reduced however not significant difference between groups during 5 days trial.
- 21. Percentage of time spent in target quadrant Percentage of time spent in target quadrant in all groups. #, p < 0.05, compared between C+N and REMsd ??, p < 0.05, compared between C and REMsd There was significantly least time spent in target quadrant for REMsd group.
- 22. DREAM protein expression DREAM protein expression of A) control group (C); B) nicotine-treated control (C+N); C) sleep-deprived group (REMsd); and D) nicotine-treated sleep-deprived group (REMsd+N). (CA1= CA1 area of hippocampus; CA3= CA3 area of the hippocampus; DG= dentate gyrus)
- 23. DISCUSSIONS • Data for food and water intake and body weight – REMsd and REMsd+N rats showed constant food intake but significant decrease in body weight and water intake during wet condition – Similar result found in previous study in our lab (Siran et al., 2014) – Decrease in body weight with constant food intake represent negative energy state by a number of changes to physiological systems – Sleep will usually limit metabolic rate i.e. there was an increase in energy expenditure • These results suggests the accomplishment of induction of REM sleep-deprivation in rats in this study
- 24. Morris Water Maze • Result show gradual decrease of escape latency recorded by all groups within 5 days of MWM trial. • C+N group has the fastest time in finding the platform especially in days 3 to 5 compared to other groups. – However a study (Alzoubi et al., 2006) reported nicotine treatment may have no effects on normal subjects. – Our results could suggest that rats experienced mild stress during some part of the experiment.
- 25. Probe test • REMsd group has the least percentage of time spent in target quadrant. – This supports our understanding that REM sleep-deprived rats have impaired some of their learning and memory processes. • However the nicotine treatment showed some of the beneficial effects by abolishing this effect (as shown by REMsd+N groups) • Meanwhile the percentage of time spent in target quadrant in REMsd+N was found to be not significantly different compared to C and C+N groups. • Transdermal nicotine treatment has also been showed to abolish mild cognitive impairment in Alzheimer patients (Roh & Evins, 2012)
- 26. Immunohistochemistry analysis • Preliminary results showed that DREAM protein was expressed in all regions of hippocampus in all groups. – This propose that DREAM protein could be involved in spatial and learning processes in this study. • However, the mechanism how DREAM protein modulated the spatial learning and memory processes are still unclear.
- 27. CONCLUSION • This study shows that REM sleep deprivation partially affected spatial learning and memory performance. • Treatment of nicotine has improved this effect. • The contribution of DREAM protein modulation in this effect is still unclear and required further study to elucidate the mechanism.
- 28. LIMITATION • Time constraints – Sleep deprivation using wide platform cannot be done. • Technical problems – Immunohistochemistry study cannot be finished – Comparison of DREAM protein expression between groups cannot be seen.
- 29. FUTURE DIRECTIONS • Further studies are needed to link between the expression of DREAM protein and spatial learning and memory performance. This could be done by – Quantifying DREAM protein expression in the hippocampus and compare between groups. – Quantifying another protein which involved in learning and memory such as CREB and BDNF protein. – Prolonging the period of MWM study by addition of reverse test after the probe test where platform is positioned at opposing quadrant than that of original position and trial is done for another 5 days.
- 30. REFERENCES • Aleisa, A. M., Helal, G., Alhaider, I. A., Alzoubi, K. H., Srivareerat, M., Tran, T. T., Al-Rejaie, S. S. Alkadhi, K. A. (2011). Acute nicotine treatment prevents REM sleep deprivation-induced learning and memory impairment in rat. Hippocampus, 21(8), 899-909. • Alzoubi, K. H., Aleisa, A. M., Gerges, N. Z., & Alkadhi, K. A. (2006). Nicotine reverses adult-onset hypothyroidism-induced impairment of learning and memory: Behavioral and electrophysiological studies. Journal of Neuroscience Research, 84(5), 944-953. • Campbell, I. G. (2009). EEG Recording and Analysis for Sleep Research. Current Protocols in Neuroscience, 49. • Chase, M. W., Knapp, S., Cox, A. V., Clarkson, J. J., Butsko, Y., Joseph, J., Savolainen, V., Parokonny, A. S. (2003). Molecular Systematics, GISH and the Origin of Hybrid Taxa in Nicotiana (Solanaceae). Annals of Botany, 92(1), 107-127 • Fontán-Lozano, Á., Romero-Granados, R., del-Pozo-Martín, Y., Suárez-Pereira, I., Delgado-García, J. M., Penninger, J. M., & Carrión, Á. M. (2009). Lack of DREAM Protein Enhances Learning and Memory and Slows Brain Aging. Current Biology, 19(1), 54-60. • Le Houezec, J., Jacob, P., III, & Benowitz, N. L. (1993). A clinical pharmacological study of subcutaneous nicotine. European Journal of Clinical Pharmacology, 44(3), 225-230. • Poorthuis, R. B., Goriounova, N. A., Couey, J. J., & Mansvelder, H. D. (2009). Nicotinic actions on neuronal networks for cognition: General principles and long-term consequences. Biochemical Pharmacology, 78(7), 668– 676. • Roh, S., Evins, A. E. (2012). Possible role of nicotine for the treatment of mild cognitive impairment. Expert Review of Neurotherapeutics 12(5), 531–533 • Siran, R., Ahmad, A. H., Abdul Aziz, C. B., & Ismail, Z. (2014). REM sleep deprivation induces changes of down regulatory antagonist modulator (DREAM) expression in the ventrobasal thalamic nuclei of Sprague-Dawley rats. Journal of Physiology and Biochemistry, 70(4), 877-889. • Siran, R., Ahmad, A. H., & Ismail, Z. (2009). Down Regulatory Antagonist Modulator (DREAM) expression in relation to formalin-induced pain in rapid eye movement (REM) sleep deprived rats. European Journal of Pain, 13(S1), S54. • Stickgold, R., & Walker, M. P. (2007). Sleep-dependent memory consolidation and reconsolidation. Sleep Medicine, 8(4), 331–343. • Yang, R. H., J., H. S., Wang, Y., Zhang, W. B., Luo, W. J., & Chen, J. Y. (2008). Paradoxical sleep deprivation impairs spatial learning and affects membrane excitability and mitochondrial protein in the hippocampus. Brain Research, 1230, 224–232.
- 31. THANK YOU
Editor's Notes
- #17: immunoperoxidase three-step avidin-biotin peroxidase method