The Effect Of Dopamine D₁Receptor In The Prefrontal Cortex On Learning And Memory Of Chronic Sleep Deprivation Rats

With the fast living style, fierce competition and more pressure of modern society, people spend more time in work, entertainment and social contact. As a result, the sleep time is gradually decreasing, and more people suffer from insomnia and other sleep disorders, notably sleep deprivation (SD). SD, especially the incidence of the chronic sleep deprivation (CSD) is more frequently and serious in special occupational groups such as pilots, seamen, doctors, drivers, etc. and in some unusual cases, for example, disease, war, floods and earthquakes. SD can cause cognitive dysfunction, reduce alertness and behavior control ability. More serious is, due to the errors of judgment and decision-making, the safety accidents happen more frequently. So far, the exact mechanism of chronic sleep deprivation induced learning, memory and cognitive dysfunction are not fully understood.Functional imaging studies have shown that SD induces the metabolic activity changes in parietal lobe, prefrontal lobe and cuneate lobe. Applying different kinds of drugs can improve the metabolic activity of these brain regions and the cognitive impairments caused by SD. These evidence showed that the neocortex plays an important role in SD induces neurobehavioral changes. Prefrontal cortex (PFC) is neocortex, which located in the anterior part of the frontal lobe. PFC is involved in learning, memory, planning, decision-making, attention and other higher nervous activity. It also plays an irreplaceable role in maintaining alertness; inhibiting inappropriate behavior and emotional reactions, reducing the decision-making errors. So, PFC may play critical function in SD induced learning and memory dysfunction, which provides evidence for the relationship between SD and PFC cognitive function.Different neurotransmitters and receptors in the CNS may regulate the synaptic plasticity changes caused by SD, and then affects the learning and memory function. Dopamine (DA) in PFC comes mainly from the midbrain ventral tegmental area, which involves in the regulation of sleep-awakening process and motivation, cognition, learning and memory consolidation and other higher nervous function. Therefore, SD induced DA and D1R disorder in PFC may be a key factor of learning, memory and cognitive dysfunction. DA is released from the presynaptic and plays a physiological effect via DA receptors. DA receptors, including5subtypes (D1～D5), D1and D5belong to the D1-like receptors; D2, D3and D4are D2-like receptors. In the PFC of monkeys and rodents,D1 receptor binding sites and mRNA are significantly more than other types of receptors. Moreover, D1receptor in PFC may have a role to promote learning and memory. So in this study, we focused the effect of CSD on DA and D1receptor and their effect on learning and memory impairment after CSD.The mechanisms of learning and memory dysfunction after CSD are not understood clearly. In particular, the role of prefrontal cortex D1receptor in this process is still unknown. So this study established rats long term REM sleep deprivation model via depriving sleep for consecutive21d (18h/d) to detect the underlying mechanisms. To observe how CSD affected the general state, learning and memory abilities, PFC neurons, synaptic structure, content of DA and the expression of D1receptor were determined. After administration of D1receptor agonist SKF38393, we observed the above indicators again to clarify the D1and its receptor in PFC, which may play an import role in the cognitive dysfunction caused by CSD. This study may provide new evidence that SD could decrease work efficiency and increase work accident. As well, this study may provide some possible target and strategy to develop some drugs aiming at reducing sleep disorder.Part1Chronic sleep deprivation could impair rats general state, learning and memory abilitiesObjective:To establish chronic sleep deprivation model and detect the changes of general state and learning memory abilities after CSD.Methods:110male Sprague Dawle rats were screened by Morris water maze and swimming training, then90rats were selected and randomly divided into3groups:blank control (BC, n=30), treatment control (TC, n=30) and chronic sleep deprivation (CSD, n=30). Use the modified multiple platform sleep deprivation (MMPM) for CSD model. Rats were deprived sleep for18h a day in consecutive21d. Observed the CSD rats weight, fur, general state and reaction ability. Morris water maze and open field test were used to determine rats learning, memory and autonomic activity ability.Results:Compared with BC and TC group, CSD group was listless and irritated, with hair color matte. CSD group body weight decreased obviously than the other2groups in any time points and showed a decline during the whole sleep deprivation (P<0.01). CSD rats exhaustive time of weight loading swimming test significantly reduced compaired with other2groups (P<0.01). Morris water maze latency was significantly prolonged, the number of platform crossings significantly reduced (P<0.01), and quadrant dwell time percent reduced compared with the other2groups (P<0.05). The auto motion distance and times were decreased in CSD group (P<0.05). There was no statistical difference between TC group and BC group (P>0.05).Conclusion:Chronic sleep deprivation changed the rats general state and damaged the rats learning, memory and the autonomic activity ability.Part2The impact of chronic sleep deprivation on prefrontal cortex neurons and ultrastructureObjective:To detect the changes of PFC neural structures in CSD via morphological methods and to discuss the histological mechanisms of CSD induced learning and memory dysfunction.Methods:At the end of the CSD protocol, selected6rats randomly from each group, rats were rapidly infused with4℃physiological saline by cardiac perfusion after anesthesia to flush blood, then infused with4℃4%paraformaldehyde-PBS, and fixed the PFC tissues in4%paraformaldehyde-PBS for24h. The apoptosis of PFC was evaluated by TUNEL assay.4rats per group were infused with4℃physiological saline by cardiac perfusion to flush blood after anesthesia, then infused with4℃4%paraformaldehyde-2.5%glutaraldehyde-PBS, at last fixedthe PFC tissues in4℃mixture fixation buffer above for24h. Prepared the PFC tissue as1×1×1mm3and observed the ultrastructural changes by transmission electron microscope.Results:TUNEL assay showed that PFC neuronal apoptosis in CSD group increased compared with BC and TC group (P<0.01). The electron microscopy results showed that, compared with BC and TC group, the number of PFC neuronal mitochondria in CSD group decreased and the structure of mitochondrial cristae was fuzzy and ruptured. Some synapses were edematous; the number of synaptic vesicles decreased and the postsynaptic dense zone were much thin or disappeared. Rough endoplasmic reticulum structures became fuzzy and the ribosome significantly decreased or disappeared. The neuronal morphology, the number of TUNEL stain positive neurons showed no difference between TC group and BC group (P>0.05). The ultrastructure was also normal.Conclusion:CSD could cause more PFC neurons apoptosis and induce ultrastructural damage. The neuronal morphology changes may affect PFC physiological functions, consequently, may impair rats learning and memory.Part3The impact of chronic sleep deprivation on the prefrontal cortex dopamine content and D1receptor expressionObjective:To determine the changes of DA content and D1receptors in rats PFC after chronic sleep deprivation, and to elucidate the likely molecular mechanisms of learning and memory dysfunction caused by CSD.Methods:At the end of the CSD21d protocol,14rats were infused with4℃normal saline by cardiac perfusion after anesthesia, and the PFC tissues were isolated on ice.6PFC tissues per group were used in Western blot to analyze the expression of D1receptor.8PFC tissues per group were used in high performance liquid chromatography-electrochemical detectors (HPLC-ECD) detection. Another6rats per group were anesthetized and accepted4%paraformaldehyde-PBS solution cardiac perfusion, then PFC tissues were fixed in4%paraformaldehyde-PBS solution for24h. D1receptor location and expression were determined by immunohistochemistry (IHC).Results:Compared with BC group and TC group, CSD group DA content was significantly decreased (P<0.01), and the expression of D1receptors in PFC was significantly decreased (P<0.05) as well. IHC showed D1receptor positive stained areas in PFC mainly located on neurons. When compared CSD group with the other two groups, D1receptors positive stained cells were significantly lower (P<0.01). TC group and BC group had no significant statistical difference in the above indicators (P>0.05).Conclusion:CSD could reduce DA content and D1receptor expression in PFC. The results suggested that decreased DA content and D1receptor expression in PFC may down-regulated neurons excitability, which may be a crucial regulator of learning and memory dysfunction induced by CSD.Part4D1receptor agonist improved learning and memory abilities, prefrontal cortex neurons structure, dopamine content and D1receptor expression in chronic sleep deprivation ratsObjective:To evaluated learning memory abilities, PFC neurons structure, DA content and D1receptor expression after administration of D1receptor agonist SKF38393on CSD rats.Methods:110male male Sprague Dawle rats were screened by Morris water maze and swimming training, then90rats were selected and randomly divided into3groups:big platform treatment control group (TC+PBS, n=30), chronic sleep deprivation group (CSD+PBS, n=30) and chronic sleep deprivation treated with D1receptor agonist SKF38393group (CSD+drug, n=30). Rats underwent sleep deprivation18h a day in consecutive21d. Besides CSD, the CSD+drug group rats were also administered SKF38393(1mg/kg) from15d to21d in CSD protocol, while TC+PBS group and CSD+PBS group were treated with equal volume PBS. Use Morris water maze and open field test to determine rats learning memory abilities and autonomic activity ability. The apoptosis was evaluated by TUNEL assay. Tissues were observed ultrastructural changes by transmission electron microscope. Western blot and immunohistochemistry were used to analyze the expression of D1receptor.Results:Compared with CSD+PBS group, CSD+drug group did not show significant differences in hair colour, body weight and exhaustive time of weight loading swimming test (P>0.05), but compared with TC+PBS group, the CSD+drug group body weight decreased at every time point during CSD protocol (P<0.01), and the exhaustive time of weight loading swimming test also significantly reduced (P<0.01). CSD+drug group and CSD+PBS group didn’t show differences in latency, platform crossings and quadrant dwell time percent at7d and14d after CSD (P>0.05). When treated with SKF38393for7d, that was21d after CSD, CSD+drug group rats showed decreased latency, increased platform crossings and quadrant dwell time percent compared with CSD+PBS group (P<0.05). While CSD+drug group still showed higher latency, lower platform crossings and quadrant dwell time percent in comparison with TC+PBS group at any time point (P<0.01). When treated with SKF38393for7d, that was21d after CSD, CSD+drug group rats showed increased auto motion distance and times compared with CSD+PBS group (P<0.05). While compared with TC+PBS group, CSD+drug group still showed lower auto motion distance and times (P<0.01). CSD+drug group showed decreased neuronal apoptosis compared with CSD+PBS group in TUNEL stain (P<0.01). While compared with TC+PBS group, CSD+drug group still showed more TUNEL stain positive neurons (P<0.01). The electron microscopy results showed that, compared with CSD+PBS group, the number of PFC neuronal mitochondria in CSD+drug group increased and the structure of mitochondrial cristae were clearer. Synaptic vesicles increased, and the postsynaptic dense zones were much thicker. Rough endoplasmic reticulum structures became clearer and the ribosome significantly increased. While compared with TC+PBS group, all results above were still worse in CSD+drug group. Compared with CSD+PBS group, CSD+drug group DA content was significantly increased (P<0.01), but compared with TC+PBS group, CSD+drug group DA content was still decreased (P<0.01). Compared with CSD+PBS group, CSD+drug group D1receptor expression in PFC was significantly increased (P<0.05); but compared with TC+PBS group, CSD+drug group D1receptor expression in PFC was still decreased (P<0.01). IHC showed, compared with CSD+PBS group, CSD+drug group D1receptor positive stained cells were significantly increased (P<0.01); but compared with TC+PBS group, CSD+drug group D1receptor positive stained positive cells were still decreased (P<0.01).Conclusion:After treated with D1receptor agonist SKF38393, the CSD rats general state improved and learning memory abilities partially restored. DA content and the expression of D1receptor in PFC significantly increased. The PFC neuronal apoptosis and ultrastructure also improved. Thus, activation of D1receptor may improve DA content and D1receptor function, which restored learning and memory dysfunction caused by chronic sleep deprivation.