The Exploration For The Molecular Mechanism Of The Marginal Division Of Striatum Contributing To Learning And Memory

The marginal division of the striatum (MrD) is a newly identified structure made up of spindle-shaped neurons at the caudal-most edge of the neostriatum of the rat brain. It was first discovered by Shu et al in 1987. Ethology research in animal and auditory digital working memory research with fMRI in human have identified that MrD had intense relationship with learning and memory function of brain. Using immunohistochemical and in situ hybridization methods, many neuropeptides, neurotrophic factor, GABA, GABAR_B1, NMDAR1, NMDAR2A, NMDAR2B and DA were found densely distributed in the MrD. Protein kinase A(PKA) , phosphorylated cAMP responsive element binding protein(CREB)and its downstream moleculars: immediate early gene c-fos and c-jun were all identified being involved in the learning and memory related signal transduction in MrD. But these work were extremely not enough to understand the molecular mechanism of MrD contributing to learning and memory.Both protein phosphstase 1(PP1) and Calcineurin(CN) are opposive modulatingcomponents of learning and memory signaling pathway. PP1 is a major eukaryotic serine/threonine protein phosphatase and CN is an ubiquitously ditributing Ca +/CaM dependent serine/threonine protein phosphatase. In brain, they mainly distribute in neurons of cerebral cortex s hippocampus and striatum. PP1 could dephosphorylate phospho-CREB in nucleus and make it deactivated. Activation of PP1 needs the activation of its upstream phosphatase CN, which in turn results in the dephosphorylation inhibitor-1. In recent 10 years, research has identified varietal functions of PP1 and CN in learning and memory. They were involved in depotentiatio^ long-term potentiation(LTP)> long-term depression(LTD)of synaptic effect and cognitive memory s switch from short-term memory to long-term memory ^ brain aging et al.NR2B subunit of NMDA receptor plays a pivotal role in modulating synaptic plasticity and learning and memory. Mice over-expressing NR2B has improved ability in synaptic potentials information acquisition and maintaining. They became more "clever". However, NR2B knockout mice showed defect in memory formation. The open of NMDA receptor channel including NR2B subunit is necessary for the NMDA-dependant ERK1/2 activation. ERK is one kind of MAPK. When cells received stimulus from transmitter such as NMDA ,ERKl/2 could be activated by a series cascades to be phopho-ERKl/2 and translocated from cytosol into nucleus where it can activate neucleoprotein such as transcription factors Elk-K c-Myc and CREB and then regulate transcription activity of many IEGs. ERK may be a point of convergence integrating PKC, PKA, CaMKH signals, in addition to the activation of other kinase-selective target substrates. The ultimate results are changes in neurotransmitter release > gene transcription ^ protein synthesis^ ethology ,the formation of LTPs LTD> learning and memory.Elk-1 is a member of Ets domain transcription factor family. It has a MAP kinasedocking site and sites for phosphorylation by MAPK. ERK-mediated Elk-1 phosphorylation promotes the formation of ternary complex with SRF and potentiates its transcriptional activation activity to activate the IEGs c-fos > Zif268 transcription. Elk-1 mRNA has redundant distribution in rat brain, but the pElk-1 protein distribution in rat brain and the temporal expression change after electrical Y-maze active avoidance learning are not clear till now.Aiming at above mentioned, this paper will be divided into 2 parts. The first part will be explained in 3 aspects: 1 > The distribution of Calcineurin in MrD of striatum and the effect of FK506 MrD injection on long-term avoidance memory in rats. 2> The distribution of PP1 in MrD of striatum and the effect of okadaic acid MrD injection on long-term avoidance memory in rats. 3^ The effect of cerebral ventrical injection of OA on the expression of pCREB after Y-maze training. The second part will be demonstrated in 3 aspects: 1 -. The distribution of pElk-1 in rat brain and the temporal expression change after electrical Y-maze active avoidance learning. 2> The distribution of pERKl/2 in rat brain and the temporal expression changes after electrical Y-maze active avoidance learning. 3> The effect of ifenprodil on the Y-maze memory and memory reconsolidation of rat and the involvement of NR2B-pERKl/2-pElk-l signaling in Y-maze learning and memory. Methods and resultsPart One: The investigation on the suppressive signal transduction during avoidance learning and memory in the MrD.1> The expression of Calcineurin in MrD of striatum and its function in long-term avoidance memory in rats: 29 qualified adult SD rats were divided into 3 groups: normal group(n=8) -. FK506 injection group(n=ll) > saline injection group(n=10). Y-maze training was used as a memory enhanced physiological stimulus and the score of Y-maze test one week after Y-maze training was used as an index oflong term memory. The normal group was used for calcineurin western blot and animals in this group did not receive Y maze training . In the FK506 and saline injection groups,FK506(1.6jig /ul) or 0.9% saline in a 0.5 ul volume was injected into bilateral MrD immediately after the third training and Nissl staining was done after the test to verifying the injection accuracy. Results: 5 rats (2 of FK506 group and 3 of saline group) were rejected because the injection point was deviated from marginal division (MrD). Calcineurin redundantly distributed in rat brain. In the detected 6 regions: olfactory bulb, prefrontal lobe, caudate putamen, MrD> hippocampus> cerebellum, we found fairly intensive CN immuno-positive bands.The third Y-maze training score was 21.78±3.07 in the FK506 injection group and 21.57±2.64 in the saline injection group (P=0.S9). But after one week of the injection ,the Y-maze test score was 24.44±2.13 in the FK506 group and 20.00±2.94 in the saline group (P= 0.003) . The difference was significant.2> The expression of PPl in MrD of striatum and its function in long-term avoidance memory in rats: 26 adult SD rats were divided into normal group(n=7)> OA injection group (n=10) and saline injection group(n=9). Normal group did not receive any training and were used for PPl immunohistochemical staining. In the OA injection group and the saline injection group, Y-maze training was used as an intensive learning stimulus. OA (0.5ul, 40ng/ul) or the same volume saline were injected bilaterally into the MrD immediately after the third training. One week later rats were tested for their Y-maze long-term memory. Nissl staining was used to verify the injection accuracy. Results: PPl immuno-positive neurons could be detected in the whole brain. In cortex > hippocampus> caudate putamen> pyriform area, PPl had the most redundant expression. At MrD, PPl positive neurons had stripped expression. Before the drug injection, the average score of the OA and saline injection groups in the Y-maze training were 22.00±3.10 and 21.83±2.92 respectively. The difference wasnot significant(P=0.923) ; but one week after the injection, the average score of the OA and saline groups in the Y-maze test were 24.71 ±2.2land 20.00±3.46 respectively. The appearance of the OA group rats in Y-maze test was better than the saline group (P=0.013) .3^ Protein phosphatase 1 is involved in the learning and memory related suppressive signal transduction in the marginal division of the rat neostriatum: 18 adult SD rats were divided into two groups: OA injection group (n=10) and the saline injection group (n=8). Y-maze training was used as a memory enhancing physiological stimulus. They received bilateral cerebral ventricles injection of OA (0.8ul, 40ng/|xl) or saline (0.8ul) immediately after the third training and executed 12 hours later. The expression level of pCREB were observed by immuno- histochemical staining. Results: In the saline injection group, pCREB only had slight expression in hippocampus, cortex and pyriform area 12 hours after the Y-maze training. Striatum and MrD had no pCREB expression. In the OA injection group, fairly intensive pCREB expression could be observed in in hippocampus^ cortex ^ pyriform area and amygdalal2 hours after the Y-maze training . In MrD, stripped pCREB expression still could be observed.Part Two: An investigation of the contribution of NR2B-pERKl/2-pElk-l signaling in Y-maze learning and memory1> The distribution of pElk-1 in rat brain and the temporal expression change after electrical Y-maze active avoidance learning: 55 adult SD rats were divided into normal group > Y-maze training group > pseudo-training group. The latter two groups were divided into Ohoius lhour> 3hours, 6hours> 24hours after training subgroups. The Y-maze training group received light-electricity integrated training and the pseudo-training group received light-electricity non-integrated training. pElk-1 expression was detected with immuno- histochemical method. Results: pElk-1immuno-positive neurons distributed diffusely in rat brain. pElk-1 had intensive expression in hippocampus, cortex, amymgdala, caudate putamen> MrD, cerebellum, some nucleuses of hypothalamus. In the Y-maze training group, the pElk-1 expression level increased immediately after the training in hippocampus, cortex, amymgdala, caudate putamenN MrD, cerebellum, some nucleuses of hypothalamus and this increase was maintained in lhour, 3hours, 6hours after training. 24hours after the training, pElk-1 expression returned to normal level in all groups. The pseudo-training group had increased pElk-1 expression in cortex and amygdale at Ohour, lhour, 3hours, 6hours after pseudo-training too. But compared with the training group, the expression increase in hippocampus > caudate putamen and MrD was slight in the pseudo-training groups and these differences were statisticaly significant(/><0.01).2, The distribution of pERKl/2 in rat brain and the temporal expression change after electrical Y-maze active avoidance learning: 55 adult SD rats were divided into normal group, Y-maze training group, pseudo-training group. The latter two groups were divided into Ohour, lhour, 3hours, 6hours, 24hours after training subgroups. The Y-maze training group received light- electricity integrated training and the pseudo-training group received light-electricity non-integrated training. pERKl/2 expression was detected with immuno-histochemical method. Results: immuno-positive neurons distributed only in some areas in rat brain. Supraoptic nucleus of hypothalamus, paraventricular nucleus of hypothalamus had the most intensive pERKl/2 expression. Then were the molecular layer of cerebellum , Purkinje's layer, amygdala, striate cortex. In hippocampus, only a few neurons and some positive fabric were found. In caudate putamen and MrD,no positive neuron was found. 0 hour after the Y-maze training, pERKl/2 positive neurons emerged in the caudate putamen , MrD, CA2, CA3 of hippocampus. Most of cortex, amygdalahad enhanced pERKl/2. At lhour, 3hours, 6hours after training, pERKl/2 expression enhancement was remained. The pseudo-training group had enhancement in cerebellum, amygdala, striate cortex ,but only few positive pERKl/2 expression in hippocampus, caudate putamen and MrD at every time point.3,NR2B-pERKl/2-pElk-l signaling contributes to the Y-maze learning and memory: 45 adult male SD rats were divided into 4 groups: 1 > ifenprodil pretraining peritoneal injection group (ifenprodil ip group, n=14), 2, vehicle pretraining peritoneal injection group (DMSO ip group, n=15) .7 animals from the first two groups repectively were executed after training for immuno- histochemical staining of pERKl/2 and pElk-1.3, ifenprodil post-testing cerebral ventricle injection group (ifenprodil ic group, n=8),4, vehicle post-testing cerebral ventricle injection group (DMSO ip group, n=8). The last two groups were undertaken Western blotting of pERKl/2 and pElk-1 after memory reconsolidation test. Y-maze training and test were used as an intensive learning and memory enhancing stimulus. Results: Compared with the DMSO ip group, ifenprodil ip group 15 minutes before training did not change Y-maze learning score(P=0.864), but impaired Y-maze memory tested 24 after learning(P=0.033). Ifenprodil ip injection made brain pERKl/2 and pElk-1 expression decreased generally. In hippocampus, caudate putamen, MrD, amygdala, most of cortex, the decrease was more significant. Compared with the DMSO ic group,the reconsolidation of Y-maze memory tested 6hours after drug injection was impaired in ifenprodil ic group(P=0.025). The OD value of pERKl/2 and pElk-1 positive bands in ifenprodil ic group attenuated in 6 detected regions: hippocampus, amygdala, prefrontal lobe, olfactory bulb, caudate putamen, MrD. And in MrD and caudate putamen, the pElk-1 positive bands almost disappeared in ifenprodil ic group. ConclusionsPart One : The investigation on the suppressive signal transduction during avoidancelearning and memory in the MrD.K Calcineurin redundantly expresses in learning-related brain regions. In MrD,calcineurin has intense expression.Injection of FK506 in MrD can enhance long-termavoidance memory of rats.2> PPl has widespread distribution in rat brain. In MrD, PPl shows intensive stripeddistribution.The injection of OA in MrD causes Y-maze long-term memorypotentiation.3 s Inhibition of PPl activity can enhance pCREB expression of MrD after Y-mazelearning.4, Based on the evidences mentioned above,it is reasonable to believe thatCalcineurin and PPl are involved in the Y-maze avoidance long term memory of MrD,and they may function as the suppressive modulators in the signaling associated withY-maze avoidance long term memory formation of MrD.Part Two: An investigation of the contribution of NR2B-pERKl/2-pElk-l signaling inY-maze learning and memory1> pElk-1 has widespread distribution in rat brain. Y-maze learning potentializespElk-1 expression in hippocampus n caudate putamen> MrD. The activation of Elk-1contributes to acquisition n short-term memory > switch from short-term memory tolong-term memory > long-term memory of Y-maze.2, pERKl/2 has widespread but slight distribution in rat brain. Y-maze learninginduces pERKl/2 expression in hippocampusn caudate putamen^ MrD. The activationof ERK1/2 contributes to acquisition , short-term memory > switch from short-termmemory to long-term memory > long-term memory of Y-maze.3^ NR2B is essential for the formation of long term memory ^ reconsolidation ofY-maze memory. The deactivation of NR2B by ifenprodil via peritoneal injection orcerebral ventricle injection will impair these courses. Meanwhile, the deactivation of NR2B attenuates pERKl/2 and pElk-1 expression in learning and memory related regions after Y-maze learning and memory reconsolidation test. In MrD and CPu, the pElk-1 expression are completely blocked by ifenprodil after memory reconsolidation test.4> Based on the evidences mentioned above, it is reasonable to believe that NR2B-pERKl/2-pElk-l signaling contributes to the acquisition formation of long term memory^ memory reconsolidation. This signaling is essential for long term memory formation and reconsolidation of Y-maze memory. In MrD and CPu, this signaling may have unique function in Y-maze learning and memory.