Forebrain Overexpression Of α CaMKⅡ Abolishes Amygdala Long Term Depression And Disrupts Behavioral Flexibility

It is widely accepted that Ca²⁺/calmodulin-dependent kinaseâ…¡(CaMKII) plays critical roles in regulating learning and memory and synaptic plasticity.By employing an inducible,reversible and region-specific protein knockout technique,Dr Tsien's group has establishedαCaMKII-F89G transgenic mice to manipulate the activity ofαCaMKII in the forebrain and has found that the forebrain expression ofαCaMKII inαCaMKII-F89G transgenic mice altered the bidirectional plasticity response curve in the CA1 region of the hippocampus.Moreover,they have revealed the first post-learning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.In addition,it has reported that in anterior cingulate cortex(ACC),the overexpression of CaMKII abolishes long term depression(LTD),but doesn't affect long term potentiation(LTP).Recently,Cao et al(2008) has investigated that transientαCaMKII overexpression at the time of recall impairs the retrieval of both object recognition memory and fear memories.Further experiments have shown that the recall deficits in these transgenic mice are due to the active erasure of retrieved memories.Meanwhile,it has been proposed by Wang et al that the initial 10 min of memory formation and long term potentiation are sensitive to inducible genetic upregulation ofαCaMKII activity.However,little is known regarding the roles ofαCaMKII in the amygdala synaptic plasticity and amygdala-dependent memory and emotion.Behavioral flexibility plays critical roles in adaptation to unstable environment for animals.Previous studies focused on the function ofαCaMKII in the fixed rule of behavioral tests.However,little work on the role ofαCaMKII in behavioral flexibility is investigated.It is reported that LTD is the synaptic mechanisms underlying behavioral flexibility.Furthermore,αCaMKII is critical for the induction and expression of LTD.So,some questions come up.For example,dose forebrain overexpression ofαCaMKII affect amygdala depedent behavioral flexibility? What is the cellular and molecular mechanism underlying behavioral flexibility? By employing in vitro electrophysiology and behavioral techniques,we investigate the synaptic plasticity at thalamic input synapses onto the LA(T-LA synapses) in vitro, amygdala and hippocampus dependent behavioral flexibility and some emotional behaviors.The main objectives are as follows:1.Investigation of synaptic plasticity at T-LA synapses inαCaMKII-F89G transgenic mice1.1 The basic synaptic transmissionWe have investigated the basic synaptic transmission function of T-LA pathway inαCaMKII-F89G transgenic mice using the brain slice field potential recording technique.The results from the Input-Output and Paired pulse depression curves suggest that presynaptic function and postsynaptic AMPA receptors are normal inαCaMKII-F89G transgenic mice.1.2 Investigation of synaptic plasticity at T-LA synapses inαCaMKII-F89G transgenic miceBy employing the same technique,we have studied the effect ofαCaMKII overexpression specific in forebrain on synaptic plasticity in thalamic input synapses onto the lateral amygdale(LA)pathway.The results show that the low frequency stimulation(1Hz,15min and 3Hz,5min) can induce the significant LTD in wild-type slices but not in transgenic slices.Furthermore,the specific inhibitors ofαCaMKII-F89G(NM-PP1,0.5μM) increased LTD in the transgenic slices to normal level.NM-PP1 and APV(the inhibitor of NMD A receptor;50μM),which are applied to brain slice at the same time,abolish LTD in both groups When middle frequency stimulations are used(10Hz,1.5min),there is no significant difference in synaptic plasticity at T-LA pathway between two groups.We find that high frequency stimulations(2trains of 100Hz,ls and theta bust stimulation) evoke significant LTP in both types of mice,but without significant difference between them.These results suggest that forebrain overexpression ofαCaMKII disrupts the NMDA-dependent LTD inαCaMKII-F89G transgenic slices,and NM-PP1 treatment can reverse this deficit in transgenic slices.However,overexpression ofαCaMKII has no effect on the induction and expression of LTP in amygdala.2.Investigation of behavioral flexibility inαCaMKII-F89G transgenic miceMany previous studies have shown that CaMKII is critical for learning and memory.However,the role ofαCaMKII in behavioral flexibility has been seldom studied.The present study investigates the effects of forebrain overexpression ofαCaMKII on behavioral flexibility using Morris water maze test,fear extinction, conditioned taste aversion extinction.(1) TheαCaMKII-F89G transgenic mice show the normal perception,motivation and motor ability in the visible platform test.In addition,αCaMKII-F89G transgenic mice perform normally in both place navigation training and the 1st spatial probe test.However,compared with wild type mice,αCaMKII-F89G transgenic mice spend significantly more time and swim longer distance to reach hidden platform in the 2nd and 3rd day of spatial reversal learning.(2) In the fear extinction test,NM-PP1 is injected into both types of mice 15 min before retrieval.Compared with wild type mice,αCaMKII-F89G transgenic mice exhibit significantly more freezing responses in the 3rd,4th and 5th trail of contextual extinction test.What's more,in the 2nd,3rd,4th and 5th trail of cued extinction test,significantly more freezing responses also is observed inαCaMKII-F89G transgenic mice.When mice receive NM-PP1 during the 1st to 5th trail of extinction by intraperitoneal(i.p.) injection and oral treatment,no significant difference is found in the freezing responses between two groups of mice.In the foot shock sensitivity threshold test,both groups of mice show flinching, vocalization and jumping at the similar intensities of electrical shock.(3) In the conditioned taste aversion test,αCaMKII-F89G transgenic mice show retrieval deficits.In order to make the same level of acquisition and consolidation of taste memory for both groups of mice,NM-PP1 is injected 15 min before retrieval in the extinction of conditioned taste aversion test.Compared with wild type mice,αCaMKII-F89G transgenic mice exhibit significantly more aversion to saccharin in the 2nd and 3rd trail of the extinction test.NM-PPl application during the 1st to 5th trail of extinction by i.p.injection and oral treatment reverses performance deficits ofαCaMKII-F89G transgenic mice.Baseline saccharin preference and quinine avoidance without uncoditoned stimulation are normal inαCaMKII-F89G transgenic mice,indicating that transgenic mice have normal saccharin preference and taste sensitivity.These results suggest thatαCaMKII overexpression in the forebrain impair behavioral flexibility in different tasks.3.Research on aversive motivated learning tasks inαCaMKII-F89G transgenic micePrevious studies have shown thatαCaMKII overexpression in the forebrain disrupt fear conditioning memory and novel object recognition memory.However, little attention has been paid on the role ofαCaMKII in others aversive motivated memory.We conduct step-down and step-through inhibitory tasks to test somatically-based aversive memory.The results show thatαCaMKII-F89G transgenic mice have significantly shorter escape latency than wild type mice in step-through and step-down inhibitory tasks.Furthermore,these performance deficits can be readily reversed by NM-PP1 treatment before the recall.In addition, in the light-dark transition test,αCaMKII-F89G transgenic mice exhibit normal dark preference naturally.Therefore,our results suggest thatαCaMKII in the forebrain plays a critical role in the retrieval of aversive motivated memory.4.Research on basic and emotional behavior ofαCaMKII-F89G transgenic miceIn order to identify whether the performance deficits ofαCaMKII-F89G transgenic mice in behavioral flexibility and aversive motivated learning tasks have caused by abnormality ofαCaMKII-F89G transgenic mice in basic and emotional behavior,we conduct open field,rotarod test,elevated plus maze test,forced swimming test and tail suspension test.Transgenic animals show normal locomotor activity,exploratory ability,motor coordination and ability in open field and rotarod test.In addition,both groups of mice exhibit the comparable level of anxiety and depression in elevated plus maze test,forced swimming test and tail suspension test. These results demonstrate that forebrain overexpression ofαCaMKII does not affect the locomotor activity and emotional behavior ofαCaMKII-F89G transgenic mice.5.Forebrain morphology ofαCaMKII-F89G transgenic miceBy employing Nissl staining technique,light microscopy show no gross structural abnormalities in the forebrain of theαCaMKII-F89G transgenic mice(3-4 months).Coronal sections show no detectable morphological differences in the insular cortex,hippocampus and amygdala.Results suggest thatαCaMKII overexpression in the forebrain dosen't affect the structure of hippocampus, amygdale and insular cortex.In summary,forebrain overexpression ofαCaMKII disrupts behavioral flexibility and NMDAR dependent LTD in amygdala ofαCaMKII-F89G transgenic mice, suggesting that the abolished amydala LTD may underlie the performance deficits in behavioral flexibility.In addition,αCaMKII-F89G transgenic mice show normal locomotor activity and emotional behavior.Our research is helpful to understand cellular and molecular mechanism of behavioral flexibility.