Involvement Of Actin Rearrangement In The Formation And Extinction Of Conditioned Taste Aversion Memory

Background:A recent World Health Organization report predicts that mental disorder such as fear disorder, depression will be the leading cause of disability and premature death in the worldwide and predicted to grow significantly in the coming years. Despite progress in studies of the etiology and treatment of mental disorder, a clear understanding of the detailed molecular and cellular mechanisms underlying this disorder and the identification of potentially novel drugs are still lacking. It has been reported that the causes of some mental diseases are related to the slower rate of memory extinction. Thus understanding the molecule mechanisms of memory formation and extinction is of great important.The structural remodeling of synapses and formation of new synaptic contacts is a possible mechanism underlying the late phase of long-term potentiation, a form of plasticity which is involved in learning and memory. Actin is the major structural component of synapse, which has been shown to play an important role in synaptic plasticity by maintaining characteristically and highly dynamic transformation between G-actin and F-actin through actin rearrangement. Actin dynamics plays an essential role in learning and memory. It has been demonstrated that synaptic remodeling mediated by actin rearrangements is important for consolidation and extinction of contextual fear conditioning in hippocampus. In addition, consolidation of auditory fear conditioning depends on actin polymerization in the lateral amygdala. Recent study supports that actin rearrangements in the amygdala and dorsal hippocampus are required for the acquisition and consolidation of the aversive memories of drug withdrawal.However, it is still unknown how actin dynamics plays its roles in learning and memory process and what's the structural base of actin rearrangement. In addition, the temporal and spatial regulation of actin dynamics related synaptic structure in different phases of associative memory has not been fully understood.Conditioned taste aversion (CTA) is a type of associative memory in which the subject learns to associate a taste with delayed malaise. CTA has a number of unique temporal properties that distinguish it from other forms of learning, which is formed by single-trial training and is a long-lasting memory with little or no loss of memory months after conditioning. In addition, a strong taste aversion can be induced even if there is a long delay between the CS and the US. These characteristics make CTA a useful model to study the different phases of memory, such as acquisition, consolidation, retrieval and extinction. The neural circuitry of CTA formation includes the insular cortex (IC), amygdala, thalamus, parabrachial nucleus, and nucleus of the solitary tract. The neural circuitry of CTA extinction includes the insular cortex (IC), amygdala, and ventromedial prefrontal cortex (vmPFC).In the present study, using the CTA paradigm and transmission electron microscope technique, we try to address the temporal and spatial involvement of actin rearrangement related synaptic structure alterations in different phases of CTA memory.Objective:1. The synapse structure alterations in different brain areas during CTA short-term memory formation, CTA long-term memory formation and CTA extinction.2. The effect of actin rearrangement within different brain areas during CTA short-term memory formation, CTA long-term memory formation and CTA extinction.3. The involvement of actin rearrangement in different CTA memory processes within a specific brain area.Methods:1. Conditioned taste aversion (CTA)CTA acquisition:In CTA procedure, saccharin (0.1% w/v, sodium salt) was used as an unfamiliar taste and intraperitoneal LiCI (0.15 M,2% body weight) was used as a malaise-inducing agent. The CTA protocol was essentially as described as followed:In brief, rats were trained over 3 days to get their daily water ration within 10 min from 2 pipettes, each containing 10 ml of tap water. On conditioning day, water was replaced with tastant solution (5 ml of 0.1% saccharin). This was followed 40 min later by an intraperitoneal injection of LiCI.4 h or 72 h after the injection of LiCI rats were presented with an array of six pipettes, three containing 5 ml of saccharin and three containing 5 ml of water for 10 min, and their liquid consumption was recorded to test the acquisition of short-term and long-term CTA memory respectively. The aversion index (Al) was defined as [milliliters of water/(milliliters of water+milliliters of saccharin)]×100% consumed in the test.CTA extinction:For extinction, three days after training, the conditioned rats were presented once a day with the choice situation, for four to six consecutive days. The aversion index is {[ml of water/(ml of water+ml of saccharin)]×100} consumed in the test. For the no-extinction control, rats were conditioned but not extinction-trained, that is three days after training, these conditioned rats were presented once a day with 2 pipettes, each containing 10 ml of tap water for three to six consecutive days. The extinction trials were performed in 24 hr intervals and consisted of nonreinforced exposures to LiCl.2. Transmission electron microscopyRats were deeply anesthetized with 5% Chloral Hydrate (0.6 ml/100g) and perfused with 100-200 ml 0.9% sodium chloride (PH 7.4), followed by 100-200 ml phosphate-buffered fixative (PH 7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde at the times indicated in behavioral procedure. Brains were rapidly removed and BLA, IC, PrL or IL was taken, followed by 1% osmium tetroxide treatment, dehydrated, and were then embedded. The serially 65-nm-thick ultrathin sections were then cut and collected on formvar-coated, single-slot grids, stained with uranyl acetate and lead citrate. The synapse ultrastructure was observed using TEM. The detection of synapse density:The density of synapses per unit volume (μm3) was estimated on electron micrographs using the formula Nv=NA/d, where NA is the number of synaptic profiles per unit area and d is the average cross-sectional length of synaptic junctions.The detection of postsynaptic density (PSD) length:the cross-sectional length of synaptic junctions were determined on electron micrographs using MetaMorph software.3. Subcellular functionation and immunoblotting techniquesAfter CTA training 72 h, rats were presented once a day with the choice situation, for four consecutive days. On the fourth day,2 h after CTA extinction test, brains were rapidly removed. Both sides of the PL and IL punched from brain slices. Subcellular fractionation (synaptosome) and actin analysis of the dissected brain tissue was performed. Equal volumes of samples were used to detect immunoreactivity of total actin, F-actin, and G-actin by Western blot analysis. The immunopositive signals were quantified by Metamorph software.4. Surgery and microinjectionMicroinjection was performed via chronically implanted cannulas. Rats were anesthetized and restrained in a stereotaxic apparatus and implanted bilaterally with guide cannulas (stainless steel,23 gauge) aimed at the PrL, BLA or IC. A stylus was placed in the guide cannula to prevent clogging. The injection cannula was extended 1.5 mm from the tip of the guide cannula. Animals were allowed 1 week to recuperate before being subjected to experimental manipulations. Microinjection of cytochalasin D or latrunculin A was performed at the times indicated in behavioral procedures.Results:1. Involvement of actin rearrangement in CTA memory formation1.1 Synapse alterations during CTA short-term memory formationFirst, we try to examine synapse alterations in related brain regions (BLA, IC and PrL) during CTA short-term memory formation using electron microscopy. We found there were no obvious morphological changes of synapses in the BLA or PrL between the CTA training and control groups at 4 h after CTA training. However, increased vesicles assembled near the active zone of synapses were found in the IC of training group. More importantly, quantitative analysis revealed an increased PSD length in the IC, but not in the BLA or PrL compared with other control groups during STM formation (F(4,16)=52.127, p<0.01). In contrast, the synapse density in the BLA, IC, and PrL did not change significantly compared with their control groups during STM formation.1.2 Synapse changes during CTA long-term memory formation Next we investigated the synapse changes during CTA LTM formation in different brain regions. At 72 h after CTA conditioning, post hoc comparisons revealed that both PSD length and synapse density in the IC and PrL were significantly increased during LTM formation compared with other control groups [IC (PSD length:F(4,16)=11.342, p<0.01; synapse density:F(4,17)= 3.887, p<0.05). PrL (PSD length:F(4,16)=8.997, p<0.01; synapse density: F(4,15)=5.028, p<0.01)], which suggested that both the IC and PrL might be involved in CTA LTM formation. However, there were no significant changes in the synapse morphology and density in the BLA during LTM formation.1.3 Actin rearrangement in the IC, but not in the BLA or PrL, is necessary for CTA acquisitionThe previous experiment demonstrated the increased PSD length within the IC during CTA STM formation, it is still unclear whether actin rearrangement is functional necessary for CTA acquisition. To address this question, the inhibitor of actin polymerization was microinjected into the BLA, IC, or PrL 30 min before CTA training, and the aversion index was tested 4 h after conditioning. Injection of the inhibitors into the IC produced a significant reduction of the aversion index tested 4 h after training (F(2,21)= 16.651, p< 0.01). When inhibitors were microinjected into the BLA or PrL, there were no significant differences of STM among the inhibitors and vehicle groups.1.4 Actin rearrangements in the IC and PrL are required in CTA consolidation but not retrievalTo investigate the function role of spatial actin rearrangements in CTA consolidation, cytochalasin D or latrunculin A was infused 4 h after conditioning and LTM was tested 72 h post-conditioning to measure the specific effect of actin dynamics on memory consolidation. Significant differences in aversion index were found among the groups of IC injection (F(2,23)=33.125, p< 0.01) or PrL injection (F(2,27)= 33.605, p<0.01). However, microinjection of cytochalasin D or latrunculin A into the BLA has no effect on the formation of CTA LTM. These results demonstrate that actin dynamics in the IC and PrL but not in the BLA are required for CTA LTM formation. LTM impairment could also be due to memory retrieval deficit. In an additional experiment, we microinjected cytochalasin D or latrunculin A into the BLA, IC, or PrL 30 min before LTM test. All groups were able to acquire similar levels of LTM compared with vehicle group. Thus, inhibition of actin rearrangement in the BLA, IC, or PrL did not interfere with the animals' ability to retrieve acquired CTA memory. In all, these results demonstrate that blocking of actin dynamics in the IC or PrL produces impairment in CTA memory consolidation but not in memory retrieval, implying that there are different roles for actin rearrangement in these two fundamental processes of memory.2. Involvement of actin rearrangement in CTA memory extinction.2.1 Synapse alterations within vmPFC during CTA extinctionRats were divided into three groups:the naive, the no-extinction control and CTA extinction groups. For the CTA extinction group, rats were presented once a day with the choice situation after CTA training 72 h for four consecutive days. For the no-extinction control group, rats were conditioned but not extinction-trained, that is three days after training, these conditioned rats were presented once a day with 2 pipettes, each containing 10 ml of tap water for four consecutive days. On the fourth day,2 h after CTA extinction test, the tissues of PrL and IL were then dissected for electron microscopic (EM) analysis. There were no obvious changes of synapse density in the PrL between the CTA extinction and no-extinction control groups. However, increased synapse density was found in the IL of CTA extinction group compared with their no-extinction control (F(2,8)=26.455, p<0.01). The CTA extinction learning induced synapse density in the IL but not PrL, which suggests the actin dynamics in the IL but not PrL may be involved in CTA extinction.2.2 CTA extinction was accompanied by increased F-actin/G-actin ratio within the IL but not PrL The above results demonstrated that the CTA learning induced the increasement of synapse density in the IL but not PrL. Actin is the major structural component of synapse. To make it clear whether actin rearrangements were involved in the extinction of aversive memories, using immunoblotting technique, we compared the levels of the monomeric actin (G-actin) and the polymerized actin (F-actin) in the PrL and IL in the CTA extinction group with those in the same regions of no-extinction group. A significant elevation of the ratio of F-actin to G-actin was observed in the IL 2 h after CTA extinction on test day 4 (F(1,5)=71.974,p<0.01). However, the ratio of F-actin to G-actin didn't increase in the PrL at 2 h after CTA extinction on test day 4.2.3 Actin rearrangement in the IL is necessary for CTA extinction behaviorAlthough the previous experiment demonstrated the increased synapse density and F-actin/G-actin ratio within the IL but not Prl during CTA extinction, it is still unclear whether actin rearrangement is functional necessary for this period. To address this question,72 h after training, the conditioned rats were presented once a day with the choice situation for six consecutive days. The inhibitor of actin polymerization, cytochalasin D or vehicle was microinjected into the IL immediately after CTA extinction test. The cytochalasin D injection intra IL after each CTA extinction test prevented the extinction of conditioned taste aversion (compared with the vehicle group) Day1,F(1,10)=0.019,p>0.05,Day2,F(1,9)=10.832,p<0.05,Day3,F(1,9)=37.895,p<0 .01,Day4,F(1,9)=15.488,p<0.01,Day5,F(1,10)=56.723,p<0.01,Day6,F(1,5)=29.79 4,p<0.01).2.4 Actin rearrangement in the IL is necessary for CTA extinction acquisition but not extinction consolidationJust like memory formation, memory extinction also includes acquisition, consolidation and retrieval. To further investigate which one of memory process actin rearrangement involved in, we did the following experiment.72 h after CTA training, rats were subjected to a retrieval trial in the absence of reinforcer (t=0). This was followed by a second retrieval trial at the indicated times (t=1,2,3,resectively). Each group of rats was tested on a single time point only in addition to t=0. Extinction becomes apparent at 2 h after time zero. For detection of the acquisition of CTA extinction, inhibitor or vehicle was administered into brain immediately after test of time point zero. And CTA extinction acquisition was tested at t=1,2,3, resectively. We found that cytochalasin D injected intra IL immediately after extinction test prevented the extinction acquisition at 2 h and 3 h (2h,F(i,9)=55.349, p<0.01, 3h,F(1,8)=3.885,p<0.05)but not at 1 h post time point zero. However, cytochalasin D injected intra IL 2 h after extinction test did not prevent the extinction consolidation at 24 h later. Taken together, the results indicate that Actin rearrangement in the IL is necessary for CTA extinction acquisition but not extinction consolidation. Conclusion:1. Different synaptic plasticity during STM and LTM formation of CTA.2. Region-specific involvement of actin rearrangement in conditioned taste aversion.3. The cellular mechanisms of different memory processes in a specific brain area are different.4. CTA extinction is a new learning process and need de novo synapse formation.5. The PrL and IL play different roles in CTA memory formation and extinction.