Regional synapse gain and loss accompany memory formation in larval zebrafish

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

It is widely believed that memories are formed as a result of alterations in synaptic connections between axons and dendrites, an idea first proposed by Ramon y Cajal (1–4). Although synapse changes have been extensively studied in brain slices in the context of long-term potentiation (5, 6), less is known about how synapses in a living vertebrate are modified when a memory is formed.Memory formation has been widely studied using classical conditioning (CC), a robust and straightforward form of learning in which an animal is exposed to a neutral stimulus (conditioned stimulus, CS) paired with an appetitive or aversive stimulus (unconditioned stimulus, US) that evokes a specific behavioral response (UR, unconditioned response) (7, 8). As a result of the pairing, animals learn to associate the CS with the US, causing them to respond to the CS with a conditioned response (CR) identical to the UR, signifying memory retrieval (9, 10). Memory retrieval is also evoked by activating a cellular engram, a group of neurons active during memory formation and retrieval (11–18). The central locus of CC in mammals, the amygdala (19), is located in a relatively inaccessible area beneath the cortex (20). Thus, although numerous longitudinal imaging studies have documented experience-dependent changes in the structure of spines of cortical and hippocampal neurons (21, 22), few imaging studies have directly examined synaptic changes that occur in the amygdala during associative memory formation.Instead, synaptic changes that occur in the amygdala during CC (23) have been studied primarily using indirect measures of synaptic strength, such as the ratio of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor/N-methyl D-aspartate (AMPA/NMDA) currents in excitatory postsynaptic currents (EPSCs). Increases in AMPA/NMDA ratio in amygdalar neurons following auditory fear conditioning (FC), a type of CC (24–27), indicate that associative memory formation coincides with increases in synaptic strength. In addition, imaging experiments in brain regions beyond the amygdala have shown diverse effects following CC. For example, following contextual fear conditioning, engram neurons in the CA1 region of the hippocampus that receive inputs from CA3 engram neurons displayed spines that were larger and more densely packed than nonengram cells (28). Furthermore, experiments in which neuronal morphology was directly observed before and after FC found that neurons in the frontal association (29) and primary motor cortex (30) showed a decrease in the number of spines, whereas neurons in the auditory cortex showed an increase in spine number with memory formation (31).To obtain previously unavailable insight into memory formation within the central locus of associative memory storage, we developed a paradigm combining in vivo labeling and imaging with informatics and analysis tools. We used this paradigm to map synaptic changes that occur over time in the intact brain of a living vertebrate during memory formation. We imaged the pallium of teleost fish, which contains the putative homolog of the mammalian amygdala based on anatomy (32), gene expression (33), and function (34). The pallium is on the surface of the brain (35), and zebrafish larvae are highly transparent, allowing for intact, whole-brain imaging using selective plane illumination microscopy (SPIM) without the need for invasive intervention (36). In addition, while most studies of learning in zebrafish have used adults (37–40), at least one study showed that larval zebrafish can learn to associate a place with a positive valence US (41). These attributes suggest that larval zebrafish may be an ideal model organism for studying synaptic changes during memory formation due to CC. We have engaged this challenge by combining purpose-built experimental tools with data management software that enables transparent analyses of large and heterogeneous datasets. All data were characterized and stored at the time of creation in a customized data management system designed to conform to findability, accessibility, interoperability, and reusability (i.e., FAIR principles) (see Materials and Methods) (42).     

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.     

Nitric oxide and redox mechanisms in the immune response

The role of redox molecules, such as NO and ROS, as key mediators of immunity has recently garnered renewed interest and appreciation. To regulate immune responses, these species trigger the eradication of pathogens on the one hand and modulate immunosuppression during tissue-restoration and wound-healing processes on the other. In the acidic environment of the phagosome, a variety of RNS and ROS is produced, thereby providing a cauldron of redox chemistry, which is the first line in fighting infection. Interestingly, fluctuations in the levels of these same reactive intermediates orchestrate other phases of the immune response. NO activates specific signal transduction pathways in tumor cells, endothelial cells, and monocytes in a concentration-dependent manner. As ROS can react directly with NO-forming RNS, NO bioavailability and therefore, NO response(s) are changed. The NO/ROS balance is also important during Th1 to Th2 transition. In this review, we discuss the chemistry of NO and ROS in the context of antipathogen activity and immune regulation and also discuss similarities and differences between murine and human production of these intermediates.     

鼻咽刷片细胞学检查和EB病毒检测在鼻咽癌诊断中的意义

目的：探讨鼻咽细胞学检查结合DNA核型判断和细胞EB病毒检测在可疑鼻咽癌病人筛查中的作用。方法：分别对66例可疑鼻咽癌就诊病人作鼻咽刷片细胞学诊断和应用CAS200图像分析仪测定涂片细胞DNA含量,细胞学癌阳性病例同时作EB病毒编码RNA（EBERs)原位杂交。结果：与组织学诊断相比,细胞学诊断和DNA非二倍体诊断癌的敏感性分别为66％和55％,两者结合判断不能提高敏感性,并且假阴性率高;细胞学癌阳性病例的癌细胞核EBERs阳性率92．1％,其中6例DNA二部体核型病例均呈EBERs阳性,可诊断为鼻咽癌。结论：鼻咽细胞学检查结合DNA核型判断不能提高可凝鼻咽癌病人的诊断率,不适用于鼻咽癌筛查。细胞涂片的EB病毒原位杂交方法,在鼻咽癌可疑病人的诊断和鉴别诊断上具有重大实用价值,在鼻咽癌筛查的作用有待进一步研究。     

Use of the Optomap with lid retraction and its sensitivity and specificity#

Background: Optomap uses the ultra‐wide field scanning laser ophthalmoscopy to provide retinal examination. It permits fundus examination without the use of a mydriatic, which is more comfortable for the patients. This paper determines the sensitivity and specificity of the Optomap for detecting retinal signs under non‐mydriatic conditions. Methods: Fifty‐four eyes identified with retinal/choroidal signs and eight normal eyes were recruited from 31 Hong Kong Chinese subjects. Photo‐documentation of fundal changes was obtained with the Optomap under non‐mydriatic conditions before a dilated fundus examination by a clinician using standard procedures. The eyelid was retracted using a cotton bud when necessary. Dilated fundus examinations were performed by another clinician using binocular indirect ophthalmoscopy and slitlamp biomicroscopy with a fundus lens. The Optomap images were evaluated by four other investigators under masked condition. The International Classification of Disease, Ninth Revision (ICD‐9‐CM) was adopted for recording retinal features. Screening results were compared with those obtained using the dilated fundus examination as the gold standard. Results: The cotton bud method for eyelid retraction showed an improvement in the area of retina that could be visualised. The sensitivity and specificity of the Optomap averaged 76.4 and 71.9 per cent, respectively. Some fundal signs were missed by all observers in the Optomap but not with the biomicroscope. These included white‐without‐pressure, lattice degeneration, paramacular drusen and pigmentary changes at central fundus. Conclusion: Optomap serves as a reliable screening tool for fundus examination especially because it covers a much wider area of the peripheral retina than other digital instruments for fundus photography.     

Dysphonia in the aging: physiology versus disease.

A chart review from 151 dysphonic patients over the age of 60 was done to define aging related voice disorders. Overwhelmingly, patients suffered from dysphonia due to disease processes associated with aging rather than to physiologic aging alone. These include: 1. central neurological disorders affecting laryngeal function (e.g., stroke, Parkinson's disease, essential tremor, Alzheimer's disease); 2. benign vocal fold lesions (e.g., Reinke's edema, benign and dysplastic epithelial lesions); 3. inflammatory disorders (e.g., laryngitis sicca, medication effect); 4. laryngeal neoplasia; and 5. laryngeal paralysis. Typical laryngeal findings of vocal fold bowing and breathiness consistent with presbylarynges were present in only six patients. Presbylarynges is not a common disorder and should be a diagnosis of exclusion made only after careful medical and speech evaluation.     

Efficacy of recombinant human erythropoietin in critically ill patients: a randomized controlled trial

Context  Anemia is common in critically ill patients and results in a large number of red blood cell (RBC) transfusions. Recent data have raised the concern that RBC transfusions may be associated with worse clinical outcomes in some patients. Objective  To assess the efficacy in critically ill patients of a weekly dosing schedule of recombinant human erythropoietin (rHuEPO) to decrease the occurrence of RBC transfusion. Design  A prospective, randomized, double-blind, placebo-controlled, multicenter trial conducted between December 1998 and June 2001. Setting  A medical, surgical, or a medical/surgical intensive care unit (ICU) in each of 65 participating institutions in the United States. Patients  A total of 1302 patients who had been in the ICU for 2 days and were expected to be in the ICU at least 2 more days and who met eligibility criteria were enrolled in the study; 650 patients were randomized to rHuEPO and 652 to placebo. Intervention  Study drug (40 000 units of rHuEPO) or placebo was administered by subcutaneous injection on ICU day 3 and continued weekly for patients who remained in the hospital, for a total of 3 doses. Patients in the ICU on study day 21 received a fourth dose. Main Outcome Measures  The primary efficacy end point was transfusion independence, assessed by comparing the percentage of patients in each treatment group who received any RBC transfusion between study days 1 and 28. Secondary efficacy end points identified prospectively included cumulative RBC units transfused per patient through study day 28; cumulative mortality through study day 28; change in hemoglobin from baseline; and time to first transfusion or death. Results  Patients receiving rHuEPO were less likely to undergo transfusion (60.4% placebo vs 50.5% rHuEPO; P<.001; odds ratio, 0.67; 95% confidence interval [CI], 0.54-0.83). There was a 19% reduction in the total units of RBCs transfused in the rHuEPO group (1963 units for placebo vs 1590 units for rHuEPO) and reduction in RBC units transfused per day alive (ratio of transfusion rates, 0.81; 95% CI, 0.79-0.83; P = .04). Increase in hemoglobin from baseline to study end was greater in the rHuEPO group (mean [SD], 1.32 [2] g/dL vs 0.94 [1.9] g/dL; P<.001). Mortality (14% for rHuEPO and 15% for placebo) and adverse clinical events were not significantly different. Conclusions  In critically ill patients, weekly administration of 40 000 units of rHuEPO reduces allogeneic RBC transfusion and increases hemoglobin. Further study is needed to determine whether this reduction in RBC transfusion results in improved clinical outcomes.      

中国流行性乙型脑炎防制策略

流行性乙型脑炎(简称乙脑)是由携带乙脑病毒的蚊子叮咬人体引起的急性中枢神经系统传染病,是一种人畜共患的自然疫源性疾病.临床以高热、嗜睡、惊厥、昏迷、抽搐为主要表现,严重者可致死亡,部分病例可留有后遗症.近年来,我国每年的乙脑病例数波动于5000例～8000例之间,在法定传染病排序中位于前10位以外,其死亡数和病死率分别排在第2位～6位和第4位～8位,在我国局部地区时有暴发,依旧是严重威胁儿童健康的主要传染病之一.本文在对我国乙脑防治工作现况、存在的问题进行分析的基础上,提出了相应的防制策略.……