RESUMO
Electrical excitability-the ability to fire and propagate action potentials-is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage-gated sodium channels, and to fire action potential trains. Inactivating this signaling pathway in Schwann cells impairs somatosensory neuron maturation, causing multimodal sensory defects that persist into adulthood. Collectively, our studies uncover a neurodevelopmental role for prostaglandin E2 distinct from its established role in inflammation, revealing a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.
Assuntos
Potenciais de Ação , Dinoprostona , Células de Schwann , Células Receptoras Sensoriais , Animais , Células de Schwann/metabolismo , Dinoprostona/metabolismo , Camundongos , Células Receptoras Sensoriais/metabolismo , Transdução de SinaisRESUMO
The canonical model of striatal function predicts that animal locomotion is associated with the opposing regulation of protein kinase A (PKA) in direct and indirect pathway striatal spiny projection neurons (SPNs) by dopamine1-7. However, the precise dynamics of PKA in dorsolateral SPNs during locomotion remain to be determined. It is also unclear whether other neuromodulators are involved. Here we show that PKA activity in both types of SPNs is essential for normal locomotion. Using two-photon fluorescence lifetime imaging8-10 of a PKA sensor10 through gradient index lenses, we measured PKA activity within individual SPNs of the mouse dorsolateral striatum during locomotion. Consistent with the canonical view, dopamine activated PKA activity in direct pathway SPNs during locomotion through the dopamine D1 receptor. However, indirect pathway SPNs exhibited a greater increase in PKA activity, which was largely abolished through the blockade of adenosine A2A receptors. In agreement with these results, fibre photometry measurements of an adenosine sensor11 revealed an acute increase in extracellular adenosine during locomotion. Functionally, antagonism of dopamine or adenosine receptors resulted in distinct changes in SPN PKA activity, neuronal activity and locomotion. Together, our results suggest that acute adenosine accumulation interplays with dopamine release to orchestrate PKA activity in SPNs and proper striatal function during animal locomotion.
Assuntos
Adenosina , Corpo Estriado , Proteínas Quinases Dependentes de AMP Cíclico , Dopamina , Locomoção , Neurônios , Animais , Camundongos , Adenosina/metabolismo , Corpo Estriado/citologia , Corpo Estriado/enzimologia , Corpo Estriado/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Locomoção/fisiologia , Neurônios/enzimologia , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo , Receptor A2A de Adenosina/metabolismoRESUMO
Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.
Assuntos
Técnicas Biossensoriais , Animais , Camundongos , Técnicas Biossensoriais/métodos , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Drosophila/metabolismoRESUMO
Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.
Assuntos
Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácidos Mirísticos/metabolismo , Neurônios/metabolismo , Potenciais de Ação , Motivos de Aminoácidos , Animais , Membrana Celular/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/química , Neurônios/fisiologia , Processamento de Proteína Pós-Traducional , RatosRESUMO
The brain relies on many forms of dynamic activities in individual neurons, from synaptic transmission to electrical activity and intracellular signaling events. Monitoring these neuronal activities with high spatiotemporal resolution in the context of animal behavior is a necessary step to achieve a mechanistic understanding of brain function. With the rapid development and dissemination of highly optimized genetically encoded fluorescent sensors, a growing number of brain activities can now be visualized in vivo. To date, cellular calcium imaging, which has been largely used as a proxy for electrical activity, has become a mainstay in systems neuroscience. While challenges remain, voltage imaging of neural populations is now possible. In addition, it is becoming increasingly practical to image over half a dozen neurotransmitters, as well as certain intracellular signaling and metabolic activities. These new capabilities enable neuroscientists to test previously unattainable hypotheses and questions. This review summarizes recent progress in the development and delivery of genetically encoded fluorescent sensors, and highlights example applications in the context of in vivo imaging.
Assuntos
Encéfalo , Corantes , Animais , Encéfalo/metabolismo , Corantes/metabolismo , Neurônios/metabolismoRESUMO
Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.
Assuntos
Corantes Fluorescentes/análise , Guanilato Quinases/análise , Proteínas de Membrana/análise , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Região CA1 Hipocampal/química , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Espinhas Dendríticas/química , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Corantes Fluorescentes/metabolismo , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de ÓrgãosRESUMO
Proper brain function requires the precise localization of proteins and signaling molecules on a nanometer scale. The examination of molecular organization at this scale has been difficult in part because it is beyond the reach of conventional, diffraction-limited light microscopy. The recently developed method of superresolution, localization-based fluorescent microscopy (LBM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), has demonstrated a resolving power at a 10 nm scale and is poised to become a vital tool in modern neuroscience research. Indeed, LBM has revealed previously unknown cellular architectures and organizational principles in neurons. Here, we discuss the principles of LBM, its current applications in neuroscience, and the challenges that must be met before its full potential is achieved. We also present the unpublished results of our own experiments to establish a sample preparation procedure for applying LBM to study brain tissue.
Assuntos
Limite de Detecção , Microscopia de Fluorescência/métodos , Neurônios/ultraestrutura , Animais , Humanos , Microscopia Eletrônica/métodos , Neurônios/metabolismo , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
A subset of retinal ganglion cells has recently been discovered to be intrinsically photosensitive, with melanopsin as the pigment. These cells project primarily to brain centres for non-image-forming visual functions such as the pupillary light reflex and circadian photoentrainment. How well they signal intrinsic light absorption to drive behaviour remains unclear. Here we report fundamental parameters governing their intrinsic light responses and associated spike generation. The membrane density of melanopsin is 10(4)-fold lower than that of rod and cone pigments, resulting in a very low photon catch and a phototransducing role only in relatively bright light. Nonetheless, each captured photon elicits a large and extraordinarily prolonged response, with a unique shape among known photoreceptors. Notably, like rods, these cells are capable of signalling single-photon absorption. A flash causing a few hundred isomerized melanopsin molecules in a retina is sufficient for reaching threshold for the pupillary light reflex.
Assuntos
Fótons , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Opsinas de Bastonetes/metabolismo , Potenciais de Ação/efeitos da radiação , Animais , Encéfalo/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pupila/fisiologia , Pupila/efeitos da radiação , Reflexo Pupilar/efeitos da radiaçãoRESUMO
Several neuronal populations in the brain transmit both the excitatory and inhibitory neurotransmitters, glutamate, and GABA, to downstream neurons. However, it remains largely unknown whether these opposing neurotransmitters are co-released onto the same postsynaptic neuron simultaneously or are independently transmitted at different time and locations (called co-transmission). Here, using whole-cell patch-clamp recording on acute mouse brain slices, we observed biphasic miniature postsynaptic currents, i.e., minis with time-locked excitatory and inhibitory currents, in striatal spiny projection neurons (SPNs). This observation cannot be explained by accidental coincidence of monophasic miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively), arguing for the co-release of glutamate and GABA. Interestingly, these biphasic minis could either be an mEPSC leading an mIPSC or vice versa. Although dopaminergic axons release both glutamate and GABA in the striatum, deletion of dopamine neurons did not eliminate biphasic minis, indicating that the co-release originates from another neuronal type. Importantly, we found that both types of biphasic minis were detected in other neuronal subtypes in the striatum as well as in nine out of ten additionally tested brain regions. Our results suggest that co-release of glutamate and GABA is a prevalent mode of neurotransmission in the brain.
RESUMO
We report a genetically encoded fluorescence lifetime sensor for protein kinase C (PKC) activity, named CKAR3, based on Förster resonance energy transfer. CKAR3 exhibits a 10-fold increased dynamic range compared to its parental sensors and enables in vivo imaging of PKC activity during animal behavior. Our results reveal robust PKC activity in a sparse neuronal subset in the motor cortex during locomotion, in part mediated by muscarinic acetylcholine receptors.
RESUMO
Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the ß-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.
RESUMO
Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific specializations in the composition of glutamatergic synapses, identifying Btbd11 as an inhibitory interneuron-specific, synapse-enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins, including Psd-95. Intriguingly, we show that Btbd11 can undergo liquid-liquid phase separation when expressed with Psd-95, supporting the idea that the glutamatergic postsynaptic density in synapses in inhibitory interneurons exists in a phase-separated state. Knockout of Btbd11 decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons alters exploratory behavior, measures of anxiety, and sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific mechanism that supports glutamatergic synapse function in inhibitory interneurons-with implications for circuit function and animal behavior.
Assuntos
Sinapses , Transmissão Sináptica , Animais , Camundongos , Proteína 4 Homóloga a Disks-Large/metabolismo , Interneurônios/metabolismo , Camundongos Knockout , Células Piramidais/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Fatores de Transcrição/metabolismoRESUMO
The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the protein coding sequence often suffer from insertions/deletions (INDELs) resulting from the efficient non-homologous end joining pathway (NHEJ). To overcome this limitation, we have developed CRISPR-mediated insertion of exon (CRISPIE). CRISPIE circumvents INDELs and other editing errors by inserting a designer exon flanked by adjacent intron sequences into an appropriate intronic location of the targeted gene. Because INDELs at the insertion junction can be spliced out, "CRISPIEd" genes produce precisely edited mRNA transcripts that are virtually error-free. In part due to the elimination of INDELs, high-efficiency labeling can be achieved in vivo. CRISPIE is compatible with both N- and C-terminal labels, and with all common transfection methods. Importantly, CRISPIE allows for later removal of the protein modification by including exogenous single-guide RNA (sgRNA) sites in the intronic region of the donor module. This protocol provides the detailed CRISPIE methodology, using endogenous labeling of ß-actin in human U-2 OS cells with enhanced green fluorescent protein (EGFP) as an example. When combined with the appropriate gene delivery methods, the same methodology can be applied to label post-mitotic neurons in culture and in vivo. This methodology can also be readily adapted for use in other gene editing contexts.
RESUMO
cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 µM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted ß-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.
Assuntos
Diagnóstico por Imagem , Sistemas do Segundo Mensageiro , Animais , Corantes , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , CamundongosRESUMO
The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Sistema de Sinalização das MAP Quinases , Células Piramidais/enzimologia , Potenciais de Ação/fisiologia , Animais , Artefatos , Proteínas de Bactérias/genética , Linhagem Celular , Dendritos/enzimologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Rim/citologia , Proteínas Luminescentes/genética , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , RatosRESUMO
Cyclic adenosine monophosphate (cAMP) is a universal second messenger that plays a crucial role in diverse biological functions, ranging from transcription to neuronal plasticity, and from development to learning and memory. In the nervous system, cAMP integrates inputs from many neuromodulators across a wide range of timescales - from seconds to hours - to modulate neuronal excitability and plasticity in brain circuits during different animal behavioral states. cAMP signaling events are both cell-specific and subcellularly compartmentalized. The same stimulus may result in different, sometimes opposite, cAMP dynamics in different cells or subcellular compartments. Additionally, the activity of protein kinase A (PKA), a major cAMP effector, is also spatiotemporally regulated. For these reasons, many laboratories have made great strides toward visualizing the intracellular dynamics of cAMP and PKA. To date, more than 80 genetically encoded sensors, including original and improved variants, have been published. It is starting to become possible to visualize cAMP and PKA signaling events in vivo, which is required to study behaviorally relevant cAMP/PKA signaling mechanisms. Despite significant progress, further developments are needed to enhance the signal-to-noise ratio and practical utility of these sensors. This review summarizes the recent advances and challenges in genetically encoded cAMP and PKA sensors with an emphasis on in vivo imaging in the brain during behavior.
Assuntos
Encéfalo , Transdução de Sinais , Animais , Encéfalo/diagnóstico por imagem , Aprendizagem , Plasticidade Neuronal , FosforilaçãoRESUMO
Cortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons. Here, we use the excitatory postsynaptic marker PSD-95, fluorescently labeled at endogenous levels, as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) interneurons in the barrel cortex of adult mice. Longitudinal in vivo imaging under baseline conditions reveals that, although synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell-type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, although PV+ synapses also exhibit an additive component. These results reveal that cell-type-specific processes govern cortical synaptic strengths and dynamics.
Assuntos
Proteína 4 Homóloga a Disks-Large/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/fisiologia , Inibição Neural , Parvalbuminas/metabolismo , Células Piramidais/fisiologia , Sinapses/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade NeuronalRESUMO
Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Mutagênese Insercional/genética , Proteínas/análise , Proteínas/genética , Animais , Linhagem Celular Tumoral , Éxons/genética , Humanos , Camundongos , Neurônios/citologia , Proteínas/química , Proteínas/metabolismoRESUMO
To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein-based sensors for the activation of the small GTPase protein Ras with slow (FRas) and fast (FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear (Hill coefficient approximately 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca(2+)) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca(2+) concentration.
Assuntos
Espinhas Dendríticas/fisiologia , Diagnóstico por Imagem , Hipocampo/fisiologia , Proteínas ras/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Diagnóstico por Imagem/instrumentação , Diagnóstico por Imagem/métodos , Ativação Enzimática , Humanos , Microscopia Confocal , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Fótons , RatosRESUMO
Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer's disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications.