Surface charge manipulation and electrostatic immobilization of synaptosomes for super-resolution imaging: a study on tau compartmentalization

Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

Thalamic bursts modulate cortical synchrony locally to switch between states of global functional connectivity in a cognitive task

Performing a cognitive task requires going through a sequence of functionally diverse stages. Although it is typically assumed that these stages are characterized by distinct states of cortical synchrony that are triggered by sub-cortical events, little reported evidence supports this hypothesis. To test this hypothesis, we first identified cognitive stages in single-trial MEG data of an associative recognition task, showing with a novel method that each stage begins with local modulations of synchrony followed by a state of directed functional connectivity. Second, we developed the first whole-brain model that can simulate cortical synchrony throughout a task. The model suggests that the observed synchrony is caused by thalamocortical bursts at the onset of each stage, targeted at cortical synapses and interacting with the structural anatomical connectivity. These findings confirm that cognitive stages are defined by distinct states of cortical synchrony and explains the network-level mechanisms necessary for reaching stage-dependent synchrony states.

Modeling the role of gap junctions between excitatory neurons in the developing visual cortex

Recent experiments in the developing mammalian visual cortex have revealed that gap junctions couple excitatory cells and potentially influence the formation of chemical synapses. In particular, cells that were coupled by a gap junction during development tend to share an orientation preference and are preferentially coupled by a chemical synapse in the adult cortex, a property that is diminished when gap junctions are blocked. In this work, we construct a simplified model of the developing mouse visual cortex including spike-timing-dependent plasticity of both the feedforward synaptic inputs and recurrent cortical synapses. We use this model to show that synchrony among gap-junction-coupled cells underlies their preference to form strong recurrent synapses and develop similar orientation preference; this effect decreases with an increase in coupling density. Additionally, we demonstrate that gap-junction coupling works, together with the relative timing of synaptic development of the feedforward and recurrent synapses, to determine the resulting cortical map of orientation preference.

Selective Regional Loss of Cortical Synapses Lacking Presynaptic Mitochondria in the 5xFAD Mouse Model

Synaptic loss in Alzheimer's disease (AD) is strongly correlated with cognitive impairment. Accumulating evidence indicates that amyloid pathology leads to synaptic degeneration and mitochondrial damage in AD. However, it remains unclear whether synapses and presynaptic mitochondria are differentially affected in various cortical regions of the AD brain at the ultrastructural level. Using serial block-face scanning electron microscopy, we assessed synaptic structures in the medial prefrontal cortex (mPFC) and primary visual cortex (V1) of the 5xFAD mouse model of AD. At 6 months of age, 5xFAD mice exhibited significantly elevated levels of amyloid deposition in layer 2/3 of the mPFC but not V1. Accordingly, three-dimensional reconstruction of synaptic connectivity revealed a significant reduction in excitatory synaptic density in layer 2 of the mPFC, but not V1, of male transgenic mice. Notably, the density of synapses lacking presynaptic mitochondria was selectively decreased in the mPFC of 5xFAD mice, with no change in the density of mitochondria-containing synapses. Further classification of spines into shape categories confirmed a preferential loss of thin spines whose presynaptic boutons were largely devoid of mitochondria in the 5xFAD mPFC. Furthermore, the number of mitochondria per bouton in spared mitochondria-containing boutons was reduced in the mPFC, but not V1, of 5xFAD mice. Collectively, these results highlight region-specific vulnerability of cortical synapses to amyloid deposition and suggest that the presence of presynaptic mitochondria may affect synaptic degeneration in AD.

Dopaminergic Neuromodulation of Spike Timing Dependent Plasticity in Mature Adult Rodent and Human Cortical Neurons

Synapses in the cerebral cortex constantly change and this dynamic property regulated by the action of neuromodulators such as dopamine (DA), is essential for reward learning and memory. DA modulates spike-timing-dependent plasticity (STDP), a cellular model of learning and memory, in juvenile rodent cortical neurons. However, it is unknown whether this neuromodulation also occurs at excitatory synapses of cortical neurons in mature adult mice or in humans. Cortical layer V pyramidal neurons were recorded with whole cell patch clamp electrophysiology and an extracellular stimulating electrode was used to induce STDP. DA was either bath-applied or optogenetically released in slices from mice. Classical STDP induction protocols triggered non-hebbian excitatory synaptic depression in the mouse or no plasticity at human cortical synapses. DA reverted long term synaptic depression to baseline in mouse via dopamine 2 type receptors or elicited long term synaptic potentiation in human cortical synapses. Furthermore, when DA was applied during an STDP protocol it depressed presynaptic inhibition in the mouse but not in the human cortex. Thus, DA modulates excitatory synaptic plasticity differently in human vs. mouse cortex. The data strengthens the importance of DA in gating cognition in humans, and may inform on therapeutic interventions to recover brain function from diseases.

Surface Charge Manipulation and Electrostatic Immobilization of Synaptosomes for Super-resolution Imaging: A Study on Tau Compartmentalization

Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a simple and facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

Neuroserpin Is Strongly Expressed in the Developing and Adult Mouse Neocortex but Its Absence Does Not Perturb Cortical Lamination and Synaptic Proteome

Neuroserpin is a serine protease inhibitor that regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin is strongly expressed during nervous system development as well as during adulthood, when it is predominantly found in regions eliciting synaptic plasticity. In the hippocampus, neuroserpin regulates developmental neurogenesis, synaptic maturation and in adult mice it modulates synaptic plasticity and controls cognitive and social behavior. High expression levels of neuroserpin in the neocortex starting from prenatal stage and persisting during adulthood suggest an important role for the serpin in the formation of this brain region and in the maintenance of cortical functions. In order to uncover neuroserpin function in the murine neocortex, in this work we performed a comprehensive investigation of its expression pattern during development and in the adulthood. Moreover, we assessed the role of neuroserpin in cortex formation by comparing cortical lamination and neuronal maturation between neuroserpin-deficient and control mice. Finally, we evaluated a possible regulatory role of neuroserpin at cortical synapses in neuroserpin-deficient mice. We observed that neuroserpin is expressed starting from the beginning of corticogenesis until adulthood throughout the neocortex in several classes of glutamatergic projection neurons and GABA-ergic interneurons. However, in the absence of neuroserpin we did not detect any alteration either in cortical layer formation, or in neuronal soma size and dendritic length. Furthermore, no significant quantitative changes were observed in the proteome of cortical synapses upon neuroserpin deficiency. We conclude that, although strongly expressed in the neocortex, absence of neuroserpin does not lead to gross developmental abnormalities, and does not perturb the composition of the cortical synaptic proteome.

Prolonged anesthesia alters brain synaptic architecture

Prolonged medically induced coma (pMIC) is carried out routinely in intensive care medicine. pMIC leads to cognitive impairment, yet the underlying neuromorphological correlates are still unknown, as no direct studies of MIC exceeding ∼6 h on neural circuits exist. Here, we establish pMIC (up to 24 h) in adolescent and mature mice, and combine longitudinal two-photon imaging of cortical synapses with repeated behavioral object recognition assessments. We find that pMIC affects object recognition, and that it is associated with enhanced synaptic turnover, generated by enhanced synapse formation during pMIC, while the postanesthetic period is dominated by synaptic loss. Our results demonstrate major side effects of prolonged anesthesia on neural circuit structure.

Astrocyte-derived thrombospondin induces cortical synaptogenesis in a sex-specific manner

The regulation of synaptic connectivity in the brain is vital to proper functioning and development of the central nervous system (CNS). Formation of neural networks in the CNS has been shown to be heavily influenced by astrocytes, which secrete factors, including thrombospondin (TSP) family proteins, that promote synaptogenesis. However, whether this process is different between males and females has not been thoroughly investigated. In this study, we found that cortical neurons purified from newborn male rats showed a significantly more robust synaptogenic response compared to female-derived cells when exposed to factors secreted from astrocytes. This difference was driven largely by the neuronal response to TSP2, which increased synapses in male neurons while showing no effect on female neurons. Blockade of endogenous 17β-estradiol production with letrozole normalized the TSP response between male and female cells, indicating a level of regulation by estrogen signaling. Our results suggest that TSP-induced synaptogenesis is critical for the development of male but not female cortical synapses, contributing to sex differences in astrocyte-mediated synaptic connectivity.