Synaptic Distribution Research Articles

The bile acid chenodeoxycholic acid associates with reduced stroke in humans and mice

Bile acids (BAs) are liver-derived signaling molecules that can be found in the brain, but their role there remains largely unknown. We found increased brain chenodeoxycholic acid (CDCA) in mice with absent 12α-hydroxylase (Cyp8b1), a BA synthesis enzyme. In these Cyp8b1-/-, and in wild-type mice administered CDCA, stroke infarct area was reduced. Elevated glutamate-induced excitotoxicity mediated by aberrant N-methyl-D-aspartate receptor (NMDAR) over-activation contributes to neuronal death in ischemic stroke. We found reduced glutamate-induced excitotoxicity in neurons from Cyp8b1-/- and CDCA-treated wild-type mice. CDCA decreased NMDAR-mediated excitatory post-synaptic currents by reducing over-activation of NMDAR subunit GluN2B in wild-type brains. Synaptic NMDAR activity was also decreased in Cyp8b1-/- brains. Expression and synaptic distribution of GluN2B were unaltered in Cyp8b1-/- mice, suggesting CDCA may directly antagonize GluN2B-containing NMDARs. Supporting our findings, in a case-control cohort of acute ischemic stroke patients, we found lower circulatory CDCA. Together, our data suggest that CDCA, acting in the liver-brain axis, decreases GluN2B-containing NMDAR over-activation, contributing to neuroprotection in stroke.

Synaptic and extrasynaptic distribution of NMDA receptors in the cortex of Alzheimer's disease patients.

Synaptic and extrasynaptic distribution of N-methyl-D-aspartate receptors (NMDARs) has not been addressed in the brain from Alzheimer´s disease (AD) subjects, despite theircontribution to neurodegeneration. We have developed a protocol to isolate synaptic and extrasynaptic membranes from controls and AD frontal cortex. We characterized the distribution of the NMDAR subunits GluN2B, GluN2A, GluN1, and GluN3A, as well aspost-translational modifications, such as phosphorylation and glycosylation. Lower levels of synaptic GluN2B and GluN2A were found in AD fractions, while extrasynaptic GluN2B and GluN1 levels were significantly higher; GluN3A distribution remained unaffected in AD. We also identified different glycoforms of GluN2B and GluN2A in extrasynaptic membranes. Synaptic Tyr1472 GluN2B phosphorylation was significantly lower in AD fractions. Reduction of synaptic NMDAR subunits, particularly for GluN2B, is likely to contribute to synaptic transmission failure in AD. Additionally, the increment of extrasynaptic NMDAR subunits could favor the activation of excitotoxicity in AD. New protocol to isolate synaptic and extrasynaptic membranes from the human cortex. Low GluN2B and GluN2A levels in Alzheimer´s disease (AD) synaptic membranes. High GluN2B and GluN1 levels in AD extrasynaptic membranes. Specific glycoforms of extrasynaptic GluN2B and GluN2A. Low phosphorylation at Tyr1472 in synaptic GluN2B in AD.

A dendritic substrate for temporal diversity of cortical inhibition.

In the mammalian neocortex, GABAergic interneurons (INs) inhibit cortical networks in profoundly different ways. The extent to which this depends on how different INs process excitatory signals along their dendrites is poorly understood. Here, we reveal that the functional specialization of two major populations of cortical INs is determined by the unique association of different dendritic integration modes with distinct synaptic organization motifs. We found that somatostatin (SST)-INs exhibit NMDAR-dependent dendritic integration and uniform synapse density along the dendritic tree. In contrast, dendrites of parvalbumin (PV)-INs exhibit passive synaptic integration coupled with proximally enriched synaptic distributions. Theoretical analysis shows that these two dendritic configurations result in different strategies to optimize synaptic efficacy in thin dendritic structures. Yet, the two configurations lead to distinct temporal engagement of each IN during network activity. We confirmed these predictions with in vivo recordings of IN activity in the visual cortex of awake mice, revealing a rapid and linear recruitment of PV-INs as opposed to a long-lasting integrative activation of SST-INs. Our work reveals the existence of distinct dendritic strategies that confer distinct temporal representations for the two major classes of neocortical INs and thus dynamics of inhibition.

Nanoscale organization is changed in native, surface AMPARs by mouse brain region and tauopathy.

Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native surface AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex. In 6 months old PS19 model of tauopathy, synaptic and extrasynaptic distributions are disrupted in the hippocampus but not in the cortex. Thus, this optimized super-resolution imaging tool allows us to observe synaptic deterioration at the onset of tauopathy before apparent neurodegeneration.

Automatic dendritic spine segmentation in widefield fluorescence images reveal synaptic nanostructures distribution with super-resolution imaging.

Dendritic spines are the main sites for synaptic communication in neurons, and alterations in their density, size, and shapes occur in many brain disorders. Current spine segmentation methods perform poorly in conditions with low signal-to-noise and resolution, particularly in the widefield images of thick (10 μm) brain slices. Here, we combined two open-source machine-learning models to achieve automatic 3D spine segmentation in widefield diffraction-limited fluorescence images of neurons in thick brain slices. We validated the performance by comparison with manually segmented super-resolution images of spines reconstructed from direct stochastic optical reconstruction microscopy (dSTORM). Lastly, we show an application of our approach by combining spine segmentation from diffraction-limited images with dSTORM of synaptic protein PSD-95 in the same field-of-view. This allowed us to automatically analyze and quantify the nanoscale distribution of PSD-95 inside the spine. Importantly, we found the numbers, but not the average sizes, of synaptic nanomodules and nanodomains increase with spine size.

KLHL17 differentially controls the expression of AMPA- and KA-type glutamate receptors to regulate dendritic spine enlargement.

Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders. The key morphological feature of Klhl17-deficient neurons is impaired dendritic spine enlargement, resulting in the amplitude of calcium events being increased. Our previous studies have indicated an involvement of F-actin and the spine apparatus in KLHL17-mediated dendritic spine enlargement. Here, we show that KLHL17 further employs different mechanisms to control the expression of two types of glutamate receptors, that is, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KARs), to regulate dendritic spine enlargement and calcium influx. We deployed proteomics to reveal that KLHL17 interacts with N-ethylmaleimide-sensitive fusion protein (NSF) in neurons, with this interaction of KLHL17 and NSF enhancing NSF protein levels. Consistent with the function of NSF in regulating the surface expression of AMPAR, Klhl17 deficiency limits the surface expression of AMPAR, but not its total protein levels. The NSF pathway also contributes to synaptic F-actin distribution and the dendritic spine enlargement mediated by KLHL17. KLHL17 is known to act as an adaptor mediating degradation of the KAR subunit GluK2 by the CUL3 ubiquitin ligase complex, and Klhl17 deficiency impairs activity-dependent degradation of GluK2. Herein, we further demonstrate that GluK2 is critical to the increased amplitude of calcium influx in Klhl17-deficient neurons. Moreover, GluK2 is also involved in KLHL17-regulated dendritic spine enlargement. Thus, our study reveals that KLHL17 controls AMPAR and KAR expression via at least two mechanisms, consequently regulating dendritic spine enlargement. The regulatory effects of KLHL17 on these two glutamate receptors likely contribute to neuronal features in patients suffering from certain neurological disorders.

Anomalous diffusion of synaptic vesicles and its influences on spontaneous and evoked neurotransmission.

Neurons in the central nervous system communicate with each other by activating billions of tiny synaptic boutons distributed along their fine axons. These presynaptic varicosities are very crowded environments, comprising hundreds of synaptic vesicles. Only a fraction of these vesicles can be recruited in a single release episode, either spontaneous or evoked by action potentials. Since the seminal work by Fatt and Katz, spontaneous release has been modelled as a memoryless process. Nevertheless, at central synapses, experimental evidence indicates more complex features, including non-exponential distributions of release intervals and power-law behaviour in their rate. To describe these features, we developed a probabilistic model of spontaneous release based on Brownian motion of synaptic vesicles in the presynaptic environment. To account for different diffusion regimes, we based our simulations on fractional Brownian motion. We show that this model can predict both deviation from the Poisson hypothesis and power-law features in experimental quantal release series, thus suggesting that the vesicular motion by diffusion could per se explain the emergence of these properties. We demonstrate the efficacy of our modelling approach using electrophysiological recordings at single synaptic boutons and ultrastructural data. When this approach was used to simulate evoked responses, we found that the replenishment of the readily releasable pool driven by Brownian motion of vesicles can reproduce the characteristic binomial release distributions seen experimentally. We believe that our modelling approach supports the idea that vesicle diffusion and readily releasable pool dynamics are crucial factors for the physiological functioning of neuronal communication. KEY POINTS: We developed a new probabilistic model of spontaneous and evoked vesicle fusion based on simple biophysical assumptions, including the motion of vesicles before they dock to the release site. We provide closed-form equations for the interval distribution of spontaneous releases in the special case of Brownian diffusion of vesicles, showing that a power-law heavy tail is generated. Fractional Brownian motion (fBm) was exploited to simulate anomalous vesicle diffusion, including directed and non-directed motion, by varying the Hurst exponent. We show that our model predicts non-linear features observed in experimental spontaneous quantal release series as well as ultrastructural data of synaptic vesicles spatial distribution. Evoked exocytosis based on a diffusion-replenished readily releasable pool might explain the emergence of power-law behaviour in neuronal activity.

Adaptive observer and control of spatiotemporal delayed neural fields

An adaptive observer is proposed to estimate the synaptic distribution between neurons asymptotically from the measurement of a part of the neuronal activity and a delayed neural field evolution model. The convergence of the observer is proved under a persistency of excitation condition. Then, the observer is used to derive a feedback law ensuring asymptotic stabilization of the neural fields. Finally, the feedback law is modified to ensure simultaneously practical stabilization of the neural fields and asymptotic convergence of the observer under additional restrictions on the system. Numerical simulations confirm the relevance of the approach.

The spinal cord facilitates cerebellar upper limb motor learning and control; inputs from neuromusculoskeletal simulation.

Complex interactions between brain regions and the spinal cord (SC) govern body motion, which is ultimately driven by muscle activation. Motor planning or learning are mainly conducted at higher brain regions, whilst the SC acts as a brain-muscle gateway and as a motor control centre providing fast reflexes and muscle activity regulation. Thus, higher brain areas need to cope with the SC as an inherent and evolutionary older part of the body dynamics. Here, we address the question of how SC dynamics affects motor learning within the cerebellum; in particular, does the SC facilitate cerebellar motor learning or constitute a biological constraint? We provide an exploratory framework by integrating biologically plausible cerebellar and SC computational models in a musculoskeletal upper limb control loop. The cerebellar model, equipped with the main form of cerebellar plasticity, provides motor adaptation; whilst the SC model implements stretch reflex and reciprocal inhibition between antagonist muscles. The resulting spino-cerebellar model is tested performing a set of upper limb motor tasks, including external perturbation studies. A cerebellar model, lacking the implemented SC model and directly controlling the simulated muscles, was also tested in the same. The performances of the spino-cerebellar and cerebellar models were then compared, thus allowing directly addressing the SC influence on cerebellar motor adaptation and learning, and on handling external motor perturbations. Performance was assessed in both joint and muscle space, and compared with kinematic and EMG recordings from healthy participants. The differences in cerebellar synaptic adaptation between both models were also studied. We conclude that the SC facilitates cerebellar motor learning; when the SC circuits are in the loop, faster convergence in motor learning is achieved with simpler cerebellar synaptic weight distributions. The SC is also found to improve robustness against external perturbations, by better reproducing and modulating muscle cocontraction patterns.

Memory-efficient neurons and synapses for spike-timing-dependent-plasticity in large-scale spiking networks.

This paper addresses the challenges posed by frequent memory access during simulations of large-scale spiking neural networks involving synaptic plasticity. We focus on the memory accesses performed during a common synaptic plasticity rule since this can be a significant factor limiting the efficiency of the simulations. We propose neuron models that are represented by only three state variables, which are engineered to enforce the appropriate neuronal dynamics. Additionally, memory retrieval is executed solely by fetching postsynaptic variables, promoting a contiguous memory storage and leveraging the capabilities of burst mode operations to reduce the overhead associated with each access. Different plasticity rules could be implemented despite the adopted simplifications, each leading to a distinct synaptic weight distribution (i.e., unimodal and bimodal). Moreover, our method requires fewer average memory accesses compared to a naive approach. We argue that the strategy described can speed up memory transactions and reduce latencies while maintaining a small memory footprint.

Msemalign: a pipeline for serial section multibeam scanning electron microscopy volume alignment.

Serial section multibeam scanning electron microscopy (ssmSEM) is currently among the fastest technologies available for acquiring 3D anatomical data spanning relatively large neural tissue volumes, on the order of 1 mm3 or larger, at a resolution sufficient to resolve the fine detail of neuronal morphologies and synapses. These petabyte-scale volumes can be analyzed to create connectomes, datasets that contain detailed anatomical information including synaptic connectivity, neuronal morphologies and distributions of cellular organelles. The mSEM acquisition process creates hundreds of millions of individual image tiles for a single cubic-millimeter-sized dataset and these tiles must be aligned to create 3D volumes. Here we introduce msemalign, an alignment pipeline that strives for scalability and design simplicity. The pipeline can align petabyte-scale datasets such that they contain smooth transitions as the dataset is navigated in all directions, but critically that does so in a fashion that minimizes the overall magnitude of section distortions relative to the originally acquired micrographs.

Abstract 15651: Astrocytic Chordin-Like 1 Regulates AMPAR Synaptic Content and Motor Recovery in a Mouse Model of Ischemic Stroke

Introduction: Ischemic stroke triggers a series of molecular events that impairs receptor levels at excitatory synapses. AMPA receptors (AMPARs), the main excitatory receptors in the mammalian brain, are crucial in maintaining proper brain function. In response to ischemic stroke, their subunit composition and location at the synapse are deeply altered and trigger apoptotic cell death cascades. We previously found that Chordin-like 1 (Chrdl1), an astrocyte-derived protein that regulates AMPARs, is highly upregulated in the peri-infarct region at early stages after stroke (1 and 7 days post-stroke, dps). When Chrdl1 was absent, neurons were protected from stroke-induced structural degeneration. Hypothesis: Elimination of Chrdl1 prevents stroke-induced aberrant AMPAR synaptic levels, limits AMPAR-mediated apoptotic cascades and alleviates behavioral deficits. Methods: We used photothrombosis to model ischemic stroke in the motor cortex of adult male and female WT and Chrdl1 KO mice and performed analyses at 1 and 7 dps in the peri-infarct area. We used fluorescent in situ hybridization to analyze mRNA levels of Chrdl1, immunohistochemistry to determine synaptic AMPAR levels and subunit composition, TUNEL to determine apoptotic cell death, MRI to monitor the progression of injury volume, and adhesive removal and grid walk tests to assess sensorimotor and motor behaviors. Results: WT mice, after photothrombotic injury showed important impairments in the AMPAR synaptic distribution and composition. This aberrant AMPAR subunit composition and location at synapses was abolished in Chrdl1 KO mice. This resulted in reduced apoptotic cell death and faster motor recovery in Chrdl1 KO mice compared to WT in both male and female mice. Conclusions: This study shows that targeting Chrdl1 has therapeutic potential to reduce apoptotic cell death and aid motor recovery after ischemic stroke via synaptic regulation of AMPARs. Astrocyte-derived proteins, such as Chrdl1, may represent new molecular targets that can restore synapses to favor functional recovery after ischemic stroke.

Linking spontaneous and stimulated spine dynamics

Our brains continuously acquire and store memories through synaptic plasticity. However, spontaneous synaptic changes can also occur and pose a challenge for maintaining stable memories. Despite fluctuations in synapse size, recent studies have shown that key population-level synaptic properties remain stable over time. This raises the question of how local synaptic plasticity affects the global population-level synaptic size distribution and whether individual synapses undergoing plasticity escape the stable distribution to encode specific memories. To address this question, we (i) studied spontaneously evolving spines and (ii) induced synaptic potentiation at selected sites while observing the spine distribution pre- and post-stimulation. We designed a stochastic model to describe how the current size of a synapse affects its future size under baseline and stimulation conditions and how these local effects give rise to population-level synaptic shifts. Our study offers insights into how seemingly spontaneous synaptic fluctuations and local plasticity both contribute to population-level synaptic dynamics.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Machine learning using magnetic stochastic synapses

The impressive performance of artificial neural networks has come at the cost of high energy usage and CO2 emissions. Unconventional computing architectures, with magnetic systems as a candidate, have potential as alternative energy-efficient hardware, but, still face challenges, such as stochastic behaviour, in implementation. Here, we present a methodology for exploiting the traditionally detrimental stochastic effects in magnetic domain-wall motion in nanowires. We demonstrate functional binary stochastic synapses alongside a gradient learning rule that allows their training with applicability to a range of stochastic systems. The rule, utilising the mean and variance of the neuronal output distribution, finds a trade-off between synaptic stochasticity and energy efficiency depending on the number of measurements of each synapse. For single measurements, the rule results in binary synapses with minimal stochasticity, sacrificing potential performance for robustness. For multiple measurements, synaptic distributions are broad, approximating better-performing continuous synapses. This observation allows us to choose design principles depending on the desired performance and the device’s operational speed and energy cost. We verify performance on physical hardware, showing it is comparable to a standard neural network.

Cardiolipin and OPA1 Team up for Methamphetamine-Induced Locomotor Activity by Promoting Neuronal Mitochondrial Fusion in the Nucleus Accumbens of Mice.

Mitochondria are highly dynamic organelles with coordinated cycles of fission and fusion occurring continuously to satisfy the energy demands in the complex architecture of neurons. How mitochondria contribute to addicted drug-induced adaptable mitochondrial networks and neuroplasticity remains largely unknown. Through liquid chromatography-mass spectrometry-based lipidomics, we first analyzed the alteration of the mitochondrial lipidome of three mouse brain areas in methamphetamine (METH)-induced locomotor activity and conditioned place preference. The results showed that METH remodeled the mitochondrial lipidome of the hippocampus, nucleus accumbens (NAc), and striatum in both models. Notably, mitochondrial hallmark lipid cardiolipin (CL) was specifically increased in the NAc in METH-induced hyperlocomotor activity, which was accompanied by an elongated giant mitochondrial morphology. Moreover, METH significantly boosted mitochondrial respiration and ATP generation as well as the copy number of mitochondrial genome DNA in the NAc. By screening the expressions of mitochondrial dynamin-related proteins, we found that repeated METH significantly upregulated the expression of long-form optic atrophy type 1 (L-OPA1) and enhanced the interaction of L-OPA1 with CL, which may promote mitochondrial fusion in the NAc. On the contrary, neuronal OPA1 depletion in the NAc not only recovered the dysregulated mitochondrial morphology and synaptic vesicle distribution induced by METH but also attenuated the psychomotor effect of METH. Collectively, upregulated CL and OPA1 cooperate to mediate METH-induced adaptation of neuronal mitochondrial dynamics in the NAc, which correlates with the psychomotor effect of METH. These findings propose a potential therapeutic approach for METH addiction by inhibiting neuronal mitochondrial fusion.

Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43-/-), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43-/- mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy.

Strength of Excitatory Inputs to Layer 3 Pyramidal Neurons During Synaptic Pruning in the Monkey Prefrontal Cortex: Relevance for the Pathogenesis of Schizophrenia

BackgroundIn schizophrenia, layer 3 pyramidal neurons (L3PNs) of the dorsolateral prefrontal cortex exhibit deficits in markers of excitatory synaptic inputs that are thought to disrupt the patterns of neural network activity essential for cognitive function. These deficits are usually interpreted under Irwin Feinberg’s hypothesis of altered synaptic pruning, which postulates that normal periadolescent pruning, thought to preferentially eliminate weak/immature synapses, is altered in schizophrenia. However, it remains unknown whether periadolescent pruning on L3PNs in the primate dorsolateral prefrontal cortex selectively eliminates weak excitatory synapses or uniformly eliminates excitatory synapses across the full distribution of synaptic strengths. MethodsTo distinguish between these alternative models of synaptic pruning, we assessed the densities of dendritic spines, the site of most excitatory inputs to L3PNs, and the distributions of excitatory synaptic strengths in dorsolateral prefrontal cortex L3PNs from male and female monkeys across the periadolescent period of synaptic pruning. We used patch-clamp methods in acute brain slices to record miniature excitatory synaptic currents and intracellular filling with biocytin to quantify dendritic spines. ResultsOn L3PNs, dendritic spines exhibited the expected age-related decline in density, but mean synaptic strength and the shape of synaptic strength distributions remained stable with age. ConclusionsThe absence of age-related differences in mean synaptic strength and synaptic strength distributions supports the model of a uniform pattern of synaptic pruning across the full range of synaptic strengths. The implications of these findings for the pathogenesis and functional consequences of dendritic spine deficits in schizophrenia are discussed.

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Adult hippocampal neurogenesis (AHN) drops sharply during early stages of Alzheimer's disease (AD), via unknown mechanisms, and correlates with cognitive status in AD patients. Understanding AHN regulation in AD could provide a framework for innovative pharmacological interventions. We here combine molecular, behavioral, and clinical data and critically discuss the multicellular complexity of the AHN niche in relation to AD pathophysiology. We further present a roadmap toward a better understanding of the role of AHN in AD by probing the promises and caveats of the latest technological advancements in the field and addressing the conceptual and methodological challenges ahead.

Probing synaptic distribution and arrangement of native surface AMPAR in mouse brain slices with 3D super-resolution microscopy.

AMPARs are ion channels gated by the neurotransmitter Glutamate and are found in majority of excitatory neurons. Their numbers, arrangement, and diffusion on the cell-membrane of neurons act as important indicators of synaptic plasticity. Single molecule localization microscopy (SMLM) techniques have been used to extensively study the distribution of surface AMPARs and their clustering in synaptic nano-domains. However, most of these studies have been limited to dissociated neuronal cultures and hence lack the ability to probe surface AMPARs in brain tissue with intact circuitry, and as a function of aging and pathology. In this work, we utilize a recently developed small probe, CAM2, to specifically label native, surface AMPARs (specifically, GluA2-4 subunits) in mouse brain slices. This approach avoids artefacts arising due to protein overexpression and modification. We use a custom-built microscope to perform 2-color 3D dSTORM imaging of AMPARs as well as a post-synaptic density protein Homer1. By reducing light scattering caused by refractive-index mismatch, correcting for instrument-induced aberrations using a deformable mirror and reducing out-of-focus fluorescence background by using HILO illumination, we are able to achieve <20 nm (lateral) and 90 nm (axial) localization precision in 30 μm thick, fixed Thy1-YFP-H mouse brain slices. This allows us to visualize AMPAR clustering in synaptic nanodomains and their distribution in synaptic and extra-synaptic sites in the cortex, as well as the more densely crowded hippocampus. Addition of ratiometric imaging capability to our microscope will allow us to look at one more color, such as a pre-synaptic density protein, Bassoon, improving the accuracy of AMPAR classification as synaptic vs extra-synaptic.