Axonal Boutons Research Articles

Neuronal brain‐derived neurotrophic factor manipulates microglial dynamics

Brain-derived neurotrophic factor (BDNF), a main member of the neurotrophin family that is active in the brain, supports neuronal survival and growth. Microglial BDNF affects both the structural and functional properties of neurons. In contrast, whether and how neuronal BDNF affects microglial dynamics remain largely undetermined. Here, we examined the effects of BDNF on the properties of microglia in the CA3 region of the hippocampus. We chose this site because the axonal boutons of hippocampal mossy fibers, which are mostly formed in the CA3 region, contain the highest levels of BDNF in the rodent brain. We transfected mouse dentate granule cells with an adeno-associated virus that encodes both a BDNF short hairpin RNA (shRNA) and red fluorescent protein to examine the effects of mossy fiber-derived BDNF on microglia. Based on immunohistochemistry, BDNF knockdown with an shRNA resulted in an increase in microglial density in the mossy fiber pathway and increased engulfment of mossy fiber axons by microglia. In addition, we performed time-lapse imaging of microglial processes in hippocampal slice cultures to examine the effects of BDNF on microglial motility. Time-lapse imaging revealed increases in the motility of microglial processes and the engulfment of mossy fiber synapses by microglia when BDNF signaling was pharmacologically blocked. Thus, neuronal BDNF prevents microglia from engulfing mossy fiber synapses in the hippocampus.

Cortical Projection From the Premotor or Primary Motor Cortex to the Subthalamic Nucleus in Intact and Parkinsonian Adult Macaque Monkeys: A Pilot Tracing Study.

Besides the main cortical inputs to the basal ganglia, via the corticostriatal projection, there is another input via the corticosubthalamic projection (CSTP), terminating in the subthalamic nucleus (STN). The present study investigated and compared the CSTPs originating from the premotor cortex (PM) or the primary motor cortex (M1) in two groups of adult macaque monkeys. The first group includes six intact monkeys, whereas the second group was made up of four monkeys subjected to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication producing Parkinson’s disease (PD)-like symptoms and subsequently treated with an autologous neural cell ecosystem (ANCE) therapy. The CSTPs were labeled with the anterograde tracer biotinylated dextran amine (BDA), injected either in PM or in M1. BDA-labeled axonal terminal boutons in STN were charted, counted, and then normalized based on the number of labeled corticospinal axons in each monkey. In intact monkeys, the CSTP from PM was denser than that originating from M1. In two PD monkeys, the CSTP originating from PM or M1 were substantially increased, as compared to intact monkeys. In one other PD monkey, there was no obvious change, whereas the last PD monkey showed a decrease of the CSTP originating from M1. Interestingly, the linear relationship between CSTP density and PD symptoms yielded a possible dependence of the CSTP re-organization with the severity of the MPTP lesion. The higher the PD symptoms, the larger the CSTP densities, irrespective of the origin (from both M1 or PM). Plasticity of the CSTP in PD monkeys may be related to PD itself and/or to the ANCE treatment.

Synaptic remodeling in mouse motor cortex after spinal cord injury.

Spinal cord injury dramatically blocks information exchange between the central nervous system and the peripheral nervous system. The resulting fate of synapses in the motor cortex has not been well studied. To explore synaptic reorganization in the motor cortex after spinal cord injury, we established mouse models of T12 spinal cord hemi-section and then monitored the postsynaptic dendritic spines and presynaptic axonal boutons of pyramidal neurons in the hindlimb area of the motor cortex in vivo. Our results showed that spinal cord hemi-section led to the remodeling of dendritic spines bilaterally in the motor cortex and the main remodeling regions changed over time. It made previously stable spines unstable and eliminated spines more unlikely to be re-emerged. There was a significant increase in new spines in the contralateral motor cortex. However, the low survival rate of the new spines demonstrated that new spines were still fragile. Observation of presynaptic axonal boutons found no significant change. These results suggest the existence of synapse remodeling in motor cortex after spinal cord hemi-section and that spinal cord hemi-section affected postsynaptic dendritic spines rather than presynaptic axonal boutons. This study was approved by the Ethics Committee of Chinese PLA General Hospital, China (approval No. 201504168S) on April 16, 2015.

Chemical Sectioning Fluorescence Tomography: High-Throughput, High-Contrast, Multicolor, Whole-Brain Imaging at Subcellular Resolution

A thorough neuroanatomical study of the brain architecture is crucial for our understanding of its cellular compositions, connections and working mechanisms. However, the fine and multiscale features of neuron structures make it challenging for the microscopic imaging, as one cannot have both high-contrast, and high-throughput at the same time in such imaging. Here, we proposed the concept of chemical sectioning fluorescence tomography (CSFT) to solve this problem. Based on the switching characteristics of the fluorescent proteins (FPs), we introduced the chemical sectioning (CS) effect into the block-surface microscopy, turn off all FP molecules by chemical methods, and only light those in the top section as thin as submicron for imaging, so as to achieve high-throughput, high-contrast imaging without background. We developed a three-color wide-field fluorescence micro-optical sectioning tomography (fMOST) system. We proved that CSFT method was compatible with resin-embedded multicolor FPs and could be feasibly used in whole-brain imaging on diverse genetically or virally labeled long-projecting neurons and interneurons to the systematic reconstruction degree. We demonstrated mouse whole-brain imaging at the subcellular resolution, as well as the power for quantitative acquisition of synaptic connection-related pyramidal dendritic spines and axon boutons on the brain-wide scale.at the complete single-neuron level. We believe that CSFT method would greatly facilitate our understanding of the brain-wide neuron networks.

Retinal Inputs to the Thalamus Are Selectively Gated by Arousal

The brain can flexibly filter out sensory information in a manner that depends on behavioral state. In the visual thalamus and cortex, arousal and locomotion are associated with changes in the magnitude of responses to visual stimuli. Here, we asked whether such modulation of visual responses might already occur at an earlier stage in this visual pathway. We measured neural activity of retinal axons using wide-field and two-photon calcium imaging in awake mouse thalamus across arousal states associated with different pupil sizes. Surprisingly, visual responses to drifting gratings in retinal axonal boutons were robustly modulated by arousal level in a manner that varied across stimulus dimensions and across functionally distinct subsets of boutons. At low and intermediate spatial frequencies, the majority of boutons were suppressed by arousal. In contrast, at high spatial frequencies, boutons tuned to regions of visual space ahead of the mouse showed enhancement of responses. Arousal-related modulation also varied with a bouton's preference for luminance changes and direction or axis of motion, with greater response suppression in boutons tuned to luminance decrements versus increments, and in boutons preferring motion along directions or axes of optic flow. Together, our results suggest that differential modulation of distinct visual information channels by arousal state occurs at very early stages of visual processing, before the information is transmitted to neurons in visual thalamus. Such early filtering may provide an efficient means of optimizing central visual processing and perception across behavioral contexts.

Structural dynamics and stability of corticocortical and thalamocortical axon terminals during motor learning.

Synaptic plasticity is the cellular basis of learning and memory. When animals learn a novel motor skill, synaptic modifications are induced in the primary motor cortex (M1), and new postsynaptic dendritic spines relevant to motor memory are formed in the early stage of learning. However, it is poorly understood how presynaptic axonal boutons are formed, eliminated, and maintained during motor learning, and whether long-range corticocortical and thalamocortical axonal boutons show distinct structural changes during learning. In this study, we conducted two-photon imaging of presynaptic boutons of long-range axons in layer 1 (L1) of the mouse M1 during the 7-day learning of an accelerating rotarod task. The training-period-averaged rate of formation of boutons on axons projecting from the secondary motor cortical area increased, while the average rate of elimination of those from the motor thalamus (thalamic boutons) decreased. In particular, the elimination rate of thalamic boutons during days 4–7 was lower than that in untrained mice, and the fraction of pre-existing thalamic boutons that survived until day 7 was higher than that in untrained mice. Our results suggest that the late stabilization of thalamic boutons in M1 contributes to motor skill learning.

Rapid Neuronal Ultrastructure Disruption and Recovery during Spreading Depolarization-Induced Cytotoxic Edema.

Two major pathogenic events that cause acute brain damage during neurologic emergencies of stroke, head trauma, and cardiac arrest are spreading depolarizing waves and the associated brain edema that course across the cortex injuring brain cells. Virtually nothing is known about how spreading depolarization (SD)-induced cytotoxic edema evolves at the ultrastructural level immediately after insult and during recovery. In vivo 2-photon imaging followed by quantitative serial section electron microscopy was used to assess synaptic circuit integrity in the neocortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. SD triggered a rapid fragmentation of dendritic mitochondria. A large increase in the density of synapses on swollen dendritic shafts implies that some dendritic spines were overwhelmed by swelling or merely retracted. The overall synaptic density was unchanged. The postsynaptic dendritic membranes remained attached to axonal boutons, providing a structural basis for the recovery of synaptic circuits. Upon immediate reperfusion, cytotoxic edema mainly subsides as affirmed by a recovery of dendritic ultrastructure. Dendritic recuperation from swelling and reversibility of mitochondrial fragmentation suggests that neurointensive care to improve tissue perfusion should be paralleled by treatments targeting mitochondrial recovery and minimizing the occurrence of SDs.

Arousal Modulates Retinal Output

SummaryAt various stages of the visual system, visual responses are modulated by arousal. Here, we find that in mice this modulation operates as early as in the first synapse from the retina and even in retinal axons. To measure retinal activity in the awake, intact brain, we imaged the synaptic boutons of retinal axons in the superior colliculus. Their activity depended not only on vision but also on running speed and pupil size, regardless of retinal illumination. Arousal typically reduced their visual responses and selectivity for direction and orientation. Recordings from retinal axons in the optic tract revealed that arousal modulates the firing of some retinal ganglion cells. Arousal had similar effects postsynaptically in colliculus neurons, independent of activity in the other main source of visual inputs to the colliculus, the primary visual cortex. These results indicate that arousal modulates activity at every stage of the mouse visual system.

Visualization of a Distributed Synaptic Memory Code in the Drosophila Brain

During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. We analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body γ lobe, a brain structure that mediates olfactory learning. A fluorescent Ca2+ sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca2+ could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, we show that odor-evoked Ca2+ dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter.

The Effects of In Vitro Seizure‐like Activity on Structural Plasticity of Dendritic Spines

Most excitatory synapses in the mammalian brain connect to dendritic spines. These membranous protrusions function as chemical and electrical micro‐compartments, transducing individual signals of excitatory neurotransmitters from axonal boutons. Structural plasticity, the ability of spines to change their shape and thus their function in response to synaptic activity, underlies learning and memory. This shape change requires regulation of actin mesh‐works, which are regulated by actin binding proteins (ABPs) within spines. Decreased concentrations of functional ABPs induce abnormalities in the density, morphology, and dynamic nature of dendritic spines. Early‐life seizure models demonstrate altered spine morphology, and a decrease in ABPs hinders synaptic signal amplitudes. Further, neonatal seizures are linked to cognitive deficits later in life, including autistic‐like behaviors, and patients with autism spectrum disorder express depleted concentrations of endogenous ABPs and exhibit dendritic spine dysgenesis. Based upon these findings, we hypothesized that seizure‐like activity causes deficits in the structural plasticity of dendritic spines via depletion of functional ABPs. To investigate the physiological consequences of uncontrolled, synaptic excitability on intracellular actin regulation at the molecular level, we induced in vitro seizure‐like activity in primary, rat hippocampal neurons using a zero magnesium model. At multiple time points after this event, we assessed localization of ABPs using immunocytochemistry, actin turnover employing fluorescent recovery after photo bleaching, and spine and actin dynamics in response to stimulation utilizing live‐cell imaging in GFP‐actin transfected neurons. Preliminary results show a decrease in localization of ABPs to dendritic spines and an increase in spine motility after in vitro seizure‐like activity in cultured hippocampal neurons. Taken together, these data indicate that seizures may alter ABPs in a model that also shows changes in spine dynamics and structural plasticity. Ongoing work will examine this link further to determine if ABPs could be a possible therapeutic target to decrease cognitive deficits that can result from early‐life seizures and lead to intellectual disabilities.Support or Funding InformationCCDA: Center for Chronic Disorders of Aging

Neuron‐Targeted Caveolin‐1 gene therapy preserves hippocampal neuroplasticity and memory in Alzheimer’s Disease mice

Alzheimer’s disease (AD) is a neurodegenerative condition with severe cognitive deficits which is closely correlated with loss of synapses and neurons, thus, targeting neuroprotective mechanisms specifically to neuronal cells that changes the synaptic strength and resistance to toxic Ab and the following inflammation response may restore functional neuronal and synaptic plasticity independent of removing toxic amyloid species. One potential molecular target is caveolin‐1 (Cav‐1), a membrane/lipid raft (MLR) scaffolding protein that organizes synaptic proteins and associated synaptic signaling components (e.g., neurotrophin and neurotransmitter receptors). Recent pre‐clinical and clinical evidence showed that Cav‐1 and Cav‐1 associated signaling complexes (TrkA, TrkB, GluN2B, dopaminergic, cholinergic receptors, and adenylyl cyclase) were decreased in degenerating neurons from a variety of neurodegenerative conditions which included AD, CTE, and ALS. Here we show that Cav‐1 expression decreases in the hippocampus occurs at early stages of cognitive loss (6 months (m)) and correlates to the progression of pathological of AD. Furthermore, one‐time hippocampal administration of adeno‐associated virus that encodes neuron‐targeted Cav‐1 using a synapsin promoter (AAV‐SynCav1) at 3 months (m) preserves learning and memory in 9 and 11 m old amyloid‐positive APPSwePS1d9 mice (i.e., AD). At 9 month, immunoblot and proteomic analysis of hippocampus MLR confirmed decreased Cav‐1 in AD‐SynRFP and preserved Cav‐1 in AD‐SynCav1. Bioinformatics analysis identified 80 up‐regulated proteins in AD‐SynCav1 MLR and gene ontology analysis found several proteins are related to synaptic function (Shisa9, an AMPAR regulatory protein critical for memory; Piccolo, scaffolding protein in synaptic active zones) and cholesterol homeostasis (Kiaa1468, Ap2a2). At 11 m, Golgi‐Cox histology demonstrated that SynCav1 preserved CA1 dendritic arbor in AD mice compared to AD‐SynRFP mice. Electron microscopy (EM) of CA1 apical dendrites revealed preserved ultrastructural indicators of synaptic plasticity (e.g., total type I excitatory synapses, presynaptic vesicles per axonal bouton, normal dendritic spine morphology), preserved mossy fiber area, and preserved myelinated CA3 Schaffer collaterals in 11 m AD‐SynCav1 mice. These findings offer important proof‐of‐concept evidence for the use of neuron‐targeted Cav‐1 (i.e., SynCav1) gene therapy to future AAV‐9‐based clinical trials in AD as well as other neurodegenerative diseases.Support or Funding InformationVeterans Affairs Merit Award from the Department of Veterans Affairs BX003671 (B. P. Head); National Institutes of Health, Bethesda, MD, U.S.A., NS073653 (B. P. Head); and GM085179 (P. M. Patel).SynCav1gene delivery preserves fear learning and contextual memory recall in 9 month and 11 month‐old APPSwePS1d9 mice, respectively.Figure 1SynCav1gene delivery preserved hippocampal Cav‐1 expression and MLR‐localized synaptic proteins in 9 month‐old APPSwePS1d9 mice.Figure 2

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Motor cortex can directly drive the globus pallidus neurons in a projection neuron type-dependent manner in the rat.

The basal ganglia are critical for the control of motor behaviors and for reinforcement learning. Here, we demonstrate in rats that primary and secondary motor areas (M1 and M2) make functional synaptic connections in the globus pallidus (GP), not usually thought of as an input site of the basal ganglia. Morphological observation revealed that the density of axonal boutons from motor cortices in the GP was 47% and 78% of that in the subthalamic nucleus (STN) from M1 and M2, respectively. Cortical excitation of GP neurons was comparable to that of STN neurons in slice preparations. FoxP2-expressing arkypallidal neurons were preferentially innervated by the motor cortex. The connection probability of cortico-pallidal innervation was higher for M2 than M1. These results suggest that cortico-pallidal innervation is an additional excitatory input to the basal ganglia, and that it can affect behaviors via the cortex-basal ganglia-thalamus motor loop.

Urokinase-type plasminogen activator is a modulator of synaptic plasticity in the central nervous system: implications for neurorepair in the ischemic brain.

The last two decades have witnessed a rapid decrease in mortality due to acute cerebral ischemia that paradoxically has led to a rapid increase in the number of patients that survive an acute ischemic stroke with various degrees of disability. Unfortunately, the lack of an effective therapeutic strategy to promote neurological recovery among stroke survivors has led to a rapidly growing population of disabled patients. Thus, understanding the mechanisms of neurorepair in the ischemic brain is a priority with wide scientific, social and economic implications. Cerebral ischemia has a harmful effect on synaptic structure associated with the development of functional impairment. In agreement with these observations, experimental evidence indicates that synaptic repair underlies the recovery of neurological function following an ischemic stroke. Furthermore, it has become evident that synaptic plasticity is crucial not only during development and learning, but also for synaptic repair after an ischemic insult. The plasminogen activating system is assembled by a cascade of enzymes and their inhibitors initially thought to be solely involved in the generation of plasmin. However, recent work has shown that in the brain this system has an important function regulating the development of synaptic plasticity via mechanisms that not always require plasmin generation. Urokinase-type plasminogen activator (uPA) is a serine proteinase and one of the plasminogen activators, that upon binding to its receptor (uPAR) not only catalyzes the conversion of plasminogen into plasmin on the cell surface, but also activates cell signaling pathways that promote cell migration, proliferation and survival. The role of uPA is the brain is not fully understood. However, it has been reported while uPA and uPAR are abundantly found in the developing central nervous system, in the mature brain their expression is restricted to a limited group of cells. Remarkably, following an ischemic injury to the mature brain the expression of uPA and uPAR increases to levels comparable to those observed during development. More specifically, neurons release uPA during the recovery phase from an ischemic injury, and astrocytes, axonal boutons and dendritic spines recruit uPAR to their plasma membrane. Here we will review recent evidence indicating that binding of uPA to uPAR promotes the repair of synapses damaged by an ischemic injury, with the resultant recovery of neurological function. Furthermore, we will discuss data indicating that treatment with recombinant uPA is a potential therapeutic strategy to promote neurological recovery among ischemic stroke survivors.

Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review.

.Calcium imaging is emerging as a popular technique in neuroscience. A major reason is that intracellular calcium transients are reflections of electrical events in neurons. For example, calcium influx in the soma and axonal boutons accompanies spiking activity, whereas elevations in dendrites and dendritic spines are associated with synaptic inputs and local regenerative events. However, calcium transients have complex spatiotemporal dynamics, and since most optical methods visualize only one of the somatic, axonal, and dendritic compartments, a straightforward inference of the underlying electrical event is typically challenging. We highlight experiments that have directly calibrated in vivo calcium signals recorded using fluorescent indicators against electrophysiological events. We address commonly asked questions such as: Can calcium imaging be used to characterize neurons with high firing rates? Can the fluorescent signal report a decrease in spiking activity? What is the evidence that calcium transients in subcellular compartments correspond to distinct presynaptic axonal and postsynaptic dendritic events? By reviewing the empirical evidence and limitations, we suggest that, despite some caveats, calcium imaging is a versatile method to characterize a variety of neuronal events in vivo.

Dynamic Factors for Transmitter Release at Small Presynaptic Boutons Revealed by Direct Patch-Clamp Recordings.

Small size of an axon and presynaptic structures have hindered direct functional analysis of axonal signaling and transmitter release at presynaptic boutons in the central nervous system. However, recent technical advances in subcellular patch-clamp recordings and in fluorescent imagings are shedding light on the dynamic nature of axonal and presynaptic mechanisms. Here I summarize the functional design of an axon and presynaptic boutons, such as diversity and activity-dependent changes of action potential (AP) waveforms, Ca2+ influx, and kinetics of transmitter release, revealed by the technical tour de force of direct patch-clamp recordings and the leading-edge fluorescent imagings. I highlight the critical factors for dynamic modulation of transmitter release and presynaptic short-term plasticity.

Corticotectal Projections From the Premotor or Primary Motor Cortex After Cortical Lesion or Parkinsonian Symptoms in Adult Macaque Monkeys: A Pilot Tracing Study.

The corticotectal projections, together with the corticobulbar (corticoreticular) projections, work in parallel with the corticospinal tract (CST) to influence motoneurons in the spinal cord both directly and indirectly via the brainstem descending pathways. The tectospinal tract (TST) originates in the deep layers of the superior colliculus. In the present study, we analyzed the corticotectal projections from two motor cortical areas, namely the premotor cortex (PM) and the primary motor cortex (M1) in eight macaque monkeys subjected to either a cortical lesion of the hand area in M1 (n = 4) or Parkinson’s disease-like symptoms PD (n = 4). A subgroup of monkeys with cortical lesion was subjected to anti-Nogo-A antibody treatment whereas all PD monkeys were transplanted with Autologous Neural Cell Ecosystems (ANCEs). The anterograde tracer BDA was used to label the axonal boutons both en passant and terminaux in the ipsilateral superior colliculus. Individual axonal boutons were charted in the different layers of the superior colliculus. In intact animals, we previously observed that corticotectal projections were denser when originating from PM than from M1. In the present M1 lesioned monkeys, as compared to intact ones the corticotectal projection originating from PM was decreased when treated with anti-Nogo-A antibody but not in untreated monkeys. In PD-like symptoms’ monkeys, on the other hand, there was no consistent change affecting the corticotectal projection as compared to intact monkeys. The present pilot study overall suggests that the corticotectal projection is less affected by M1 lesion or PD symptoms than the corticoreticular projection previously reported in the same animals.

Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons

Synapse formation is achieved by various synaptic organizers. Although this process is highly regulated by neuronal activity, the underlying molecular mechanisms remain largely unclear. Here we show that Cbln1, a synaptic organizer of the C1q family, is released from lysosomes in axons but not dendrites of cerebellar granule cells in an activity- and Ca2+-dependent manner. Exocytosed Cbln1 was retained on axonal surfaces by binding to its presynaptic receptor neurexin. Cbln1 further diffused laterally along the axonal surface and accumulated at boutons by binding postsynaptic δ2 glutamate receptors. Cbln1 exocytosis was insensitive to tetanus neurotoxin, accompanied by cathepsin B release, and decreased by disrupting lysosomes. Furthermore, overexpression of lysosomal sialidase Neu1 not only inhibited Cbln1 and cathepsin B exocytosis invitro but also reduced axonal bouton formation invivo. Our findings imply that co-release of Cbln1 and cathepsin B from lysosomes serves as a new mechanism of activity-dependent coordinated synapse modification.

DeepBouton: Automated Identification of Single-Neuron Axonal Boutons at the Brain-Wide Scale.

Fine morphological reconstruction of individual neurons across the entire brain is essential for mapping brain circuits. Inference of presynaptic axonal boutons, as a key part of single-neuron fine reconstruction, is critical for interpreting the patterns of neural circuit wiring schemes. However, automated bouton identification remains challenging for current neuron reconstruction tools, as they focus mainly on neurite skeleton drawing and have difficulties accurately quantifying bouton morphology. Here, we developed an automated method for recognizing single-neuron axonal boutons in whole-brain fluorescence microscopy datasets. The method is based on deep convolutional neural networks and density-peak clustering. High-dimensional feature representations of bouton morphology can be learned adaptively through convolutional networks and used for bouton recognition and subtype classification. We demonstrate that the approach is effective for detecting single-neuron boutons at the brain-wide scale for both long-range pyramidal projection neurons and local interneurons.

Neuron‐Targeted Caveolin‐1 Gene Therapy Preserves Cognitive Function and Synaptic Plasticity in a Mouse Model of Alzheimer's Disease (AD)

Introduction Alzheimer's disease (AD) is a devastating neurodegenerative condition. AD patients develop severe cognitive deficits, which are closely correlated with loss of synapses and decreased plasticity. Caveolin-1 (Cav-1), a scaffolding protein within the microdomains of membrane/lipid rafts (MLRs), organizes signaling complexes that promote functional neuronal and synaptic plasticity. Previously we showed that neuron-targeted overexpression of Cav-1 using a synapsin promoter (SynCav1) promotes neuroplasticity, increases synaptic plasticity, and improves memory in aged mice. The current study investigated the impact of SynCav-1 on markers of synaptic plasticity and cognitive function in an APPSwePS1d9 mouse model of AD. Methods AAV9-SynRFP (red fluorescent protein) or AAV9-SynCav1 was delivered into the hippocampus of wild type (WT) or AD mice at 2.5 months of age. At 9 months, open field and fear conditioning test were used to evaluate learning and memory performance. After behavior tests, animal were sacrificed for immunoblot (IB) and immunofluorence microscopy (IF). Electron microscopy (EM) was used to observe the ultrastructure morphology. Results AD-SynRFP mice exhibited significant decreased fear learning (day 1) compared to WT-SynRFP mice and AD-SynCav1 mice. In contrast, AD-SynCav1 mice showed preserved fear learning (day 1), and improved contextual and cued memory recall (day 2 & 3). Both IB and IF showed decreased Cav-1 expression in 9 month-old AD-SynRFP mice. AD-SynCav1 mice showed preserved Cav-1 levels and increased expression of neuroplasticity markers (TrkB, MAP2) compared to AD-SynRFP mice. IB analysis of MLR (buoyant fractions 4 & 5) demonstrate decreased MLR-localization of TrkB, Cav-1, CT-B, synaptophysin, TrkA, PSD95, LRP in AD-SynRFP mice. In contrast, AD-SynCav1 exhibited preserved MLR-localization of TrkB, Cav-1, CT-B, synaptophysin, TrkA, PSD95 and LRP1. IF showed preserved markers of dendritic integrity (MAP2) in AD-SynCav1 mice. EM analysis of the hippocampus revealed that AD-SynRFP mice had fewer type I excitatory synapses and decreased number of presynaptic vesicles per axonal bouton, and altered spine morphology (reduced spine length and increased spine neck diameter) in CA1 apical dendrites within the stratum radiatum, which were not exhibited by AD-SynCav1 mice. Additional analysis revealed preserved myelinated Schaffer collateral axons in AD-SynCav1 mice compared to AD-SynRFP. Conclusion SynCav1 gene delivery to the hippocampus of pre-symptomatic adult AD mice (2.5 m) preserves fear learning, improves memory, and preserves ultrastructural indicators of synaptic plasticity and myelination in mild symptomatic AD mice (9 months), therefore may serve as a novel gene therapy to restore synaptic and neuroplasticity and improve brain function in individuals afflicted with Alzheimer's disease. Support or Funding Information Veterans Affairs Merit Award from the Department of Veterans Affairs BX003671 (B. P. Head), National Institutes of Health, Bethesda, MD, U.S.A., NS073653 (B. P. Head); GM085179 (P. M. Patel). (A) AD - SynRFP at 2.5 months of age exhibited learning deficits on acquisition Day 1 training compared to both WT -SynRFP and AD -SynCav1 at 9 months of age. There was no significant difference between WT - SynRFP versus AD-SynCav1. SynCav1 delivery to AD mice significantly improved hippocampal-dependent contextual memory recall on Day 2 (B) and hippocampal-independent cued memory recall on Day 3 (C). Data are presented as mean ± SEM. Significance was assumed when p < 0.05. IF microscopy revealed that, in 9 month-old age, AD-SynRFP exhibited decreased in hippocampal Cav-1 expression compared to WT-SynRFP, while SynCav1 gene delivery preserved Cav-1 expression. Scale bar = 50 mm. IB analysis of MLR fractions (buoyant fractions 4 & 5) demonstrates decreased MLR-localization of full length TrkB, Cav-1, CT-B, synaptophysin, TrkA, LRP1 in AD-SynRFP mice; while SynCav1 treatment preserve MLR-localized synaptic proteins. CA1 distal apical dendrites in the stratum radiatum were examined for ultrastructure changes of type I asymmetric synapses and presynaptic vesicles (PSVs)/axonal bouton. SynCav1 preserves total number of synapses and PSVs in the hippocampus of 9 month old AD mice. CA1 distal apical dendrites in the stratum radiatum were examined for alterations in dendritic spine morphology, i.e., spine length (mm), spine neck diameter (mm), and spine area (mm2). SynCav1 preserves dendritic spine morphology in the hippocampus of 9 month old APPSwePS1d9 mice. G ratio analysis (axon lumen diameter (Di)/fiber (axon lumen + myelin) diameter (Do)) was used to measure changes in axonal myelin fiber content of CA3 Schaffer collaterals innervating the CA1 apical dendritic arbor. SynCav1 preserves axonal myelin fiber content of hippocampal CA3 Schaffer collaterals in 9 month old APPSwePS1d9 mice. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.