Search

Sensory cortex exhibits receptive field plasticity throughout life in response to changes in sensory experience and offers the experimental possibility of aligning functional changes in receptive field properties with underpinning structural changes in synapses. We looked at the effects on structural plasticity of two different patterns of whisker deprivation in male and female mice: chessboard deprivation, which causes functional plasticity; and all deprived, which does not. Using 2-photon microscopy and chronic imaging through a cranial window over the barrel cortex, we found that layer 2/3 neurones exhibit robust structural plasticity, but only in response to whisker deprivation patterns that cause functional plasticity. Chessboard pattern deprivation caused dual-component plasticity in layer 2/3 by (1) increasing production of new spines that subsequently persisted for weeks and (2) enlarging spine head sizes in the preexisting stable spine population. Structural plasticity occurred on basal dendrites, but not apical dendrites. Both components of plasticity were absent in α CaMKII-T286A mutants that lack LTP and experience-dependent potentiation in barrel cortex, implying that αCaMKII autophosphorylation is not only important for stabilization and enlargement of spines, but also for new spine production. These studies therefore reveal the relationship between spared whisker potentiation in layer 2/3 neurones and the form and mechanisms of structural plasticity processes that underlie them. SIGNIFICANCE STATEMENT This study provides a missing link in a chain of reasoning that connects LTP to experience-dependent functional plasticity in vivo We found that increases in dendritic spine formation and spine enlargement (both of which are characteristic of LTP) only occurred in barrel cortex during sensory deprivation that produced potentiation of sensory responses. Furthermore, the dendritic spine plasticity did not occur during sensory deprivation in mice lacking LTP and experience-dependent potentiation (αCaMKII autophosphorylation mutants). We also found that the dual-component dendritic spine plasticity only occurred on basal dendrites and not on apical dendrites, thereby resolving a paradox in the literature suggesting that layer 2/3 neurones lack structural plasticity in response to sensory deprivation., (Copyright © 2020 the authors.)

The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology.

Neurons connect and transmit information via synapses. The major excitatory and inhibitory (E-I) neurotransmitters are glutamate and γ-amino butyric acid (GABA), respectively. The E-I balance plays an important role in various brain functions. In this review, we summarize the role of GABA on synaptic integration and synaptic plasticity by introducing our own recent findings. In synaptic integration, GABA is considered to inhibit depolarization induced by glutamate and suppress action potentials. We found that GABA also has a more direct role on the synaptic plasticity of excitatory inputs. GABA effectively promotes the shrinkage and elimination of synapses by suppressing local dendritic Ca(2+) signaling, while keeping the Ca(2+) domain of the NMDA receptors intact. In this manner, GABA promoted the activation of calcineurin, which in turn activated cofilin. Interestingly, shrinkage tended to spread, likely due to the spread of cofilin, and induced competitive selection of synapses via its phosphorylation and dephosphorylation. The selection of synapses is key to the reorganization of the central nervous system during development and in adulthood, and GABA plays key roles in various mental disorders, such as autism and schizophrenia. Our results account well for the in vivo GABA functions on synaptic selection, and may help to develop new therapeutic compounds.

Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice.

Functional clustering of neurons is frequently observed in the motor cortex. However, it is unknown if, when, and how fine-scale (<100 μm) functional clusters form relative to voluntary forelimb movements. In addition, the implications of clustering remain unclear. To address these issues, we conducted two-photon calcium imaging of mouse layer 2/3 motor cortex during a self-initiated lever-pull task. In the imaging session after 8-9 days of training, head-restrained mice had to pull a lever for ∼600 ms to receive a water drop, and then had to wait for >3 s to pull it again. We found two types of task-related cells in the mice: cells whose peak activities occurred during lever pulls (pull cells) and cells whose peak activities occurred after the end of lever pulls. The activity of pull cells was strongly associated with lever-pull duration. In ∼40% of imaged fields, functional clusterings were temporally detected during the lever pulls. Spatially, there were ∼70-μm-scale clusters that consisted of more than four pull cells in ∼50% of the fields. Ensemble and individual activities of pull cells within the cluster more accurately predicted lever movement trajectories than activities of pull cells outside the cluster. This was likely because clustered pull cells were more often active in the individual trials than pull cells outside the cluster. This higher fidelity of activity was related to higher trial-to-trial correlations of activities of pairs within the cluster. We propose that strong recurrent network clusters may represent the execution of voluntary movements.

To understand the fine-scale structures and functional properties of individual neurons in vivo, we developed and validated a rapid genetic technique that enables simultaneous investigation of multiple neuronal properties with single-cell resolution in the living rodent brain. Our technique PASME (promoter-assisted sparse-neuron multiple-gene labeling using in uteroelectroporation) targets specific small subsets of sparse pyramidal neurons in layer 2/3, layer 5 of the cerebral cortex and in the hippocampus with multiple fluorescent reporter proteins such as postsynaptic PSD-95-GFP and GFP-gephyrin. The technique is also applicable for targeting independently individual neurons and their presynaptic inputs derived from surrounding neurons. Targeting sparse layer 2/3 neurons, we uncovered a novel subpopulation of layer 2/3 neurons in the mouse cerebral cortex. This technique, broadly applicable for probing and manipulating neurons with single-cell resolution in vivo, should provide a robust means to uncover the basic mechanisms employed by the brain, especially when combined with in vivo two-photon laser-scanning microscopy and/or optogenetic technologies., (2011 Elsevier Inc. All rights reserved.)

Introduction: Two-photon uncaging takes advantage of the inherent optical sectioning power of two-photon excitation to generate highly localized concentration increases of neurotransmitters such as glutamate. This can be used to activate isolated clusters of receptors and, thus, produce maps of receptor densities, or activate intracellular signal transduction under these receptors, in three dimensions and in complex structures such as hippocampal brain slices. Used in combination with two-photon imaging, two-photon uncaging provides a means to study the long-term structural and functional consequences of stimulation of structures such as dendritic spines. This protocol gives an overview of the procedures used for two-photon uncaging microscopy. It includes a detailed description of the development of a microscope that enables effective two-photon release of caged neurotransmitters and provides several examples of its use in cultured and acutely isolated hippocampal neurons.

Sensory cortex exhibits receptive field plasticity throughout life in response to changes in sensory experience and offers the experimental possibility of aligning functional changes in receptive field properties with underpinning structural changes in synapses. We looked at the effects on structural plasticity of two different patterns of whisker deprivation in male and female mice: chessboard deprivation, which causes functional plasticity; and all deprived, which does not. Using 2-photon microscopy and chronic imaging through a cranial window over the barrel cortex, we found that layer 2/3 neurones exhibit robust structural plasticity, but only in response to whisker deprivation patterns that cause functional plasticity. Chessboard pattern deprivation caused dual-component plasticity in layer 2/3 by (1) increasing production of new spines that subsequently persisted for weeks and (2) enlarging spine head sizes in the preexisting stable spine population. Structural plasticity occurred on basal dendrites, but not apical dendrites. Both components of plasticity were absent in aCaMKII-T286A mutants that lack LTP and experience-dependent potentiation in barrel cortex, implying that aCaMKII autophosphorylation is not only important for stabilization and enlargement of spines, but also for new spine production. These studies therefore reveal the relationship between spared whisker potentiation in layer 2/3 neurones and the form and mechanisms of structural plasticity processes that underlie them. [ABSTRACT FROM AUTHOR]

Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble. [ABSTRACT FROM AUTHOR]

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. We used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines. [ABSTRACT FROM AUTHOR]

Long-term potentiation of synapse strength requires enlargement of dendritic spines on cerebral pyramidal neurons. Long-term depression is linked to spine shrinkage. Indeed, spines are dynamic structures: they form, change their shapes and volumes, or can disappear in the space of hours. Do all such changes result from synaptic activity, or do some changes result from intrinsic processes? How do enlargement and shrinkage of spines relate to elimination and generation of spines, and how do these processes contribute to the stationary distribution of spine volumes? To answer these questions, we recorded the volumes of many individual spines daily for several days using two-photon imaging of CA1 pyramidal neurons in cultured slices of rat hippocampus between postnatal days 17 and 23. With normal synaptic transmission, spines often changed volume or were created or eliminated, thereby showing activity-dependent plasticity. However, we found that spines changed volume even after we blocked synaptic activity, reflecting a native instability of these small structures over the long term. Such "intrinsic fluctuations" showed unique dependence on spine volume. A mathematical model constructed from these data and the theory of random fluctuations explains population behaviors of spines, such as rates of elimination and generation, stationary distribution of volumes, and the long-term persistence of large spines. Our study finds that generation and elimination of spines are more prevalent than previously believed, and spine volume shows significant correlation with its age and life expectancy. The population dynamics of spines also predict key psychological features of memory. [ABSTRACT FROM AUTHOR]

The article presents a study on the quantum yield of neurons in isolated brain slices, using the method of photochemical uncaging of neurotransmitters. A caged glutamate, 4-carboxymethoxy-5,7-dinitroindolinylglutamate (CDNI-Glu), was synthesized. The study found that the comparative uncaging with near-ultraviolet irradiation or two-photon excitation of CDNI-glu and 4-methoxy-7-nitroidolinylglutamate (MNI-Glu) on the same neurons elicited postsynaptic currents consistent with their quantum yield.

Abstract: Two-photon excitation imaging is the least invasive optical approach to study living tissues. We have established two-photon extracellular polar-tracer (TEP) imaging with which it is possible to visualize and quantify all exocytic events in the plane of focus within secretory tissues. This technology also enables estimate of the precise diameters of vesicles independently of the spatial resolution of the optical microscope, and determination of the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). TEP imaging has been applied to representative secretory glands, e.g., exocrine pancreas, endocrine pancreas, adrenal medulla and a pheochromocytoma cell line (PC12), and has revealed unexpected diversity in the spatial organization of exocytosis and endocytosis crucial for the physiology and pathology of secretory tissues and neurons. TEP imaging and TEPIQ analysis are powerful tools for elucidating the molecular and cellular mechanisms of exocytosis and certain related diseases, such as diabetes mellitus, and the development of new therapeutic agents and diagnostic tools. [Copyright &y& Elsevier]

Abstract: Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the “initial dip” in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used two-photon excitation imaging to characterize rapid activity-dependent mitochondrial responses in single neurons. This approach allowed simultaneous multicolor imaging of individual mitochondria in single mouse Purkinje neurons in culture. Mitochondrial depolarization was induced immediately when the cytosolic free Ca2+ concentration ([Ca2+]i) exceeded 15μM and was associated with oxidation of mitochondrial NAD(P)H, suggesting that Ca2+-induced mitochondrial depolarization mediated by the Ca2+ uniporter directly facilitated oxidation of NAD(P)H. With the use of a miniature oxygen electrode, we detected a burst of oxygen consumption within 0.2s after the onset of cell depolarization in single Purkinje neurons, and this rapid increase in oxygen consumption was dependent on the increase in [Ca2+]i. We have thus demonstrated a rapid Ca2+-dependent consumption of oxygen that is mediated by mitochondrial depolarization in mammalian central neurons. This process might function as a rapid feed-forward mechanism in homeostatic control of the cytosolic ATP concentration. [Copyright &y& Elsevier]

Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory. [ABSTRACT FROM AUTHOR]

We have synthesized a photosensitive (or caged) 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI) derivative of γ-aminobutyric acid (GABA). Two-photon excitation of CDNI-GABA produced rapid activation of GABAergic currents in neurons in brain slices with an axial resolution of approximately 2 μm and enabled high-resolution functional mapping of GABA-A receptors. Two-photon uncaging of GABA, the main inhibitory neurotransmitter, should allow detailed studies of receptor function and synaptic integration with subcellular precision. [ABSTRACT FROM AUTHOR]

We developed a caged GABA (γ-aminobutyric acid), which, when combined with an appropriate caged glutamate, allows bimodal control of neuronal membrane potential with subcellular resolution using optically independent two-photon uncaging of each neurotransmitter. We used two-color, two-photon uncaging to fire and block action potentials from rat hippocampal CA1 neurons in brain slices with 720-nm and 830-nm light, respectively. Our method should be generalizable to other chemical messenger pairs. [ABSTRACT FROM AUTHOR]

Abstract: We developed a method that uses Channelrhodopsin-2 (ChR2) for transcranial optogenetic stimulation. This method is based on scanning a light beam over the brain, thereby photostimulating ChR2-expressing neurons in intact mice. As a proof of principle, we applied this technique to the motor cortex of transgenic mice expressing ChR2 in cortical pyramidal cells. Photostimulation induced limb movements that were time-locked with millisecond precision and could be induced at frequencies up to 20Hz. By scanning this light beam, we could map the distribution of neurons associated with limb movement. With this approach we could simultaneously define motor maps controlling two limbs and could reproducibly generate such cortical motor maps over periods of weeks. This method allows non-invasive mapping of brain circuitry in living animals and could help define the connection between behavior and brain circuitry. [Copyright &y& Elsevier]