Three-Dimensional Mapping of Unitary Synaptic Connections by Two-Photon Macro Photolysis of Caged Glutamate

2008 ◽  
Vol 99 (3) ◽  
pp. 1535-1544 ◽  
Author(s):  
Masanori Matsuzaki ◽  
Graham C. R. Ellis-Davies ◽  
Haruo Kasai
Keyword(s):  
Visual Cortex ◽  
Single Cell ◽  
Pyramidal Neurons ◽  
Three Dimensional ◽  
Core Region ◽  
Synaptic Connectivity ◽  
Synaptic Connections ◽  
Two Photon ◽  
Layer 2 ◽  
Caged Glutamate

To understand the precise microarchitecture of the cortical circuitry, it is crucial to know the distribution of synaptic connections and their synaptic strengths at the level of a single cell, rather than a group of cells. Here, we describe a new application of two-photon photolysis of caged glutamate that enabled us to induce an action potential in only a small number (about five) of pyramidal neurons by increasing the volume of two-photon excitation by reducing the effective numerical aperture of the objective. We performed whole cell patch-clamp recordings from layer 2/3 pyramidal neurons in the rat visual cortex and stimulated many neurons in a large three-dimensional space (∼600 × 600 × 100 μm) including neurons in layers 2/3 and 4 using this new technique. We mapped the density and amplitude of unitary excitatory postsynaptic currents and found that the basic microarchitecture of excitatory synaptic connections consists of two regions: a columnar, dense core region with a radius of 150 μm and an outer, sparse region. The dense core region includes the majority of strong synaptic connections in layer 2/3. Our results reveal the columnar organization of synaptic connectivity in the rat visual cortex, where functional columns have not been clearly demonstrated. Thus this technique will be a uniquely powerful tool for quantifying synaptic connectivity and manipulating neural activity at the single-cell level.

scholarly journals Three-dimensional Two-Photon Optogenetics and Imaging of Neural Circuits in vivo

2017 ◽  
Author(s):  
Weijian Yang ◽  
Luis Carrillo-Reid ◽  
Yuki Bando ◽  
Darcy S. Peterka ◽  
Rafael Yuste
Keyword(s):  
Visual Cortex ◽  
Neural Activity ◽  
Calcium Imaging ◽  
Pyramidal Neurons ◽  
Neural Circuits ◽  
Three Dimensional ◽  
Two Photon ◽  
Cellular Resolution ◽  
Photon Excitation

We demonstrate a holographic system for simultaneous three-dimensional (3D) two-photon stimulation and imaging of neural activity in the mouse neocortex in vivo with cellular resolution. Dual two-photon excitation paths are implemented with independent 3D targeting for calcium imaging and precision optogenetics. We validate the usefulness of the microscope by photoactivating local pools of interneurons in awake mice visual cortex in 3D, which suppress the nearby pyramidal neurons’ response to visual stimuli.

Patterns of Local Connectivity in the Neocortex

1993 ◽  
Vol 5 (5) ◽  
pp. 665-680 ◽  
Author(s):  
Andrew Nicoll ◽  
Colin Blakemore
Keyword(s):  
Visual Cortex ◽  
Pyramidal Neurons ◽  
Extreme Values ◽  
Pyramidal Cells ◽  
Excitatory Synapses ◽  
Synaptic Connections ◽  
Local Connectivity ◽  
Connection Probability ◽  
Layer 2 ◽  
Deep Layers

Dual intracellular recording of nearby pairs of pyramidal cells in slices of rat visual cortex has shown that there are significant differences in functional connectivity between the superficial and deep layers (Mason et al. 1991; Nicoll and Blakemore 1993). For pairs of cells no farther than 300 μm apart, synaptic connections between layer 2/3 pyramidal neurons were individually weaker (median peak amplitude, A, of single-fiber excitatory postsynaptic potentials, EPSPs, = 0.4 mV) but more frequent (connection probability, p = 0.087) than those between layer 5 pyramidal neurons (mean A = 0.8 mV, p < 0.015). Taken in combination with plausible estimates of the density of pyramidal cells, the total numbers of synapses on them and the number of synapses formed on their intracortical axons, the present analysis of the above data suggests that roughly 70% of the excitatory synapses on any layer 2/3 pyramid, but fewer than 1% of those on a layer 5 pyramidal neuron, are derived from neighboring pyramidal neurons in its near vicinity. Even assuming very extreme values for some parameters, chosen to erode this difference, the calculated proportion of "local synapses" for layer 5 pyramids was always markedly lower than for layer 2/3 pyramidal neurons. These results imply that local excitatory connections are much more likely to provide significant "intracortical amplification" of afferent signals in layer 2/3 than in layer 5 of rat visual cortex.

scholarly journals Asymmetric Temporal Integration of Layer 4 and Layer 2/3 Inputs in Visual Cortex

2011 ◽  
Vol 105 (1) ◽  
pp. 347-355 ◽  
Author(s):  
Giao B. Hang ◽  
Yang Dan
Keyword(s):  
Visual Cortex ◽  
Visual Processing ◽  
Pyramidal Neurons ◽  
Temporal Integration ◽  
Cortical Inhibition ◽  
Multiple Sources ◽  
Layer 2 ◽  
Layer 4 ◽  
The Difference

Neocortical neurons in vivo receive concurrent synaptic inputs from multiple sources, including feedforward, horizontal, and feedback pathways. Layer 2/3 of the visual cortex receives feedforward input from layer 4 and horizontal input from layer 2/3. Firing of the pyramidal neurons, which carries the output to higher cortical areas, depends critically on the interaction of these pathways. Here we examined synaptic integration of inputs from layer 4 and layer 2/3 in rat visual cortical slices. We found that the integration is sublinear and temporally asymmetric, with larger responses if layer 2/3 input preceded layer 4 input. The sublinearity depended on inhibition, and the asymmetry was largely attributable to the difference between the two inhibitory inputs. Interestingly, the asymmetric integration was specific to pyramidal neurons, and it strongly affected their spiking output. Thus via cortical inhibition, the temporal order of activation of layer 2/3 and layer 4 pathways can exert powerful control of cortical output during visual processing.

scholarly journals Visual Deprivation Decreases Somatic GAD65 Puncta Number on Layer 2/3 Pyramidal Neurons in Mouse Visual Cortex

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Alicja Kreczko ◽  
Anubhuthi Goel ◽  
Lihua Song ◽  
Hey-Kyoung Lee
Keyword(s):  
Visual Cortex ◽  
Visual Experience ◽  
Pyramidal Neurons ◽  
Immunohistochemical Method ◽  
Inhibitory Synapses ◽  
Acid Decarboxylase ◽  
3D Analysis ◽  
Individual Layer ◽  
Inhibitory Circuitry ◽  
Layer 2

Proper functioning of the visual system depends on maturation of both excitatory and inhibitory synapses within the visual cortex. Considering that perisomatic inhibition is one of the key factors that control the critical period in visual cortex, it is pertinent to understand its regulation by visual experience. To do this, we developed an immunohistochemical method that allows three-dimensional (3D) analysis of the glutamic acid decarboxylase (GAD) 65-positive inhibitory terminals in the visual cortex. Using this method on transgenic mice expressing yellow fluorescence protein (YFP) in a subset of neurons, we found that the number of somatic GAD65-puncta on individual layer 2/3 pyramidal neurons is reduced when mice are dark-reared from birth and reverted to normal levels by re-exposure to light. There was no change in GAD65-puncta volume or intensity. These results support the reorganization of inhibitory circuitry within layer 2/3 of visual cortex in response to changes in visual experience.

Layer-specific involvement of endocannabinoid signaling in muscarinic-induced long-term depression in layer 2/3 pyramidal neurons of rat visual cortex

2019 ◽  
Vol 1712 ◽  
pp. 124-131 ◽  
Author(s):  
Kayoung Joo ◽  
Kwang-Hyun Cho ◽  
Sung-Hee Youn ◽  
Hyun-Jong Jang ◽  
Duck-Joo Rhie
Keyword(s):  
Visual Cortex ◽  
Pyramidal Neurons ◽  
Long Term Depression ◽  
Layer 2

scholarly journals Divergent excitation two photon microscopy for 3D random access mesoscale imaging at single cell resolution

2019 ◽  
Author(s):  
FK Janiak ◽  
P Bartel ◽  
MR Bale ◽  
T Yoshimatsu ◽  
E Komulainen ◽  
...  
Keyword(s):  
Single Cell ◽  
Neuronal Activity ◽  
Low Cost ◽  
Three Dimensional ◽  
Random Access ◽  
Field Of View ◽  
Three Dimensions ◽  
Two Photon ◽  
Wide Range ◽  
Effective Excitation

ABSTACTIn neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing ‘divergent beam optics’ (DBO), we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation increases both the space-bandwidth product and effective excitation power, and allows for straight-forward tailoring of the point-spread-function. Moreover, rapid laser-focus control via an electrically tunable lens now allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and fully compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in mice, zebrafish and fruit flies.

scholarly journals Stability of excitatory structural connectivity predicts the probability of CA1 pyramidal neurons to become engram neurons

2019 ◽  
Author(s):  
Tim P. Castello-Waldow ◽  
Ghabiba Weston ◽  
Alireza Chenani ◽  
Yonatan Loewenstein ◽  
Alon Chen ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Structural Connectivity ◽  
Synaptic Connectivity ◽  
Two Photon ◽  
Ca1 Pyramidal Neurons ◽  
Trace Fear Conditioning ◽  
Hippocampal Ca1 ◽  
Trace Fear ◽  
Activity Dependent ◽  
Deep Brain

SUMMARYNeurons undergoing activity-dependent plasticity represent experience and are functional for learning and recall thus they are considered cellular engrams of memory. Although increase in excitability and stability of structural synaptic connectivity have been implicated in the formation and persistance of engrams, the mechanisms bringing engrams into existence are still largely unknown. To investigate this issue, we tracked the dynamics of structural excitatory synaptic connectivity of hippocampal CA1 pyramidal neurons over two weeks using deep-brain two-photon imaging in live mice. We found that neurons that will prospectively become part of an engram display higher stability of connectivity than neurons that will not. A novel experience significantly stabilizes the connectivity of non-engram neurons. Finally, the density and survival of dendritic spines negatively correlates to freezing to the context but not to the tone in a trace fear conditioning learning paradigm.

scholarly journals Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Christopher A Baker ◽  
Yishai M Elyada ◽  
Andres Parra ◽  
M McLean Bolton
Keyword(s):  
Action Potentials ◽  
Single Neuron ◽  
Synaptic Connectivity ◽  
Electrophysiological Recording ◽  
Synaptic Connections ◽  
Spike Generation ◽  
Two Photon ◽  
Cellular Resolution ◽  
Temporal Focusing ◽  
Imaging Plane

We describe refinements in optogenetic methods for circuit mapping that enable measurements of functional synaptic connectivity with single-neuron resolution. By expanding a two-photon beam in the imaging plane using the temporal focusing method and restricting channelrhodopsin to the soma and proximal dendrites, we are able to reliably evoke action potentials in individual neurons, verify spike generation with GCaMP6s, and determine the presence or absence of synaptic connections with patch-clamp electrophysiological recording.

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