scholarly journals Ex vivo and in vivo imaging of mouse parietal association cortex activity in episodes of cued fear memory formation and retrieval

2019 ◽  
Author(s):  
Olga I. Ivashkina ◽  
Anna M. Gruzdeva ◽  
Marina A. Roshchina ◽  
Ksenia A. Toropova ◽  
Konstantin V. Anokhin
Keyword(s):  
Parietal Cortex ◽  
Ex Vivo ◽  
Anterior Part ◽  
Fear Memory ◽  
Memory Formation ◽  
Movement Planning ◽  
Association Cortex ◽  
Memory Processing ◽  
Two Photon

AbstractThe parietal cortex in rodents has an integrative function and participates in sensory and spatial processing, movement planning and decision-making. However, much less is known about its functions in associative memory processing. Here using Fos immunohistochemical mapping of neuronal activity and two-photon imaging in Fos-eGFP mice we show an involvement of anterior part of the parietal cortex (PtA) in the formation and retrieval of recent fear memory in mice. Using ex vivo c-fos imaging we demonstrate the specific activation of the PtA during recent memory retrieval. In vivo two-photon c-fos imaging confirms these results as well as establishes the activation of the PtA neurons during fear memory formation. Additionally, we describe a design of Fos-Cre-GCaMP transgenic mice to investigate long-term changes of calcium dynamics in neurons captured with Fos-TRAP technique during fear conditioning training.

scholarly journals Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory

2021 ◽  
Vol 22 (15) ◽  
pp. 8244
Author(s):  
Olga I. Ivashkina ◽  
Anna M. Gruzdeva ◽  
Marina A. Roshchina ◽  
Ksenia A. Toropova ◽  
Konstantin V. Anokhin
Keyword(s):  
Ex Vivo ◽  
Fear Memory ◽  
Experimental Models ◽  
Movement Planning ◽  
Fear Learning ◽  
Association Cortex ◽  
Transgenic Mouse Line ◽  
Two Photon ◽  
Parietal Association Cortex

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis

2011 ◽  
Vol 106 (11) ◽  
pp. 939-946 ◽  
Author(s):  
Mirjam oude Egbrink ◽  
Viviane Heijnen ◽  
Remco Megens ◽  
Wim Engels ◽  
Hans Vink ◽  
...  
Keyword(s):  
Vessel Wall ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Endothelial Glycocalyx ◽  
Knock Out ◽  
Two Photon ◽  
Common Carotid Arteries ◽  
Wall Interactions

SummaryThe endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 – 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 – 80), which was significantly higher than in B6 bifurcations (0 (0 – 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 μm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 μm in young mice to 3.0 μm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 μm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

Absence of a Prevalent Laminar Distribution of IPSPs in Association Cortical Neurons of Cat

1997 ◽  
Vol 78 (5) ◽  
pp. 2742-2753 ◽  
Author(s):  
Diego Contreras ◽  
Niklaus Dürmüller ◽  
Mircea Steriade
Keyword(s):  
Cortical Neurons ◽  
Feedback Inhibition ◽  
Anterior Part ◽  
Cortical Stimulation ◽  
Association Cortex ◽  
Postsynaptic Potentials ◽  
Thalamic Stimulation ◽  
Laminar Distribution ◽  
Deep Layers

Contreras, Diego, Niklaus Dürmüller, and Mircea Steriade. Absence of a prevalent laminar distribution of IPSPs in association cortical neurons of cat. J. Neurophysiol. 78: 2742–2753, 1997. The depth distribution of inhibitory postsynaptic potentials (IPSPs) was studied in cat suprasylvian (association) cortex in vivo. Single and dual simultaneous intracellular recordings from cortical neurons were performed in the anterior part of suprasylvian gyrus (area 5). Synaptic responses were obtained by stimulating the suprasylvian cortex, 2–3 mm anterior to the recording site, as well as the thalamic lateral posterior (LP) nucleus. Neurons were recorded from layers 2 to 6 and were classified as regular spiking (RS, n = 132), intrinsically bursting (IB, n = 24), and fast spiking (FS, n = 4). Most IB cells were located in deep layers (below 0.7 mm, n = 19), but we also found some IB cells more superficially (between 0.2 and 0.5 mm, n = 5). Deeply lying corticothalamic neurons were identified by their antidromic invasion on thalamic stimulation. Neurons responded with a combination of excitatory postsynaptic potentials (EPSPs) and IPSPs to both cortical and thalamic stimulation. No consistent relation was found between cell type or cell depth and the amplitude or duration of the IPSPs. In response to thalamic stimulation, RS cells had IPSPs of 7.9 ± 0.9 (SE) mV amplitude and 88.9 ± 6.4 ms duration. In IB cells, IPSPs elicited by thalamic stimulation had 7.4 ± 1.3 mV amplitude and 84.7 ± 14.3 ms duration. The differences between the two (RS and IB) groups were not statistically significant. Compared with thalamically elicited inhibitory responses, cortical stimulation evoked IPSPs with higher amplitude (12.3 ± 1.7 mV) and longer duration (117 ± 17.3 ms) at all depths. Both cortically and thalamically evoked IPSPs were predominantly monophasic. Injections of Cl− fully reversed thalamically as well as cortically evoked IPSPs and revealed additional late synaptic components in response to cortical stimulation. These data show that the amount of feed forward and feedback inhibition to cat's cortical association cells is not orderly distributed to distinct layers. Thus local cortical microcircuitry goes beyond the simplified structure determined by cortical layers.

scholarly journals Ultrasound-assisted gatifloxacin delivery in mouse cornea, in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Uk Jegal ◽  
Jun Ho Lee ◽  
Jungbin Lee ◽  
Hyerin Jeong ◽  
Myoung Joon Kim ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Ophthalmic Solution ◽  
Ultrasound Treatment ◽  
Ocular Infection ◽  
Therapeutic Effects ◽  
Corneal Surface ◽  
Ultrasound Waves ◽  
Two Photon Microscopy ◽  
Two Photon

Abstract Gatifloxacin is a 4th generation fluoroquinolone antibiotic used in the clinic to treat ocular infection. One limitation of gatifloxacin is its relatively poor corneal penetration, and the increase of its trans-corneal delivery would be beneficial to reduce the amount or frequency of daily dose. In this study, ultrasound treatment was applied to enhance the trans-corneal delivery of gatifloxacin without damage. Experiments were conducted on mouse eyes in ex vivo and in vivo conditions. Ultrasound waves with 1 MHz in frequency, 1.3 W/cm2 in intensity were applied onto the mouse cornea for 5 minutes, and then gatifloxacin ophthalmic solution was instilled and left there for 10 minutes. 3D gatifloxacin distribution in the cornea was measured by two-photon microscopy (TPM) imaging based on its intrinsic fluorescence. Longitudinal TPM imaging of ultrasound treated mouse corneas showed the increase of initial gatifloxacin intensities on the corneal surface compared to untreated mouse corneas by 67%, and then the increased gatifloxacin delivery into the cornea from the surface at later time. The delivered gatifloxacin in the corneal epithelium stayed longer in the ultrasound treated corneas than in the untreated corneas. The enhanced trans-corneal delivery and extended stay of gatifloxacin in the mouse cornea by ultrasound treatment could be beneficial for therapeutic effects. This study demonstrated the detail process of enhanced trans-corneal gatifloxacin delivery by ultrasound treatment.

scholarly journals Two-Photon Microscopy Allows Imaging and Characterization of Cochlear Microvasculature In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Friedrich Ihler ◽  
Mattis Bertlich ◽  
Bernhard Weiss ◽  
Steffen Dietzel ◽  
Martin Canis
Keyword(s):  
Fluorescence Microscopy ◽  
Inner Ear ◽  
Ex Vivo ◽  
Small Scale ◽  
Published Data ◽  
Cochlear Blood Flow ◽  
Two Photon ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanate- or Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of90.0±22.7 μm. Imaging yielded a mean contrast-to-noise ratio (CNR) of3.3±1.7. Mean diameter in vivo was16.5±6.0 μm for arterioles and8.0±2.4 μm for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with12.2±1.6 μm and6.6±1.0 μm, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P<0.001andP=0.022, two-way ANOVA). Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.

Two-Photon Imaging of Cellular Activities in Oxygen Sensing Tissues

2008 ◽  
Vol 14 (6) ◽  
pp. 519-525 ◽  
Author(s):  
Christoph Wotzlaw ◽  
Utta Berchner-Pfannschmidt ◽  
Joachim Fandrey ◽  
Helmut Acker
Keyword(s):  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Oxygen Sensing ◽  
Rat Kidney ◽  
Hypoxia Inducible Factor ◽  
The Body ◽  
Cellular Functions ◽  
Protein Protein Interaction ◽  
Two Photon

AbstractThe cellular oxygen sensing system of the body ensures appropriate adaptation of cellular functions toward hypoxia by regulating gene expression and ion channel activity. Two-photon laser microscopy is an ideal tool to study and prove the relevance of the molecular mechanisms within oxygen sensing pathways on the cellular and complex tissue or organ level. Images of hypoxia inducible factor 1 (HIF-1) subunit nuclear mobility and protein-protein interaction in living cells, of hypoxia-induced changes in membrane potential and intracellular calcium of live ex vivo carotid bodies as well as of rat kidney proximal tubulus function in vivo, will be shown.

scholarly journals Emergent cortical circuit dynamics contain dense, interwoven ensembles of spike sequences

2017 ◽  
Vol 118 (3) ◽  
pp. 1914-1925 ◽  
Author(s):  
Joseph B. Dechery ◽  
Jason N. MacLean
Keyword(s):  
Ex Vivo ◽  
Temporal Coding ◽  
Population Activity ◽  
Flexible Assembly ◽  
Two Photon ◽  
Phase Sequence ◽  
Spike Sequences ◽  
Neuronal Assembly ◽  
Large Repertoire

Temporal codes are theoretically powerful encoding schemes, but their precise form in the neocortex remains unknown in part because of the large number of possible codes and the difficulty in disambiguating informative spikes from statistical noise. A biologically plausible and computationally powerful temporal coding scheme is the Hebbian assembly phase sequence (APS), which predicts reliable propagation of spikes between functionally related assemblies of neurons. Here, we sought to measure the inherent capacity of neocortical networks to produce reliable sequences of spikes, as would be predicted by an APS code. To record microcircuit activity, the scale at which computation is implemented, we used two-photon calcium imaging to densely sample spontaneous activity in murine neocortical networks ex vivo. We show that the population spike histogram is sufficient to produce a spatiotemporal progression of activity across the population. To more comprehensively evaluate the capacity for sequential spiking that cannot be explained by the overall population spiking, we identify statistically significant spike sequences. We found a large repertoire of sequence spikes that collectively comprise the majority of spiking in the circuit. Sequences manifest probabilistically and share neuron membership, resulting in unique ensembles of interwoven sequences characterizing individual spatiotemporal progressions of activity. Distillation of population dynamics into its constituent sequences provides a way to capture trial-to-trial variability and may prove to be a powerful decoding substrate in vivo. Informed by these data, we suggest that the Hebbian APS be reformulated as interwoven sequences with flexible assembly membership due to shared overlapping neurons. NEW & NOTEWORTHY Neocortical computation occurs largely within microcircuits comprised of individual neurons and their connections within small volumes (<500 μm3). We found evidence for a long-postulated temporal code, the Hebbian assembly phase sequence, by identifying repeated and co-occurring sequences of spikes. Variance in population activity across trials was explained in part by the ensemble of active sequences. The presence of interwoven sequences suggests that neuronal assembly structure can be variable and is determined by previous activity.

scholarly journals Calcium Imaging of Living Astrocytes in the Mouse Spinal Cord following Sensory Stimulation

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Giovanni Cirillo ◽  
Daniele De Luca ◽  
Michele Papa
Keyword(s):  
Spinal Cord ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Sensory Stimulation ◽  
Widespread Application ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Heart Beating ◽  
Sensory Activity

Astrocytic Ca2+dynamics have been extensively studied inex vivomodels; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca2+signaling in living central nervous system. Ca2+waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca2+levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca2+levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca2+levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion,in vivomorphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

scholarly journals PSD-95 in dorsal CA1 contributes to the persistence of fear memory

2020 ◽  
Author(s):  
Magdalena Ziółkowska ◽  
Malgorzata Borczyk ◽  
Agata Nowacka ◽  
Maria Nalberczak-Skóra ◽  
Małgorzata Alicja Śliwińska ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Fear Memory ◽  
Fear Extinction ◽  
Glutamatergic Synapses ◽  
Contextual Fear ◽  
Genetic Manipulations ◽  
3D Electron Microscopy ◽  
Ca1 Area ◽  
And Function

ABSTRACTThe ability to update and extinguish fearful memories is essential for survival. Accumulating data indicate that the dorsal CA1 area (dCA1) contributes to this process. However, the cellular and molecular basis of fear memory updating remains poorly understood. Postsynaptic density protein 95 (PSD-95) regulates the structure and function of glutamatergic synapses. Here, we investigated the role of dCA1 PSD-95-driven synaptic plasticity in contextual fear extinction. Using dCA1-targeted genetic manipulations in vivo combined with PSD-95 immunostaining and 3D electron microscopy ex vivo, we demonstrate that phosphorylation of PSD-95 at serine 73 PSD-95(S73) is necessary for contextual fear extinction-induced expression of PSD-95 and remodeling of glutamatergic synapses. Surprisingly, PSD-95 phosphorylation is not necessary for fear memory formation or early extinction but is required for updating a partly extinguished fear memory, affecting its persistence. Using a chemogenetic manipulation, we confirm that updating of the partly extinguished fear requires PSD-95 expression and dCA1 activity during a prior extinction session. Overall, our data indicate that dCA1 synapses are remodeled upon the extinction of contextual fear memories; this process relies on PSD-95(S73) phosphorylation and enables future updating of a partly extinguished contextual fear memory. These findings show how the hippocampus may contribute to the persistence of fear memories.

scholarly journals Deep tissue imaging by enhanced photon collection

2014 ◽  
Vol 07 (05) ◽  
pp. 1450034 ◽  
Author(s):  
Viera Crosignani ◽  
Sohail Jahid ◽  
Alexander Dvornikov ◽  
Enrico Gratton
Keyword(s):  
Ex Vivo ◽  
Second Harmonic ◽  
Fold Increase ◽  
Turbid Media ◽  
Tissue Imaging ◽  
Fluorescence Lifetime Imaging ◽  
Detection Scheme ◽  
Two Photon ◽  
Tissue Phantoms

We have developed a two-photon fluorescence microscope capable of imaging up to 4mm in turbid media with micron resolution. The key feature of this instrument is the innovative detector, capable of collecting emission photons from a wider surface area of the sample than detectors in traditional two-photon microscopes. This detection scheme is extremely efficient in the collection of emitted photons scattered by turbid media which allows eight fold increase in the imaging depth when compared with conventional two-photon microscopes. Furthermore, this system also has in-depth fluorescence lifetime imaging microscopy (FLIM) imaging capability which increases image contrast. The detection scheme captures emission light in a transmission configuration, making it extremely efficient for the detection of second harmonic generation (SHG) signals, which is generally forward propagating. Here we present imaging experiments of tissue phantoms and in vivo and ex vivo biological tissue performed with this microscope.

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