Fiberoptic fluorescence microscopy (FFM) employs optical fibers as small as 300 micrometers in diameter and offers the ability to image cellular and subcellular processes in deep brain structures including the Ventral Tegmental Area (VTA) and the substantia nigra (Sn).
Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.
This document contains three annotated bibliographies summarizing research articles:
The first summarizes a study finding that intracellular gold nanoparticles increase neuronal excitability and seizure activity in mice brains. The second describes research using an automated maze to assess hippocampus-sensitive memory in mice. The third summarizes a study using magnetic resonance imaging to find age-dependent axonal transport deficits in a mouse model of frontotemporal dementia.
1. The document summarizes three research papers on the effects of nanoparticles, memory tests in mice, and axonal transport deficits.
2. One study found that intracellular gold nanoparticles can increase neuronal excitability and aggravate seizures. Another developed an automated maze to assess hippocampus-dependent memory without human interference.
3. The third study used MRI to show age-dependent axonal transport deficits in a mouse model of tauopathy, linking the deficits to hyperphosphorylated tau protein in the brain.
Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Loco...InsideScientific
In this webinar sponsored by Neurotar, experts present their research utilizing the Mobile HomeCage®, an experimental tool which ensures the stability required for high-precision neurophysiological techniques while allowing mice to navigate and explore their environment.
Case Study #1:
Dr. Sarah Stuart and Dr. Jon Palacios-Filardo of the University of Bristol present their studies combining analysis of goal-directed behavior with whole-cell recordings from the hippocampus of awake mice. The researchers share useful tips for the surgery protocol and for adjusting the head fixation angle in order to facilitate mouse motility and exploratory behavior.
Case Study #2:
Dr. Alexander Dityatev and Weilun Sun from the German Center for Neurodegenerative Diseases (DZNE) discuss 2-photon imaging of fluorescently labeled microglia in vivo in the context of neurodegenerative disease. They also present their recent data on the effects of different anesthetics on the microglial response to localized laser injury.
Case Study #3:
Dr. Norbert Hájos from the Hungarian Academy of Sciences presents his lab’s research into the amygdala’s role in reward-driven behavior. He shares the challenges of making single-unit recordings using silicon probes during mouse locomotion and subsequent morphological identification of active neurons in the amygdala.
Key topics covered during this webinar include:
- Requirements for stable single-cell recordings and 2-photon imaging in behaving mice
- Challenges of combining high-precision techniques with behavioral research
- Methodological considerations for improving exploratory behavior in head-fixed mice
- Quantitative analysis of microglial function using 2-photon microscopy in awake mice
- Recording neuronal activity in the amygdala of awake mice followed by morphological identification of recorded neurons
Making Optical and Electrophysiological Measurements in the Brain of Head-Fix...InsideScientific
A growing number of researchers are moving from reduced preparations such as dissociated cultured neurons or brain slices, to experimentation in live animals - in vivo - using advanced methods such as two-photon microscopy or combined optogenetics and patch-clamp recordings. In order to immobilize the animal during these challenging applications general anesthesia is often administered; however, the use of anesthetics greatly distorts brain function.
Is there a better way?
In this exclusive webinar sponsored by Neurotar Ltd, leading experts in the technology will discuss methodology, best-practices and show attendees how to immobilize the rodent’s head without restraining its body using the Mobile HomeCage™. The result is a controlled research environment for studying brain function in awake, freely-moving subjects with no stress to the animal. Discussion around how this technique can be applied to the study of neuronal plasticity, neurodegeneration, addiction, brain trauma and other pathophysiological conditions in longitudinal experiments will be included. Furthermore, presenters will demonstrate how this methodology is best combined with microscopy and electrophysiology techniques – all in vivo.
Nanotechnology offers solutions to challenges in genetic engineering. MEMS chips can be used for nanoinjection, delivering DNA to fertilized eggs through a nanometer-scale lance charged with DNA. This overcomes issues with pronuclear microinjection in agricultural animals. Nanoinjection uses electroporation, generating a localized electric field to insert DNA without fluid. Nanoparticles can also deliver therapeutic genes for conditions like retinopathies, protecting against viruses while targeting delivery and limiting expression time. Various nanoparticle types including polymers, gold, and magnetic varieties show promise for gene therapy applications.
How To Study Structural and Functional Properties of TendonInsideScientific
WATCH THE VIDEO: http://bit.ly/2nl7Nx6
In this webinar presented by Aurora Scientific, Matthew Borkowski and Dylan Sarver discuss how to characterize the structural and functional properties of tendon.
Specifically, Mr. Borkowski describes the engineering behind the multi-purpose Aurora Scientific Dual Mode Lever — a fast actuator and sensitive force transducer in one — and how this device can be used to study connective tissue.
Following, Mr. Sarver discusses his current research focused on sex-related differences in the structural and functional status of Tendon, from macromolecular structural properties to transcriptomic, proteomic, and cell biology of resident tendon fibroblasts. He explains why tendon research is important, reviews methodology for investigating tendon structure and function, and discusses research findings supporting sex-related differences in tendon.
Key topics covered during this webinar will include…
- How to use The Dual-Mode Lever to perfom demanding stress/strain assays
- What is a tendon, and why is tendon research important
- How to characterize the structural and functional status of tendon
- Case Study: investigating sex-related differences in tendon
Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.
This document contains three annotated bibliographies summarizing research articles:
The first summarizes a study finding that intracellular gold nanoparticles increase neuronal excitability and seizure activity in mice brains. The second describes research using an automated maze to assess hippocampus-sensitive memory in mice. The third summarizes a study using magnetic resonance imaging to find age-dependent axonal transport deficits in a mouse model of frontotemporal dementia.
1. The document summarizes three research papers on the effects of nanoparticles, memory tests in mice, and axonal transport deficits.
2. One study found that intracellular gold nanoparticles can increase neuronal excitability and aggravate seizures. Another developed an automated maze to assess hippocampus-dependent memory without human interference.
3. The third study used MRI to show age-dependent axonal transport deficits in a mouse model of tauopathy, linking the deficits to hyperphosphorylated tau protein in the brain.
Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Loco...InsideScientific
In this webinar sponsored by Neurotar, experts present their research utilizing the Mobile HomeCage®, an experimental tool which ensures the stability required for high-precision neurophysiological techniques while allowing mice to navigate and explore their environment.
Case Study #1:
Dr. Sarah Stuart and Dr. Jon Palacios-Filardo of the University of Bristol present their studies combining analysis of goal-directed behavior with whole-cell recordings from the hippocampus of awake mice. The researchers share useful tips for the surgery protocol and for adjusting the head fixation angle in order to facilitate mouse motility and exploratory behavior.
Case Study #2:
Dr. Alexander Dityatev and Weilun Sun from the German Center for Neurodegenerative Diseases (DZNE) discuss 2-photon imaging of fluorescently labeled microglia in vivo in the context of neurodegenerative disease. They also present their recent data on the effects of different anesthetics on the microglial response to localized laser injury.
Case Study #3:
Dr. Norbert Hájos from the Hungarian Academy of Sciences presents his lab’s research into the amygdala’s role in reward-driven behavior. He shares the challenges of making single-unit recordings using silicon probes during mouse locomotion and subsequent morphological identification of active neurons in the amygdala.
Key topics covered during this webinar include:
- Requirements for stable single-cell recordings and 2-photon imaging in behaving mice
- Challenges of combining high-precision techniques with behavioral research
- Methodological considerations for improving exploratory behavior in head-fixed mice
- Quantitative analysis of microglial function using 2-photon microscopy in awake mice
- Recording neuronal activity in the amygdala of awake mice followed by morphological identification of recorded neurons
Making Optical and Electrophysiological Measurements in the Brain of Head-Fix...InsideScientific
A growing number of researchers are moving from reduced preparations such as dissociated cultured neurons or brain slices, to experimentation in live animals - in vivo - using advanced methods such as two-photon microscopy or combined optogenetics and patch-clamp recordings. In order to immobilize the animal during these challenging applications general anesthesia is often administered; however, the use of anesthetics greatly distorts brain function.
Is there a better way?
In this exclusive webinar sponsored by Neurotar Ltd, leading experts in the technology will discuss methodology, best-practices and show attendees how to immobilize the rodent’s head without restraining its body using the Mobile HomeCage™. The result is a controlled research environment for studying brain function in awake, freely-moving subjects with no stress to the animal. Discussion around how this technique can be applied to the study of neuronal plasticity, neurodegeneration, addiction, brain trauma and other pathophysiological conditions in longitudinal experiments will be included. Furthermore, presenters will demonstrate how this methodology is best combined with microscopy and electrophysiology techniques – all in vivo.
Nanotechnology offers solutions to challenges in genetic engineering. MEMS chips can be used for nanoinjection, delivering DNA to fertilized eggs through a nanometer-scale lance charged with DNA. This overcomes issues with pronuclear microinjection in agricultural animals. Nanoinjection uses electroporation, generating a localized electric field to insert DNA without fluid. Nanoparticles can also deliver therapeutic genes for conditions like retinopathies, protecting against viruses while targeting delivery and limiting expression time. Various nanoparticle types including polymers, gold, and magnetic varieties show promise for gene therapy applications.
How To Study Structural and Functional Properties of TendonInsideScientific
WATCH THE VIDEO: http://bit.ly/2nl7Nx6
In this webinar presented by Aurora Scientific, Matthew Borkowski and Dylan Sarver discuss how to characterize the structural and functional properties of tendon.
Specifically, Mr. Borkowski describes the engineering behind the multi-purpose Aurora Scientific Dual Mode Lever — a fast actuator and sensitive force transducer in one — and how this device can be used to study connective tissue.
Following, Mr. Sarver discusses his current research focused on sex-related differences in the structural and functional status of Tendon, from macromolecular structural properties to transcriptomic, proteomic, and cell biology of resident tendon fibroblasts. He explains why tendon research is important, reviews methodology for investigating tendon structure and function, and discusses research findings supporting sex-related differences in tendon.
Key topics covered during this webinar will include…
- How to use The Dual-Mode Lever to perfom demanding stress/strain assays
- What is a tendon, and why is tendon research important
- How to characterize the structural and functional status of tendon
- Case Study: investigating sex-related differences in tendon
Tracking times in temporal patterns embodied in intra-cortical data for cont...IJECEIAES
Brain-machines capture brain signals in order to restore communication and movement to disabled people who suffer from brain palsy or motor disorders. In brain regions, the ensemble firing of populations of neurons represents spatio-temporal patterns that are transformed into outgoing spatio-temporal patterns which encode complex cognitive task. This transformation is dynamic, non-stationary (time-varying) and highly nonlinear. Hence, modeling such complex biological patterns requires specific model structures to uncover the underlying physiological mechanisms and their influences on system behavior. In this study, a recent multi-electrode technology allows the record of the simultaneous neuron activities in behaving animals. Intra-cortical data are processed according to these steps: spike detection and sorting, than desired action extraction from the rate of the obtained signal. We focus on the following important questions about (i) the possibility of linking the brain signal time events with some time-delayed mapping tools; (ii) the use of some suitable inputs than others for the decoder; (iii) a consideration of separated data or a special representation founded on multi-dimensional statistics. This paper concentrates mostly on the analysis of parallel spike train when certain critical hypotheses are ignored by the data for the working method. We have made efforts to define explicitly whether the underlying hypotheses are actually achieved. In this paper, we propose an algorithm to define the embedded memory order of NARX recurrent neural networks to the hand trajectory tracking process. We also demonstrate that this algorithm can improve performance on inference tasks.
Self Head Fixation Training for the Study of Perceptual Decisions in MiceInsideScientific
In this webinar, Andrea Benucci, PhD will discuss a setup developed in his laboratory for high-throughput behavioral training of mice based on voluntary head fixation. He will describe its flexible use for behavioral training and concurrent neural recordings, delving into some technical considerations related to user-specific customizations as well.
In Andrea’s lab, they study the neural substrate of visual processing and vision-based decision making. To this end, they aim to define a research framework capable of linking neural architectures to the underlying computations. The solution they have developed is to integrate experimental methods for all-optical dissection of neuronal circuits with large-scale dynamical network models based on artificial neural networks (aNNs). The connectivity architecture of aNNs closely mirror that of biological neural networks, thus representing an effective theoretical framework to unify computational, algorithmic, and implementation levels of analysis.
Finally, Andrea will present some examples of unique research achievements made possible by the use of this setup.
SuperArgus PET/CT: Advanced Pre-Clinical Imaging for Small to Medium AnimalsInsideScientific
Positron Emission Tomography (PET) is the gold standard in metabolic imaging, providing high sensitivity to radiotracers used to detect metabolic activity or biomarkers in vivo. The most common uses for PET imaging in pre-clinical research include oncology, neurobiology, cardiology and dynamic imaging.
In this Scintica Instrumentation webinar, Tonya Coulthard discusses how Position Emission Tomography is used for preclinical imaging. Tonya provides an overview of applications including real-time imaging of awake animals, self-gated cardiac imaging and multiplexed PET imaging of standard and non-standard isotopes.
She also intruduces the SuperArgus PET/CT system, which is ideally suited for preclinical imaging of small animals like mice up to medium-sized animals like swine and non human primates.
Key Discussion Topics Include:
- Overview of Pre-Clinical PET/CT imaging
- Examples from applications including oncology, neurology, cardiology and dynamic imaging
- An introduction to SuperArgus PET/CT and what makes it unique
Studying Retinal Function in Large Animals: Laser-Induced Choroidal Neovascul...InsideScientific
The document describes a laser-induced choroidal neovascularization model in pigs for studying retinal diseases. It discusses the Micron X imaging system for obtaining high-quality images of the retina and fluorescein angiograms in large animals like pigs. The model involves using laser shots to induce CNV lesions and then treating them with drugs like aflibercept to evaluate effectiveness. Quantitative analysis of the lesions is done using OCT, fluorescein angiography, and flat mounts, showing that aflibercept significantly reduces CNV development. The pig model is said to be more clinically relevant and translatable than rodent models for testing new therapies.
Cerebral Open Flow Microperfusion (cOFM) for in vivo Cerebral Fluid Sampling ...InsideScientific
Cerebral open flow microperfusion (cOFM) is a minimally invasive, in vivo sampling technology that allows continuous long-term sampling of cerebral fluid in living animals. The decisive advantage of cOFM is that the cOFM probe is membrane–free and comprises macroscopic openings which offer the possibility for a multitude of applications without restriction regarding size, lipophilicity or protein binding effects of the collected substances. The cOFM probe is designed to elicit minimal tissue reactions and allows for reconstitution of the blood-brain barrier (BBB). Thus, cOFM can sample cerebral fluids in living and freely moving animals with intact BBB.
During this webinar, Dr. Joanna Hummer introduces cOFM and presents how cOFM is used as an in vivo sampling technology in neuroscience for drug development.
Dr. Florie Le Prieult, presents data her team collected using cOFM during a pharmacokinetic studies of therapeutic antibodies. Her study includes head-to-head comparison of cOFM and microdialysis.
1. The document discusses combining optogenetics techniques with electrophysiology recording using Andor Mosaic and Axon pCLAMP.
2. Optogenetics allows control of neuronal firing and silencing using light-sensitive ion channels inserted into neurons via genetic targeting.
3. Andor Mosaic enables precise spatial and temporal light patterning for optogenetic stimulation while Axon pCLAMP allows electrophysiology recording.
4. Combining these techniques allows controlling and monitoring neuronal activity at single cell level for studying neural circuits.
Noninvasive, Automated Measurement of Sleep, Wake and Breathing in RodentsInsideScientific
In this exclusive webinar sponsored by Signal Solutions LLC, Dr. Bruce O’Hara discusses methodology, best-practices and use studies of the PiezoSleep system. Discussion focuses on how these techniques can answer questions about animal behavior, phenotyping and relationships between sleep and disease. Dr. O’Hara also highlights the benefits of the PiezoSleep system that can assess sleep, wake and breathing variables.
Employing Electrophysiology and Optogenetics to Measure and Manipulate Neuron...InsideScientific
In this webinar, Dr. Tahl Holtzman, Founder of Cambridge NeuroTech, describes a new generation of silicon neural probes offering dozens of recording channels in precisely spaced, high-resolution arrays, built using sophisticated fabrication techniques borrowed from the electronics industry, along with simple-to-follow surgical implantation schemes for both acute and chronic animals.
Watch to learn how to take advantage of ultra-small chronic drives to open up scalability to span multiple brain areas in parallel and to achieve excellent chronic stability. In addition, Dr. Holtzman demonstrates integration of novel probes and drives offered by Cambridge NeuroTech with optogenetics that thereby enable your experiments to have the combined capability for measurement AND manipulation of neuronal activity in both acute and freely behaving settings.
This webinar will benefit both established electrophysiologists who wish to increase their data yield and experimental reach as well as those investigators whose expertise is centred in and around the animal behavioural, neuropharmacological, and optogenetics arenas. Viewers will learn what silicon neural probes are and how to use them in both acute and chronic experiments, best-practice techniques for surgical implantation in species ranging from mice to monkeys and how to integrate fibre optic cannulas with your probes to enable simultaneous opto-electrophysiology.
The Brain as a Whole: Executive Neurons and Sustaining Homeostatic GliaInsideScientific
Carl Petersen and Alexei Verkhratsky share their research on homeostatic neuroglia and imaging of neuronal network function. This webinar is brought to you by APS’ new journal, Function, and part of their Physiology in Focus learning series.
During this exclusive live webinar, Carl Petersen and Alexei Verkhratsky discuss astrocyte-mediated homeostatic control of the central nervous system, and how optical and 2-photon microscopy can be used for functional neuroimaging.
Imaging Neuronal Function
Carl Petersen, PhD
Highly dynamic and spatially distributed neuronal circuits in the brain control mammalian behavior. Through technological advances, optical measurements of neuronal function can now be carried out in behaving mice at multiple scales. Wide-field imaging allows the dynamic interactions between different brain areas to be studied as sensory information is processed and transformed into behavioral output. Within a brain region, two-photon microscopy can be used to image the neuronal network activity with cellular resolution allowing different types of projection neurons to be distinguished. Together optical methods provide versatile tools for causal mechanistic understanding of neuronal network function in mice.
Astrocytes: indispensable neuronal supporters in sickness and in health
Alexei Verkhratsky, MD, PhD, DSc
The nervous system is composed of two arms: the executive neurons and the homeostatic neuroglia. The neurons require energy, support, and protection, all of which is provided by the neuroglia. Astrocytes, the principal homeostatic cells of the brain and spinal cord, are tightly integrated into the neural networks and act within the context of the neural tissue. As astrocytes control the homeostasis of the central nervous system at all levels of organization, from the molecular to the whole organ level, we can begin to define and understand brain vulnerabilities to aging and diseases.
Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air...InsideScientific
In this webinar sponsored by Neurotar, experts present their research on 2-photon imaging of hippocampal place cells and on stress monitoring in head-fixed awake behaving mice. Dr. Konrad Juczewski from the National Institutes of Health (NIH)/National Institute on Alcohol Abuse and Alcoholism (NIAAA) discusses the impact of head fixation on animal’s stress, locomotion and performance in classical behavioral paradigms.
Dr. Mary Ann Go from the Laboratory of Neural Coding and Neurodegenerative Disease at Imperial College London led by Prof. Simon Schultz presents her research using 2-photon microscopy aimed at place cell mapping in the hippocampus during exploration and navigation of a circular linear track.
Key Discussion Topics Include:
- Stress reduction in head-fixed rodents
- Improving data reproducibility and translational value of the data acquired from head-fixed rodents
- Effects of head fixation on blood corticosterone concentration, locomotion patterns and performance in stress-associated behavioral tests
- Optimizing habituation protocol for head-fixed mice
- Monitoring neural activity and mapping of place cells using 2-photon microscopy during navigation and exploration behavior
- Automating the experiments using a closed-loop approach and behavior-triggered reward systems
Monitoring neural activities by optical imagingMd Kafiul Islam
Monitoring neural activities by optical imaging along with the use of genetic modification provides better spatio-temporal resolution to study single neural firing and hence very useful in understanding the neural process and dynamics. This is just a glimpse of few articles reported their outcome of such imaging.
Presentation of the book: Magnetic Resonance Imaging of the Rhesus Monkey Brain by, Roland Tammer, Sabine Hofer, Klaus-Dietmar Merboldt and Jens Frahm.
http://www.v-r.de/de/Magnetic-Resonance-Imaging-of-the-Rhesus-Monkey-Brain/t/1001003576/
The basics for symbiosis of Optics and Genetics have been explained in this presentation. " How light can change the very way of life?" .This question has been addressed using relevant web content, consultations from book and through nature videos. This presentation was awarded the highest score in PHM805 at Dayalbagh Educational Institute, Agra.
Netrin signaling through DCC modulates retinotectal synaptic connectivity in Xenopus laevis by influencing axon branching and synapse formation in the developing brain. Live imaging of retinal ganglion cell axons expressing GFP-synaptobrevin and DsRed showed that injecting netrin-1 into the tectum increased presynaptic site addition, branch addition and total branch number over 24 hours. In contrast, injecting DCC blocking antibodies prevented the normal increase in presynaptic sites and branch number. Dynamic analysis revealed the antibodies specifically decreased new synapse and branch additions without affecting stability of existing structures, indicating netrin is required for branching and synaptic differentiation through DCC signaling.
This document summarizes a study that used picosecond optical tomography with a white laser and streak camera to measure changes in oxyhemoglobin and deoxyhemoglobin concentration in the brains of zebra finches in response to auditory stimulation. The technique showed submicromolar sensitivity and was able to resolve fast changes in the hippocampus and auditory forebrain with 250 μm resolution. Stimulation resulted in an early decrease in hemoglobin and oxyhemoglobin levels, followed by an increase in blood oxygen availability and pronounced vasodilation after stimulus end. The findings provide direct evidence linking blood oxygen level-dependent signals to changes in oxygen transport in birds.
Hippocampal Place Cells in Echolocating Bats stanfordneuro
1) Hippocampal place cells in bats rapidly update their spatial firing based on incoming sensory information from echolocation calls. Place cell spikes occurring later after an echolocation call show reduced spatial selectivity compared to earlier spikes.
2) This demonstrates that place cells can integrate new sensory information on a fast time scale of hundreds of milliseconds to tune their spatial firing based on the acuity of available sensory information.
3) Previous studies in rodents found slower dynamics of place cell remapping in response to changes in sensory information, but bats provide a system to study rapid updating of spatial signals.
High Precision And Fast Functional Mapping Of Cortical Circuitry Through A No...Taruna Ikrar
Taruna Ikrar, MD., PhD. Author at (High Precision and Fast Functional Mapping of Cortical Circuitry Through a Novel Combination of Voltage Sensitive Dye Imaging and Laser Scanning Photostimulation)
This study examined the role of the Sox2 gene in regulating astrocyte processes density in the mouse retina. Conditional knockout (cKO) mice lacking Sox2 specifically in astrocytes were compared to conditional wild-type (cWT) mice. Retinal images were analyzed to measure astrocyte processes frequency. While cKO mice generally showed lower average processes counts, the differences were not statistically significant due to large standard errors overlapping between groups. Further analysis found Sox2 may be more involved in regulating astrocyte processes density in the retinal periphery compared to central and middle areas, though results were still highly variable. Therefore, this study was unable to definitively determine the role of Sox2 in astrocyte networks due to significant
Studying Epilepsy in Awake Head-Fixed Mice Using Microscopy, Electrophysiolog...InsideScientific
Epilepsy research employs sophisticated research methods such as fluorescence optical imaging and optogenetics, as well as novel electrophysiological techniques, to address unresolved questions about seizure generation and propagation on the cellular and circuitry levels. Since epilepsy research is most relevant when performed in non-anesthetized mice, it requires specialized tools that ensure stable head fixation during high-precision imaging and recordings.
In this webinar, Dr. Anthony Umpierre (Prof. LongJun Wu group, Mayo Clinic, USA) and Prof. Rob Wykes (UCL, UK) present their research on microglial calcium signaling and epileptic networks carried out in awake head-fixed mice. In addition to sharing exciting new findings, the presenters address the challenges of working with awake mice.
Key topics will include…
- Mesoscopic investigations of seizure dynamics and propagation using widefield calcium imaging
- Generating full-bandwidth electrophysiological recordings enabled by graphene micro-transistors to detect spreading depolarizations and seizures
- On-demand optogenetic induction of spreading depolarizations to investigate pharmacological suppression in the awake brain
- The impact of acute versus chronic window preparations on microglial calcium activity
- The use of genetically encoded calcium indicators to study calcium dynamics in microglia
- The effects of bi-directional shifts in neuronal activity caused by kainate-triggered status epilepticus and isoflurane anesthesia on microglial calcium
1) The document discusses preliminary studies using two-photon microscopy to image brain areas of zebra finches through their thin skin and hollow skull structure for non-invasive monitoring of brain activity.
2) Experiments were conducted imaging hollow fibers filled with Rhodamine B passed through fixed zebra finch skin and skull samples to evaluate spatial resolution and distortion. Reflectance confocal measurements were also taken to determine scattering properties of fresh and fixed skin and skull.
3) The goal is to determine if two-photon microscopy can provide sufficient resolution for in vivo brain imaging and metabolism monitoring of zebra finches as a model for studying vocal recognition, without requiring craniotomy as in other small animal studies.
Epi-Fluorescence Microscopy: Explore Its Amazing Powers and Uses | The Lifesc...The Lifesciences Magazine
Epi-fluorescence microscopy, also known as epifluorescence microscopy, is a specialized imaging technique that utilizes fluorescence to illuminate specimens of interest.
Tracking times in temporal patterns embodied in intra-cortical data for cont...IJECEIAES
Brain-machines capture brain signals in order to restore communication and movement to disabled people who suffer from brain palsy or motor disorders. In brain regions, the ensemble firing of populations of neurons represents spatio-temporal patterns that are transformed into outgoing spatio-temporal patterns which encode complex cognitive task. This transformation is dynamic, non-stationary (time-varying) and highly nonlinear. Hence, modeling such complex biological patterns requires specific model structures to uncover the underlying physiological mechanisms and their influences on system behavior. In this study, a recent multi-electrode technology allows the record of the simultaneous neuron activities in behaving animals. Intra-cortical data are processed according to these steps: spike detection and sorting, than desired action extraction from the rate of the obtained signal. We focus on the following important questions about (i) the possibility of linking the brain signal time events with some time-delayed mapping tools; (ii) the use of some suitable inputs than others for the decoder; (iii) a consideration of separated data or a special representation founded on multi-dimensional statistics. This paper concentrates mostly on the analysis of parallel spike train when certain critical hypotheses are ignored by the data for the working method. We have made efforts to define explicitly whether the underlying hypotheses are actually achieved. In this paper, we propose an algorithm to define the embedded memory order of NARX recurrent neural networks to the hand trajectory tracking process. We also demonstrate that this algorithm can improve performance on inference tasks.
Self Head Fixation Training for the Study of Perceptual Decisions in MiceInsideScientific
In this webinar, Andrea Benucci, PhD will discuss a setup developed in his laboratory for high-throughput behavioral training of mice based on voluntary head fixation. He will describe its flexible use for behavioral training and concurrent neural recordings, delving into some technical considerations related to user-specific customizations as well.
In Andrea’s lab, they study the neural substrate of visual processing and vision-based decision making. To this end, they aim to define a research framework capable of linking neural architectures to the underlying computations. The solution they have developed is to integrate experimental methods for all-optical dissection of neuronal circuits with large-scale dynamical network models based on artificial neural networks (aNNs). The connectivity architecture of aNNs closely mirror that of biological neural networks, thus representing an effective theoretical framework to unify computational, algorithmic, and implementation levels of analysis.
Finally, Andrea will present some examples of unique research achievements made possible by the use of this setup.
SuperArgus PET/CT: Advanced Pre-Clinical Imaging for Small to Medium AnimalsInsideScientific
Positron Emission Tomography (PET) is the gold standard in metabolic imaging, providing high sensitivity to radiotracers used to detect metabolic activity or biomarkers in vivo. The most common uses for PET imaging in pre-clinical research include oncology, neurobiology, cardiology and dynamic imaging.
In this Scintica Instrumentation webinar, Tonya Coulthard discusses how Position Emission Tomography is used for preclinical imaging. Tonya provides an overview of applications including real-time imaging of awake animals, self-gated cardiac imaging and multiplexed PET imaging of standard and non-standard isotopes.
She also intruduces the SuperArgus PET/CT system, which is ideally suited for preclinical imaging of small animals like mice up to medium-sized animals like swine and non human primates.
Key Discussion Topics Include:
- Overview of Pre-Clinical PET/CT imaging
- Examples from applications including oncology, neurology, cardiology and dynamic imaging
- An introduction to SuperArgus PET/CT and what makes it unique
Studying Retinal Function in Large Animals: Laser-Induced Choroidal Neovascul...InsideScientific
The document describes a laser-induced choroidal neovascularization model in pigs for studying retinal diseases. It discusses the Micron X imaging system for obtaining high-quality images of the retina and fluorescein angiograms in large animals like pigs. The model involves using laser shots to induce CNV lesions and then treating them with drugs like aflibercept to evaluate effectiveness. Quantitative analysis of the lesions is done using OCT, fluorescein angiography, and flat mounts, showing that aflibercept significantly reduces CNV development. The pig model is said to be more clinically relevant and translatable than rodent models for testing new therapies.
Cerebral Open Flow Microperfusion (cOFM) for in vivo Cerebral Fluid Sampling ...InsideScientific
Cerebral open flow microperfusion (cOFM) is a minimally invasive, in vivo sampling technology that allows continuous long-term sampling of cerebral fluid in living animals. The decisive advantage of cOFM is that the cOFM probe is membrane–free and comprises macroscopic openings which offer the possibility for a multitude of applications without restriction regarding size, lipophilicity or protein binding effects of the collected substances. The cOFM probe is designed to elicit minimal tissue reactions and allows for reconstitution of the blood-brain barrier (BBB). Thus, cOFM can sample cerebral fluids in living and freely moving animals with intact BBB.
During this webinar, Dr. Joanna Hummer introduces cOFM and presents how cOFM is used as an in vivo sampling technology in neuroscience for drug development.
Dr. Florie Le Prieult, presents data her team collected using cOFM during a pharmacokinetic studies of therapeutic antibodies. Her study includes head-to-head comparison of cOFM and microdialysis.
1. The document discusses combining optogenetics techniques with electrophysiology recording using Andor Mosaic and Axon pCLAMP.
2. Optogenetics allows control of neuronal firing and silencing using light-sensitive ion channels inserted into neurons via genetic targeting.
3. Andor Mosaic enables precise spatial and temporal light patterning for optogenetic stimulation while Axon pCLAMP allows electrophysiology recording.
4. Combining these techniques allows controlling and monitoring neuronal activity at single cell level for studying neural circuits.
Noninvasive, Automated Measurement of Sleep, Wake and Breathing in RodentsInsideScientific
In this exclusive webinar sponsored by Signal Solutions LLC, Dr. Bruce O’Hara discusses methodology, best-practices and use studies of the PiezoSleep system. Discussion focuses on how these techniques can answer questions about animal behavior, phenotyping and relationships between sleep and disease. Dr. O’Hara also highlights the benefits of the PiezoSleep system that can assess sleep, wake and breathing variables.
Employing Electrophysiology and Optogenetics to Measure and Manipulate Neuron...InsideScientific
In this webinar, Dr. Tahl Holtzman, Founder of Cambridge NeuroTech, describes a new generation of silicon neural probes offering dozens of recording channels in precisely spaced, high-resolution arrays, built using sophisticated fabrication techniques borrowed from the electronics industry, along with simple-to-follow surgical implantation schemes for both acute and chronic animals.
Watch to learn how to take advantage of ultra-small chronic drives to open up scalability to span multiple brain areas in parallel and to achieve excellent chronic stability. In addition, Dr. Holtzman demonstrates integration of novel probes and drives offered by Cambridge NeuroTech with optogenetics that thereby enable your experiments to have the combined capability for measurement AND manipulation of neuronal activity in both acute and freely behaving settings.
This webinar will benefit both established electrophysiologists who wish to increase their data yield and experimental reach as well as those investigators whose expertise is centred in and around the animal behavioural, neuropharmacological, and optogenetics arenas. Viewers will learn what silicon neural probes are and how to use them in both acute and chronic experiments, best-practice techniques for surgical implantation in species ranging from mice to monkeys and how to integrate fibre optic cannulas with your probes to enable simultaneous opto-electrophysiology.
The Brain as a Whole: Executive Neurons and Sustaining Homeostatic GliaInsideScientific
Carl Petersen and Alexei Verkhratsky share their research on homeostatic neuroglia and imaging of neuronal network function. This webinar is brought to you by APS’ new journal, Function, and part of their Physiology in Focus learning series.
During this exclusive live webinar, Carl Petersen and Alexei Verkhratsky discuss astrocyte-mediated homeostatic control of the central nervous system, and how optical and 2-photon microscopy can be used for functional neuroimaging.
Imaging Neuronal Function
Carl Petersen, PhD
Highly dynamic and spatially distributed neuronal circuits in the brain control mammalian behavior. Through technological advances, optical measurements of neuronal function can now be carried out in behaving mice at multiple scales. Wide-field imaging allows the dynamic interactions between different brain areas to be studied as sensory information is processed and transformed into behavioral output. Within a brain region, two-photon microscopy can be used to image the neuronal network activity with cellular resolution allowing different types of projection neurons to be distinguished. Together optical methods provide versatile tools for causal mechanistic understanding of neuronal network function in mice.
Astrocytes: indispensable neuronal supporters in sickness and in health
Alexei Verkhratsky, MD, PhD, DSc
The nervous system is composed of two arms: the executive neurons and the homeostatic neuroglia. The neurons require energy, support, and protection, all of which is provided by the neuroglia. Astrocytes, the principal homeostatic cells of the brain and spinal cord, are tightly integrated into the neural networks and act within the context of the neural tissue. As astrocytes control the homeostasis of the central nervous system at all levels of organization, from the molecular to the whole organ level, we can begin to define and understand brain vulnerabilities to aging and diseases.
Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air...InsideScientific
In this webinar sponsored by Neurotar, experts present their research on 2-photon imaging of hippocampal place cells and on stress monitoring in head-fixed awake behaving mice. Dr. Konrad Juczewski from the National Institutes of Health (NIH)/National Institute on Alcohol Abuse and Alcoholism (NIAAA) discusses the impact of head fixation on animal’s stress, locomotion and performance in classical behavioral paradigms.
Dr. Mary Ann Go from the Laboratory of Neural Coding and Neurodegenerative Disease at Imperial College London led by Prof. Simon Schultz presents her research using 2-photon microscopy aimed at place cell mapping in the hippocampus during exploration and navigation of a circular linear track.
Key Discussion Topics Include:
- Stress reduction in head-fixed rodents
- Improving data reproducibility and translational value of the data acquired from head-fixed rodents
- Effects of head fixation on blood corticosterone concentration, locomotion patterns and performance in stress-associated behavioral tests
- Optimizing habituation protocol for head-fixed mice
- Monitoring neural activity and mapping of place cells using 2-photon microscopy during navigation and exploration behavior
- Automating the experiments using a closed-loop approach and behavior-triggered reward systems
Monitoring neural activities by optical imagingMd Kafiul Islam
Monitoring neural activities by optical imaging along with the use of genetic modification provides better spatio-temporal resolution to study single neural firing and hence very useful in understanding the neural process and dynamics. This is just a glimpse of few articles reported their outcome of such imaging.
Presentation of the book: Magnetic Resonance Imaging of the Rhesus Monkey Brain by, Roland Tammer, Sabine Hofer, Klaus-Dietmar Merboldt and Jens Frahm.
http://www.v-r.de/de/Magnetic-Resonance-Imaging-of-the-Rhesus-Monkey-Brain/t/1001003576/
The basics for symbiosis of Optics and Genetics have been explained in this presentation. " How light can change the very way of life?" .This question has been addressed using relevant web content, consultations from book and through nature videos. This presentation was awarded the highest score in PHM805 at Dayalbagh Educational Institute, Agra.
Netrin signaling through DCC modulates retinotectal synaptic connectivity in Xenopus laevis by influencing axon branching and synapse formation in the developing brain. Live imaging of retinal ganglion cell axons expressing GFP-synaptobrevin and DsRed showed that injecting netrin-1 into the tectum increased presynaptic site addition, branch addition and total branch number over 24 hours. In contrast, injecting DCC blocking antibodies prevented the normal increase in presynaptic sites and branch number. Dynamic analysis revealed the antibodies specifically decreased new synapse and branch additions without affecting stability of existing structures, indicating netrin is required for branching and synaptic differentiation through DCC signaling.
This document summarizes a study that used picosecond optical tomography with a white laser and streak camera to measure changes in oxyhemoglobin and deoxyhemoglobin concentration in the brains of zebra finches in response to auditory stimulation. The technique showed submicromolar sensitivity and was able to resolve fast changes in the hippocampus and auditory forebrain with 250 μm resolution. Stimulation resulted in an early decrease in hemoglobin and oxyhemoglobin levels, followed by an increase in blood oxygen availability and pronounced vasodilation after stimulus end. The findings provide direct evidence linking blood oxygen level-dependent signals to changes in oxygen transport in birds.
Hippocampal Place Cells in Echolocating Bats stanfordneuro
1) Hippocampal place cells in bats rapidly update their spatial firing based on incoming sensory information from echolocation calls. Place cell spikes occurring later after an echolocation call show reduced spatial selectivity compared to earlier spikes.
2) This demonstrates that place cells can integrate new sensory information on a fast time scale of hundreds of milliseconds to tune their spatial firing based on the acuity of available sensory information.
3) Previous studies in rodents found slower dynamics of place cell remapping in response to changes in sensory information, but bats provide a system to study rapid updating of spatial signals.
High Precision And Fast Functional Mapping Of Cortical Circuitry Through A No...Taruna Ikrar
Taruna Ikrar, MD., PhD. Author at (High Precision and Fast Functional Mapping of Cortical Circuitry Through a Novel Combination of Voltage Sensitive Dye Imaging and Laser Scanning Photostimulation)
This study examined the role of the Sox2 gene in regulating astrocyte processes density in the mouse retina. Conditional knockout (cKO) mice lacking Sox2 specifically in astrocytes were compared to conditional wild-type (cWT) mice. Retinal images were analyzed to measure astrocyte processes frequency. While cKO mice generally showed lower average processes counts, the differences were not statistically significant due to large standard errors overlapping between groups. Further analysis found Sox2 may be more involved in regulating astrocyte processes density in the retinal periphery compared to central and middle areas, though results were still highly variable. Therefore, this study was unable to definitively determine the role of Sox2 in astrocyte networks due to significant
Studying Epilepsy in Awake Head-Fixed Mice Using Microscopy, Electrophysiolog...InsideScientific
Epilepsy research employs sophisticated research methods such as fluorescence optical imaging and optogenetics, as well as novel electrophysiological techniques, to address unresolved questions about seizure generation and propagation on the cellular and circuitry levels. Since epilepsy research is most relevant when performed in non-anesthetized mice, it requires specialized tools that ensure stable head fixation during high-precision imaging and recordings.
In this webinar, Dr. Anthony Umpierre (Prof. LongJun Wu group, Mayo Clinic, USA) and Prof. Rob Wykes (UCL, UK) present their research on microglial calcium signaling and epileptic networks carried out in awake head-fixed mice. In addition to sharing exciting new findings, the presenters address the challenges of working with awake mice.
Key topics will include…
- Mesoscopic investigations of seizure dynamics and propagation using widefield calcium imaging
- Generating full-bandwidth electrophysiological recordings enabled by graphene micro-transistors to detect spreading depolarizations and seizures
- On-demand optogenetic induction of spreading depolarizations to investigate pharmacological suppression in the awake brain
- The impact of acute versus chronic window preparations on microglial calcium activity
- The use of genetically encoded calcium indicators to study calcium dynamics in microglia
- The effects of bi-directional shifts in neuronal activity caused by kainate-triggered status epilepticus and isoflurane anesthesia on microglial calcium
1) The document discusses preliminary studies using two-photon microscopy to image brain areas of zebra finches through their thin skin and hollow skull structure for non-invasive monitoring of brain activity.
2) Experiments were conducted imaging hollow fibers filled with Rhodamine B passed through fixed zebra finch skin and skull samples to evaluate spatial resolution and distortion. Reflectance confocal measurements were also taken to determine scattering properties of fresh and fixed skin and skull.
3) The goal is to determine if two-photon microscopy can provide sufficient resolution for in vivo brain imaging and metabolism monitoring of zebra finches as a model for studying vocal recognition, without requiring craniotomy as in other small animal studies.
Epi-Fluorescence Microscopy: Explore Its Amazing Powers and Uses | The Lifesc...The Lifesciences Magazine
Epi-fluorescence microscopy, also known as epifluorescence microscopy, is a specialized imaging technique that utilizes fluorescence to illuminate specimens of interest.
Functional Ultrasound (fUS) Imaging in the Brain of Awake Behaving MiceInsideScientific
To watch the webinar, visit:
https://insidescientific.com/webinar/functional-ultrasound-imaging-brain-awake-behaving-mice-neurotar-iconeus
Functional ultrasound (fUS) imaging is a new kid on the block in neuroimaging. It combines high spaciotemporal resolution with deep tissue penetration, which enables non-invasive whole-brain imaging in mice.
This exciting new technology complements and extends classical imaging modalities: it enables more straightforward, unobstructed and non-invasive functional measurements in mouse models of CNS diseases. Sensitive to changes in cerebral blood volume, fUS imaging is used to characterize brain networks with functional connectivity analysis and to measure the responses to sensory stimuli and pharmacological challenges.
fUS imaging performed in the brain of awake mice removes the biases and artifacts associated with the use of general anesthesia, which is no longer a “necessary evil” of translational imaging. Besides that: fUS imaging in awake mice allows integrating functional imaging with behavioral readouts starting from open field locomotion tracking to maze navigation and sociability studies.
In this webinar, you will learn:
– Functional ultrasound (fUS) imaging methodology
– How translational fUS neuroimaging helps to advance basic neuroscience research and preclinical drug discovery
– The main advantages and limitations of using functional ultrasound compared to other techniques such as BOLD fMRI
– The benefits of imaging in awake, head-restrained but otherwise freely moving mice
– Imaging functional activation, connectivity and pharmacologically-induced changes in awake and behaving mice
– How to combine fUS imaging with behavioral observation
Monitoring live cell viability Comparative studyWerden Keeler
This document compares three live cell imaging techniques: fluorescence microscopy, oblique incidence reflection microscopy, and phase contrast microscopy. It finds that oblique incidence reflection microscopy is the simplest, least expensive, and least phototoxic method, causing the least damage to live cells during long-term monitoring of cell viability. The document describes the equipment and cell lines used, including normal and cancerous cell lines tagged with fluorescent proteins or unlabeled, to evaluate the stresses induced by different illumination techniques.
Optogenetics is a technique that uses light to control neurons that have been genetically modified to express light-sensitive ion channels. It allows scientists to precisely stimulate or silence neural activity by exposing specific neurons to light. The first demonstration of optogenetics in mammalian neurons used channelrhodopsin, a light-activated ion channel from algae, to activate neurons with light. Optogenetics holds promise for advancing understanding of brain function and developing new treatments for neurological disorders like Parkinson's disease, epilepsy, and blindness through targeted neuromodulation with light. Challenges include improving light-sensitive tools and light sources to target deeper brain regions.
This document describes an experiment that used near-infrared spectroscopy (NIRS) to noninvasively measure the optical properties of a songbird's brain. Researchers placed optical fibers on the head of anesthetized zebra finches to transmit laser light and collect the light after it passed through the brain tissue. They were able to measure the absorption and scattering coefficients of the caudal nidopallium region of the brain in vivo. This technique could help monitor brain activity and oxygenation levels in songbirds.
VisualSonics has introduced a revolutionary micro-ultrasound and photoacoustic imaging system that allows researchers to collect a plethora of important data over the lifespan of animals, thereby significantly reducing the number of animals needed.
Edgardo J. Arroyo is an Associate Research Scientist at Yale University School of Medicine who has extensive experience researching various aspects of myelin formation, degradation, and regeneration in the central and peripheral nervous systems. His research has focused on elucidating the cellular mechanisms and microanatomy of neuron-glial interactions using techniques such as immunohistochemistry, confocal microscopy, and biochemistry. He has studied topics such as the effects of spinal cord injury, stem cell transplantation, sodium channel expression after nerve damage, and how demyelination affects the molecular organization of nodes of Ranvier.
Optical clearing is a new technique that improves 3D imaging of the enteric nervous system (ENS). Researchers used optical clearing and confocal microscopy to image the human ileum in 3D. Optical clearing reduced light scattering in the tissue, allowing deeper photon penetration and higher definition images. This generated a panoramic 3D view of gut wall structures like nerves, muscles, and crypts. The researchers plan to use this method to study gastrointestinal disease mechanisms by observing microstructures, vasculature, and nerve networks in animal models and comparing healthy and diseased human tissues.
Christian Jaques Hissom proposes a project to investigate the branching patterns of corticospinal neurons using a two-viral vector tracing strategy in rats. The strategy involves injecting one virus in the spinal cord to retrogradely label corticospinal neurons, and a second virus in the motor cortex to express a fluorescent tracer only in doubly infected neurons. This will allow mapping of corticospinal tract collaterals. The proposal details experiments involving perfusion, tissue sectioning, staining, imaging and 3D reconstruction of projections to define collateralization and inform understanding of motor control. The student expresses a passion for neuroscience research inspired by a family member with paralysis.
Using the 3D Cell Explorer-fluo for fluorescence and holotomographic imagingMathieuFRECHIN
Nanolive’s 3D Cell Explorer allows for the creation of very powerful 3D images and 4D time lapses
of living cells with very high spatio-temporal resolution (x,y:180nm; z:400nm; t:1.7sec). Moreover,
imaging with the 3D Cell Explorer does not require the use of any labels since the microscope directly
measures the refractive index of the different substructures of the cell. However, in the context of
molecular or cellular biology investigations, it can be useful to follow fluorescent markers combined
with the refractive index distribution to validate specific structures or to correlate fluorescent signals
and cellular states.
For this purpose, Nanolive developed the 3D Cell Explorer-fluo (https://nanolive.ch/fluo/) – a
complete solution that combines high precision tomographic data with high quality tri- or fourchannel
epifluorescence provided by a CoolLed module (https://www.coolled.com/).
In this application note we will present the time lapse imaging of mouse embryonic stem cells
(mESCs) that have been genetically modified to express the fluorescence ubiquitination cell cycle
indicator (FUCCI), a two-color (red and green) indicator that allows to monitor the cell cycle phases.
We will explain how to use the 3D Cell Explorer-fluo to record movies that require both fluorescence
and 3D refractive index imaging and will propose a solution to analyze the resulting time lapse
experiment.
This document summarizes a study that used functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (NIRS) to measure hemodynamic changes in the brain of zebra finches in response to hypercapnia. Hypercapnia induces vasodilation and is often used to model hemodynamic responses. Both fMRI, which detects blood oxygen level-dependent (BOLD) signals, and NIRS, which measures concentrations of oxyhemoglobin and deoxyhemoglobin, clearly showed increases in blood oxygen saturation in the brain during hypercapnia. The results provide the first correlation in songbirds between hemodynamic parameter variations measured by NIRS and local BOLD signal variations measured by fMRI.
The document discusses various technologies used at the House Ear Institute including genomics, proteomics, and imaging. It describes how researchers are using these tools to study diseases like neurofibromatosis type 2 (NF2) at the molecular level in order to develop personalized treatments and therapies. Maintaining high quality biospecimens is important for enabling various types of research.
Mapping Inhibitory Neuronal Ircuits By Laser Scanning Photostimula TionTaruna Ikrar
1) The document describes a technique called laser scanning photostimulation (LSPS) combined with whole-cell patch clamp recording to map local inhibitory neuronal circuits.
2) LSPS uses laser pulses to selectively activate neurons via glutamate uncaging, allowing mapping of excitatory and inhibitory synaptic inputs onto recorded neurons from many stimulation sites.
3) An example is provided showing excitatory synaptic input maps for a fast-spiking inhibitory interneuron in mouse somatosensory cortex, revealing strong input from layer 4 and deeper layers.
This document discusses the use of fluorescent proteins in current biological research. It begins with an overview of the development of optical microscopy and fluorescence techniques. It then focuses on the green fluorescent protein (GFP) and how it has been used as a molecular tag to study protein expression and interactions in living cells through techniques like gene delivery, transfection, viral infection, FRET, and optogenetics. The document concludes that fluorescent proteins have revolutionized cell biology by enabling the real-time visualization and control of molecular pathways and signaling processes in living systems.
This document describes a micro project to develop a C. elegans tracking system using a digital microscope and tracking software. The project aims to analyze the locomotion of C. elegans to better understand the mechanisms underlying its forward movement. A Dino-Lite digital microscope will be used to record videos of C. elegans on an agar plate. Tracking software like WormLab and ImageJ will then analyze the videos to track the worm's movement and calculate metrics like number of bends and directional changes. The goal is to help elucidate the neural control of C. elegans locomotion and how it mediates the worm's foraging and avoidance behaviors.
The study analyzed Purkinje cell responses in the cerebellar flocculus of mice during visually and vestibularly driven eye movements. Recordings were made from Purkinje cells in awake, head-fixed mice undergoing optokinetic and vestibular stimulation. As in other species, the Purkinje cells carried both vestibular and nonvestibular signals related to eye and head movement. However, the mouse Purkinje cells showed a higher sensitivity to eye velocity compared to other species, likely reflecting the smaller range of eye movements in mice.
Similar to White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice (20)
This document describes a high resolution multi-modal imaging platform that combines ultrasound and photoacoustics. It provides anatomical, functional, and molecular data with superior resolution down to 30 μm. The system has a customizable touchscreen interface and is compact and portable. It can be used for applications in neurobiology, molecular biology, cardiology, and oncology to assess vascularity, perfusion, biomarkers, and response to therapy.
Vevo 3100 - The ultimate preclinical imaging experience. The Vevo 3100 is a new and innovative platform created for the future of imaging. It combines ultra high frequency ultrasound imaging, quantification and education in a convenient all-in-one touchscreen platform.
This document provides a bibliography of top cardiovascular research papers organized by topic. The topics covered include abdominal aortic aneurysm, atherosclerosis, cardiac hypertrophy, cardiac injection, cardiomyopathy, chick embryo, contrast, developmental cardiology, diastolic dysfunction, graft transplantation, Holt-Oram syndrome, Marfan syndrome, myocardial infarction, pulmonary hypertension, rabbit cardiovascular, rat cardiovascular, stem cells, stress echocardiography, and valvular flow & function. For each topic, several of the most influential papers from 2009-2010 are listed with their citation information.
This document provides a bibliography of research papers related to cancer organized by topic. It includes 3-7 references for each of 17 cancer-related topics, such as 3D tumor imaging, angiogenesis, bladder cancer, breast cancer, contrast imaging, and others. The references provided for each topic are journal articles published between 2005-2010 that describe research on imaging, molecular markers, treatment responses, and other aspects of various cancers.
This document provides a bibliography of top nephrology research papers from 2010 and earlier, focusing on topics related to polycystic kidney disease, acute kidney injury, fibrosis, cyst formation, glomerular disease, target organ damage, regional blood flow in the mouse kidney, renal ischemia, phosphate homeostasis, aortic valve calcification in rats, kidney safety in drug development, imaging the kidney with ultrasound, changes to lymphatic and blood vessel architecture from transgenic expression of Angiopoietin 1 in the liver, and a functional floxed allele of Pkd1 that can be conditionally inactivated in vivo. The bibliography contains 20 research papers published between 2004 and 2010.
This document provides a protocol for imaging the deep brain of freely moving mice. It describes:
1) Implanting a guide cannula into the mouse skull to access the brain region of interest. This involves drilling holes, inserting screws, and cementing the cannula in place.
2) Precisely positioning the cannula using stereotactic coordinates to target a specific brain structure.
3) Inserting the cannula 300-600 micrometers into the brain depending on the target depth.
Tumor angiogenesis is currently one of the key focal points in biomedical research. It is based upon the hypothesis laid out by Judah Folkman in 1971 that neovasculature is needed to support the growth and metastasis of tumors, and thus anti-angiogenic treatment might be an effective way to cure cancer. Genentech’s anti-VEGF-A drug Avastin a great demonstration of this concept, generating more than $2.7 billion of sales in 2008.
Breast cancer research in animal models has long been hindered by the lack of a fast, portable, high resolution, research and animal focused imaging system that can visualize 2D tumor size, 3D tumor volume, neoangiogenesis and blood perfusion in vivo, in real-time and most importantly, non-invasively.
1. The document describes the Vevo 2100 imaging platform from VisualSonics, which redefines preclinical ultrasound imaging with high resolution, advanced functionality, and quantitative analysis capabilities.
2. It can image a wide range of animal models from embryos to adults, provides anatomical, functional and molecular data non-invasively, and has applications across many research areas.
3. The system offers superior resolution, color and power Doppler, 3D imaging, advanced measurements, and is easy to use with one-button presets and analysis software.
Radiotherapy and chemotherapy aim at killing tumor cells or at least stopping their multiplication. Those therapies have strong limitations: first, their inherent toxicity is not limited to tumoral cells, but also affects healthy tissue; second, only the strongest and most resistant tumoral cells are able to survive, leading to increasingly aggressive tumors.
The document compares the features of the Vevo 2100 and Vevo 770 ultrasound systems, noting that the Vevo 2100 has improved high frequency transducers, multiple focal zones, higher frame rates, access to raw RF data export, improved non-linear contrast and power Doppler imaging, and faster 3D imaging compared to the Vevo 770. The Vevo 2100 also has new features like VevoStrain, color Doppler, simultaneous dual mode viewing, steerable Doppler, ECG-gating, and rabbit imaging that are not available on the Vevo 770.
The document compares the features of the Vevo 2100 and Vevo 770 ultrasound systems, noting that the Vevo 2100 has improved high frequency transducers, multiple focal zones, higher frame rates, access to raw RF data export, improved non-linear contrast and power Doppler imaging, and faster 3D imaging compared to the Vevo 770. The Vevo 2100 also has new features like VevoStrain, color Doppler, simultaneous dual mode viewing, steerable Doppler, ECG-gating, and rabbit imaging that are not available on the Vevo 770.
Development and Validation of a Combined Photoacoustic Micro-Ultrasound Syste...FUJIFILM VisualSonics Inc.
Photoacoustic (PA) Imaging can estimate the spatial distribution of oxygen saturation (sO2) and total hemoglobin concentration (HbT) in blood, and be co-registered with B-Mode ultrasound images of the surrounding anatomy. This study will focus on the development of a PA imaging mode on a commercially available array based micro-ultrasound (μUS) system that is capable of creating such images.
This 3-sentence summary provides the key details about the protocol:
The protocol describes how to implant a guide cannula in transgenic mice expressing fluorescent proteins to allow insertion of a fiber optic probe for imaging deep brain structures of freely moving mice under anesthesia. The guide cannula is implanted using stereotactic surgery and then a fiber optic probe is inserted through the cannula and lowered to the target brain region under microscope guidance for in vivo imaging of fluorescent cells over multiple time points.
In this application, Cellvizio was used to study the neuronal degeneration and regeneration processes in live, anaesthetized, adult Thy1-YFP transgenic mice.
Abdominal Aortic Aneurysm (AAA) is a serious and potentially fatal disease that is prevalent in the older population. Scientists are making use of animal models to study the progression of this disease and the effects of therapeutic interventions over longitudinal studies.
Ischemia - or the lack of blood supply to a tissue - and subsequent reperfusion induces physiological and biochemical changes in the affected tissue and is an important area of study since the damage that occurs as a result is clinically important in diabetes and stroke.
Application Brief: Tumor Microenvironment Imaging with Photoacoustic TechnologyFUJIFILM VisualSonics Inc.
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This document discusses the need for improved imaging systems for breast cancer research using animal models. It introduces a new micro-ultrasound system that allows researchers to non-invasively collect longitudinal data on tumor size, volume, vascularity and perfusion over an animal's lifespan. This significantly reduces the number of animals needed for research. The system has been adopted by leading cancer research institutions and numerous studies have been published demonstrating its ability to accurately track tumor growth and response to therapies.
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
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White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice
1. VisualSonics White Paper:
In vivo Fiberoptic Fluorescence Microscopy
in freely behaving mice
October 9, 2009
Version 1.0
2. Table of Contents
Introduction ......................................................................................................... 1
Experimental setup ............................................................................................... 3
Animals ............................................................................................................... 3
Materials.............................................................................................................. 3
Stereotaxic coordinates.......................................................................................... 4
Procedure ............................................................................................................ 5
Results.................................................................................................................. 6
Discussion ............................................................................................................ 8
References.......................................................................................................... 10
Supplemental Information .................................................................................. 11
Imaging wildtype mice infected with Adeno Associated Virus (AAV) ............................ 11
Appendix I: Implantation of the guide-cannula ........................................................ 14
Appendix II: Preparation of OGB1-AM .................................................................... 16
VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice
3. Introduction
There are significant correlations between animal/organismic behavior and cellular
processes and therefore understanding physiological and pathological brain function
requires investigation at the genetic, molecular, cellular, and behavioral levels. While in
vitro studies continue to provide useful information not otherwise attainable, they do not
adequately reflect the complexity of the in vivo environment. Furthermore, as the tissue
microenvironment plays a critical role in physiological and pathological processes alike, in
situ studies which reveal neural responses in the context of intact, dynamic, and specific
neural circuits are of fundamental importance (Chang JY et al 2008)). Until recently, in vivo
in situ imaging of the brain has been limited to either low resolution imaging of large areas
or highly invasive techniques restricted to superficial cortex. To yield powerful information
of disease etiology and progression, high resolution minimally invasive imaging of deep
brain in vivo and in situ is crucial.
Fiberoptic fluorescence microscopy (FFM) employs optical fibers as small as 300
micrometers in diameter and offers the ability to image cellular and subcellular processes in
deep brain structures including the Ventral Tegmental Area (VTA) and the substantia nigra
(Sn). With FFM, structures of the deep brain can visualized for several hours making in vivo
tracking of neuronal migration, cell division, promoter activity, and other relatively
protracted processes amenable to study. Additionally, Davenne et al. (2005) reported that
imaging throughout stereotaxic positioning of beveled microprobes into deep brain tissue
showed that no cells were fractionated or distorted, as evidenced by an absence of
fluorophore leakage from cells, and noted that cells appeared to slide along the bevel of the
fiberoptic microprobe. Further investigation by ex vivo microscopy on brain slices following
implantation of beveled microprobes revealed that tissue separation, while irremediable,
was slight. Such minimally invasive access has enabled longitudinal imaging of deep brain
structures over the course of several weeks (Crescent et al, unpublished results).
However, studies of anesthetized models obviously lack behavioral corroboration.
Furthermore, active brain states may serve to accentuate differences that only manifest
partially while an animal is in the resting state (Holschneider DP and Maarek JM 2008).The
flexible nature of fiberoptic microendoscopes employed for FFM and the ability to implant
them into live subjects offers the ability to image freely moving animals.
Simultaneous investigation of cellular and organismal behavior provides a direct and
immediate means for relating causal events with consequent responses. For example,
recording of neural responses to behaviorally effective deep brain stimulation (DBS) in
freely moving animals provides a direct means for examining how DBS modulates the basal
ganglia thalamocortical circuits and thereby improves motor function (Chang JY et al.,
2008). Indeed much of our knowledge of behavioral neuroscience and our ability to relate
cellular behavior to organismal behavior has been learned through electrophysiological
recordings in freely moving animals.
Place cells are hippocampal cells that encode spatial location. Recordings from these cells in
freely moving, genetically modified mice have further advanced our understanding of how
the actual cellular representation of space is influenced by genetic alterations that affect
long-term potentiation (Mayford M et al. 1997). High resolution imaging offers increased
confidence and deeper insight: responses of individual and small populations of neurons can
be acquired, coupling morphology and function, and events such as motility and division.
VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 1
4. However, there have remained significant limitations to widespread implementation of FFM
for study of deep brain in freely moving animals including size, functionality, image stability,
and access to the technology. The size and weight needs to be small and light enough for
use in mice. Recently, Flusberg et al 2008 employed the use of a 1.1g miniaturized
epifluorescence microscope for imaging deep brain of freely moving mice. While this is a
significant advancement over previous attempts (3.9g, Flusberg et al 2005), it remains
‘heavy’ for a 25g mouse and may influence behavioral activity (particularly for studies
involving ataxic mouse models). While epifluorescent microscopy enables fast imaging with
large fields of view, out-of-focus light reduces image quality. With respect to image stability,
sophisticated hardware, image processing software, and innovative methods for fixation of
implanted fiberoptic microprobes are required to ensure that voluntary and involuntary
spastic movements do not cause motion artifact in the acquired data.
The Cellvizio® LAB In Vivo Confocal Fluorescence Microscope overcomes these limitations
and thereby provides an opportunity to longitudinally image the deep brain in situ with
subcellular (3.3μm) resolution, enabling unique research studies with a simultaneous
correlation to behavioral performance. Importantly, Cellvizio LAB is the only commercially
available solution for such sophisticated study.
The Cellvizio LAB consists of a point-scanning confocal laser which improves image quality
by limiting out-of-focus light while still allowing a 300μm diameter field of view.
Furthermore, the system is capable of 10ms frame acquisition (200 frames per second) and
employs a single-pixel avalanche photodiode detector (APD) for superior temporal resolution
and sensitivity, respectively.
The recent development of the CerboFlex™ Probe and NeuroPak™ Deep Brain Imaging
System provide a lightweight solution for chronic imaging of in situ deep brain in freely
moving mice. The CerboFlex is a fiberoptic microprobe comprised of (tens of) thousands of
individual step-index fiber optics encased within a single bundle 300um in diameter. Non-
ordered arrangement of these fibers eliminates crosstalk between adjacent fibers and
maintains high contrast and image quality. The NeuroPak System employs surgical
implantation of a <290mg stabilization plate which allows for a sturdy, mechanical, non-
permanent connection of the CerboFlex. Because the CerboFlex itself is not permanently
implanted, it can be removed, cleaned, and recalibrated prior to every imaging session,
ensuring reliable results and quantification in longitudinal studies of the same experimental
animal.
This note explains how researchers from the Institut Pasteur (Paris, France) used the
Cellvizio LAB to image neurons in the Hippocampus (see supplemental data), Sn, and VTA in
freely behaving mice.
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5. Experimental setup
Animals
Transgenic mice (Thy1-CerTN-L15; Heim et al., 2007) expressing GFP were used for
imaging the substantia nigra while Th-gfp mice (stably transfected with a vector engineered
to express GFP under the rat tyrosine hydroxylase promoter; Sawamoto et al., 2001) were
employed for imaging the VTA.
Animals were anaesthetized by intra-peritoneal injection of Ketamine/Xylazine (0.1/0.01 mg
per gram of body weight). Alternatively, animals can be anesthetized using inhaled
isofluorane (3% in Oxygen) which ensures continuous immobilization and allows for faster
recovery.
Materials
NeuroPak Deep Brain Imaging System
Figure 1. The NeuroPak Deep Brain Imaging System includes the CerboFlex Probe, 6
implants and required screws, a guide holder for use with a stereotaxic device and complete
procedural information for performing the implant surgery. Shown on the right is the
implant itself, with the guide post and cannula. The implant weighs less than 300mg
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6. CerboFlex
Figure 2. The CerboFlex Probe mounted on the implant. The flexible fiber probe is shown
protruding from the cannula. The depth of penetration may be accurately controlled during
insertion using a stereotaxic device. The fiber is 300 microns in diameter, with a beveled tip
for minimally invasive access.
Cellvizio LAB imaging system (488nm excitation; VisualSonics, Toronto, Canada)
QuantiKit™ 488 Calibration kit (VisualSonics, Toronto, Canada)
Stereotaxic equipment (World Precision Instruments, Florida)
ImageCell™ software
Stereotaxic coordinates
The guide-cannula is stereotaxically inserted in the mouse brain above the targeted brain
area and the CerboFlex then lowered to the anatomical target according to bregma
coordinates (Paxinos and Franklin, The Mouse brain in stereotaxic coordinates; Academic
Press). Coordinates used are : AP= -3,3 mm, L= 1,3 mm, Z=-3,4 mm (Substantia nigra,
reticulata) and AP = -3,4 mm, L = 0,5 mm and Z =- 4,5 mm (VTA).
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7. Procedure
The experiment required two distinct phases – surgical implantation (1) and imaging (2).
1) At least one week prior the first imaging session the mouse underwent stereotaxic
surgery to implant a guide-cannula into the skull above the targeted structures (See
appendix II for supplemental information).
2) For imaging, mice were anesthetized and the CerboFlex imaging microprobe was
stereotaxically guided to the target structures according to Z coordinates until fluorescent
neurons are identified *. The CerboFlex was then mechanically secured to the guide-cannula
using a screw to ensure stability throughout the duration of the imaging experiments.
Animals were allowed to recover from anesthesia and images acquired for various periods of
time thereafter while freely behaving in an open field cage. At the end of individual imaging
experiments, animals were re-anesthetized to carefully remove the CerboFlex from the
guide-cannula under stereotaxic guidance.
*note that calibration steps were made according to Cellvizio LAB guidelines immediately
prior to this step.
Figure 3 : Thy1-CerTN-L15 mouse with a guide-cannula implanted above the Sn.
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8. Results
The CerboFlex microprobe was introduced into an anesthetized Th-GFP mouse through the
surgically implanted guide-cannula and into the VTA under stereotaxic guidance according
to Z coordinates until fluorescent neurons were identified (Figure 4a, t=0).
As the animal recovered from anesthetic, spastic movements did not alter the position of
the CerboFlex relative to the VTA as evidenced by the retention of the original imaging field
of view acquired (Figure 4b, t=20min). Fluorescent dopaminergic neurons within the VTA
were intermittently imaged for longer than one hour with no change in the field of view
(Figure 4). Notably, background autofluorescence levels and depreciation of image quality
due to photobleaching did not ‘appreciably affect’ detection and visualization of fluorescent
neurons in the field of view (Figure 4 c and d, t=55min and t=70min respectively).
Figure 4 : Individual frames of a Neuron within the VTA extracted from different sequences
of images acquired at several time points. The red arrows point to a brightly fluorescent
neuron in the VTA of a Th-GFP mouse. Notice that the field of view remains unchanged
throughout the experiment.
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9. Similar results have been obtained for neurons of the Substantia nigra in Thy1-CerTN-L15
mice over a much longer period of time. In order to reduce photobleaching and maintain
sensitivity, intermittent image acquisition with 100% laser intensity was limited to 10
second sequences repeated several times. As shown in Figure 5, a brightly fluorescent
neuron was observed for more than 3 hours without image distortion and with minimal
depreciation of image quality.
Figure 5: Images of fluorescent neurons within the Substantia nigra of a Thy1-CerTN-L15
mouse. The Substantia nigra was identified according to stereotaxic coordinates and
observable fluorescence. The field of view was stable for more than 3 hours, sufficient for
simultaneous study of cellular and organismal behavior.
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10. While these studies were carried out in transgenic animals, similar results have been
obtained in the dorsal part of the mouse hippocampus (figure 9) in normal mice after
injection of a modified Adeno Associated Virus (AAV) vector that induces cytoplasmic
expression of GFP (See supplemental information).
Discussion
Owing to the confocal approach, nonordered fiberoptic bundle, and advanced algorithms of
the Cellvizio LAB and CerboFlex along with the lightweight, stable and semi-permanent
design of the NeuroPak, the images of fluorescent neurons in the VTA and Sn of freely
moving mice was devoid of motion artifact.
Image stability is crucial when one considers the cellular study of epileptic events in animals
during seizure where convulsive behavior is expected. It is also important when quantifying
cellular events that occur over a longer timescale such as neuronal migration. Davenne et
al.(2005) elegantly quantified the velocity of neuroblasts migrating in vivo from the
subventricular zone along the rostral migratory steam to the olfactory bulb in anesthetized
adult mice. Changes in the number, direction, and velocity of migrating cells may be
activity-dependent in response to olfactory stimuli; indeed a clear relationship has been
shown between olfactory performance and the quantity of newborn neurons in the olfactory
bulb (Lledo and Saghatelyan (2005); Rochefort, C. et al. (2002); Gheusi, G. et al. (2000);
Enwere, E. et al. (2004). Monitoring of migrating neurons in freely moving animals and non-
invasive observation of the bulbular neuronal network through the nasal cavity may serve to
address the adaptive response required for fine adjustment of olfactory ability (Vincent et
al. 2006).
The concept of restructuring in the adult brain is, of course, not limited to the olfactory
system; importantly, evidence suggests that neurogenesis occurs in the adult mammalian
Sn (Zhao, M. et al. 2003). Monitoring of cellular responses in the Sn of unrestrained, awake
animals is imperative for a comprehensive understanding of etiology and progression of
Schizophrenia and Parkinson’s disease. Equally important for this understanding is
functional imaging of neuronal activity. Specific dyes (calcium or voltage sensors, such as
Oregon Green Bapta-1 (OGB1)) must be injected prior the imaging session. These dyes
serve as fluorescent sensors of intracellular [Ca2+], undergoing conformational changes that
change the absorption/emission spectrum of the dye when in contact with Ca2+. The
variation in fluorescent intensity upon Ca2+ influx, reflective of changes in neuronal activity,
can be quantified using the kinetic analysis tool in the Cellvizio LAB software, ImageCell.
(Note: see Appendix II for a protocol describing in vivo labeling of brain tissue with OGB1).
For the experiments described in this document, animals were sacrificed by deep anesthesia
to verify the precise position of the CerboFlex tip within the brain. However, due to the
small diameter of the microprobe and therefore consequent restricted lesion of the brain
tissue, longitudinal imaging of the same structure of the same animal can be acquired.
Since these initial experiments, researchers at Institut Pasteur have successfully acquired
images of the same freely moving animal over several weeks (unpublished data).
While image stability throughout an individual imaging session is important, so is exact
repositioning the CerboFlex for longitudinal studies. Because the position of the guide
cannula is fixed relative to the anatomical structure of interest, precision when reintroducing
the CerboFlex is ensured. Furthermore, in addition to the stereotaxic Z coordinates, images
are acquired during the positioning of the CerboFlex which thereby provides another point of
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11. reference when fluorescence is restricted to specific brain areas.
The Cellvizio LAB is a unique, commercially available imaging system that affords scientists
the opportunity to image neurons in vivo and in situ of freely moving animals, even in the
deepest parts of the brain. Due to the small diameter of the CerboFlex tip and to the
flexibility of the probe, small animals can move freely in their environment or various
behavioral mazes while images of the neuronal network are acquired over long periods of
time in longitudinal studies of the same animal.
The Cellvizio LAB In Vivo Confocal Fluorescence Microscope the CerboFlex Deep Brain
Imaging Probe, and the NeuroPak Deep Brain Imaging System are distributed globally by
VisualSonics and its distribution partners. For more information, please visit
www.visualsonics.com
VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 9
12. References
1. Michael Eisenstein (2009) Getting inside their minds
Nature Methods; 6, 773-781
2. Chang JY et al 2008
3. Enwere, E. et al. (2004) Aging results in reduced epidermal growth factor receptor
signaling, diminished olfactory neurogenesis, and deficits in fine olfactory
discrimination.
J. Neurosci. 24, 8354–8365
4. Flusberg BA, Nimmerjahn A, Cocker ED, Mukamel EA, Barretto RP, Ko TH, Burns
LD, Jung JC, Schnitzer MJ. (2008) High-speed, miniaturized fluorescence
microscopy in freely moving mice.
Nat Methods. Nov;5(11):935-8.
5. Flusberg BA, Jung JC, Cocker ED, Anderson EP, Schnitzer MJ. In vivo brain imaging
using a portable 3.9 gram two-photon fluorescence microendoscope.
Opt Lett. 2005 Sep 1;30(17):2272-4.
6. Gheusi, G. et al. (2000) Importance of newly generated neurons in the adult
olfactory bulb for odor discrimination.
PNAS 97, 1823–1828
7. Heim et al. (2007), Nature Methods, 4(2):127-129
8. Holschneider DP and Maarek JM 2008
9. Mayford M et al. 1997
10. Paxinos and Franklin, The Mouse brain in stereotaxic coordinates; Academic Press
11. Rochefort, C. et al. (2002) Enriched odor exposure increases the number of
newborn neurons in the adult olfactory bulb and improves odor memory.
J. Neurosci. 22, 2679–2689
12. Sawamoto et al. (2001), PNAS, 98(11): 6423–6428
13. Stosiek C et al. (2003)
14. Tallini et al. (2006), PNAS, 103(12): 4753-58
15. Vincent et al. 2005
16. Zhao, M. et al. (2003)
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13. Supplemental Information
Imaging wildtype mice infected with Adeno Associated Virus (AAV)
Direct intracranial injection of a modified Adeno Associated Virus (AAV) vector (Figure S1)
that induces cytoplasmic expression of GFP under the GcamP2 promoter enabled imaging of
the dorsal hippocampus in a wildtype mouse.
Prior the fixation of the headstage on the skull and guide-cannula insertion above the dorsal
hippocampus, the vector was injected into the dorsal hippocampus (0.5 μl / 5 min). A delay
of 3 weeks before the imaging sessions was required to ensure adequate expression of GFP
within cells.
Figure S1: The AAV vector used expressing GcamP2 and eGFP (From Tallini et al. 2006).
As previously described, the CerboFlex was inserted into the brain of an anesthetized mouse
and neurons were imaged in sequences of 10 sec length to reduce photobleaching. Image
stability was observed for more than 4 hours as shown in figure S2.
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14. VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 12
15. Figure S2: Numerous neurons of the dentate gyrus within the dorsal hippocampus after
expression of GFP induced by injection of a modified Adeno Associated Virus (AAV) vector.
Images were acquired at different time points.
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16. Appendix I: Implantation of the guide-cannula
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17. VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 15
18. Appendix II: Preparation of OGB1-AM
Solution preparation
Use one vial of OGB 488 BAPTA-1AM (Oregon Green 488 BAPTA AM-1, MW 1258.07 g,
available from Invitrogen #O-6807)
1. Add 4µl of 20% pluronic acid (Invitrogen #P-3000MP) in DMSO
2. Vortex for 3 mins
3. After this step, the color of the solution should be slightly yellow
4. Add 36µL of Ca2+-free ACSF
5. Add 1µL of SR101 (2.5mM or 2mM mixed in ACSF)
6. Vortex for about 3min
7. Sonicate on ice for 5min
8. If dye sits longer than 30min, sonicate again for 5min
9. Pipette dye into centrifuge filter (Ultrafree MC, available from Fisher Scientific;
UFC30GV25)
10. Centrifuge for 30 sec
11. Dilute 1: 10 in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4.
12. The final solution concentration is 1mM
13. Fill pipette with approximately 8μl
Practically – add 3.97 microliters of DMSO pluronic corresponding to a 10 mM solution) in a
vial of OGB1 and dilute the solution in 35.7 microliters of Hepes solution to obtain a 1mM
solution
Ejection Parameters
Injection is done using a perfusion pump through a 36G stainless steel needle: 1microliter in
10 minutes (0.1μl/min). Do not remove the needle for 10 minutes. Retraction should be
incremental over a 5 minute period. Following retraction, wait one hour prior to imaging.
Reference: Stosiek C et al. (2003)
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