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.
Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organizationofexcitatoryinputs.Remarkably,thespatialextentofinhibitoryinputsofexcitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.
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
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.
Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organizationofexcitatoryinputs.Remarkably,thespatialextentofinhibitoryinputsofexcitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.
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
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
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.
White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving miceFUJIFILM VisualSonics Inc.
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).
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
Electrophysiology of Human Native Receptors in Neurological and Mental DisordersInsideScientific
Dr. Agenor Limon presents research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks.
Alterations in synaptic function have been found in transcriptomic, genetic, and proteomic studies of neurological and mental disorders. Clinical and preclinical studies suggest that synaptic dysfunction and behavioral abnormalities in disorders like Alzheimer’s disease and schizophrenia may be mechanistically linked to the emergence of imbalances between excitatory (E) and inhibitory (I) receptors. However, until recently, the electrophysiological E/I synaptic ratio had only been measured in animal models.
Using pioneering methods developed in the lab including reactivation and microtransplantation of synaptic receptors from frozen human brains, Dr. Agenor Limon’s research team has obtained electrophysiological metrics of global synaptic E/I ratios in cortical brain regions of subjects that were affected by Alzheimer’s Disease and synaptic measurements in schizophrenia.
In this webinar, Dr. Agenor Limon will present recent research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks.
Key Topics Include:
- Understand the global synaptic excitation to inhibition ratio between excitatory and inhibitory synaptic receptors determined from reactivated frozen human brain tissue
- Understand the relationship between electrophysiological metrics of receptor function and multi-omic data in neurodegenerative disorders
- Understand the role of deviations of the excitation to inhibition ratio with clinical presentation in Alzheimer’s disease
Sensorimotor Network Development During Early Postnatal Life in the Awake and...InsideScientific
In the last decades, electrophysiological and imaging-based approaches provided significant new insights into the mechanisms of neuronal development. Nevertheless, many important questions remain unanswered. How does the fine control of a motor output develop? How does sensorimotor integration in the early and subsequent phases of brain development shape behavior? How does sensorimotor development evolve in awake and sleeping states? What role do myoclonic twitches play in this process?
Answering these questions requires performing high-precision tests in the brain of non-anesthetized animals across sleep and wake during the early stages of their postnatal development. Such tests require head-fixation apparatus suitable for neonatal and juvenile rodents. The Mobile HomeCage combines a stable head-fixation with an air-lifted cage that closely resembles laboratory rodents’ natural habitat – an optimal platform for studying early postnatal brain development.
In this webinar, Dr. Cavaccini (Prof. Karayannis’s lab at the Brain Research Institute, University of Zurich) and Dr. Dooley (Prof. Blumberg’s lab at the University of Iowa), share their insights into the development of rodent sensorimotor neuronal circuits during early postnatal life. They elucidate the cortical and subcortical mechanisms involved in the development of sensorimotor circuitry during wakefulness (in a mouse model) and REM sleep (in a rat model).
Key Takeaways
Dr. Anna Cavaccini:
- Anatomical and functional changes occur at the striatal level before and after the onset of different sensory modalities
- Locomotor activity changes throughout early development, and it correlates with striatal function
- Sensory information coming from whiskers affects locomotion and striatal function before and after the onset of different sensory modalities
Dr. James Dooley:
- Myoclonic twitches in REM sleep continue to trigger cortical and thalamic activity beyond the early postnatal period
- Twitch-related thalamic activity is spatiotemporally refined by the third postnatal week
- Motor thalamus activity reflects an internal model of movement produced by twitches and is dependent on the cerebellar output
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
Impact of percutaneous coronary intervention on the levels of interleukin-6 and C-reactive protein in the coronary circulation of subjects with coronary artery disease
An inhibitory pull–push circuit in frontal cortexTaruna Ikrar
Push–pull is a canonical computation of excitatory cortical circuits. By contrast, we identify a pull–push inhibitory circuit in frontal cortex that originates in vasoactive intestinal polypeptide (VIP)-expressing interneurons. During arousal, VIP cells rapidly and directly inhibit pyramidal neurons; VIP cells also indirectly excite these pyramidal neurons via parallel disinhibition. Thus, arousal exerts a feedback pull–push influence on excitatory neurons—an inversion of the canonical push–pull of feedforward input.
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
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.
White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving miceFUJIFILM VisualSonics Inc.
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).
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
Electrophysiology of Human Native Receptors in Neurological and Mental DisordersInsideScientific
Dr. Agenor Limon presents research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks.
Alterations in synaptic function have been found in transcriptomic, genetic, and proteomic studies of neurological and mental disorders. Clinical and preclinical studies suggest that synaptic dysfunction and behavioral abnormalities in disorders like Alzheimer’s disease and schizophrenia may be mechanistically linked to the emergence of imbalances between excitatory (E) and inhibitory (I) receptors. However, until recently, the electrophysiological E/I synaptic ratio had only been measured in animal models.
Using pioneering methods developed in the lab including reactivation and microtransplantation of synaptic receptors from frozen human brains, Dr. Agenor Limon’s research team has obtained electrophysiological metrics of global synaptic E/I ratios in cortical brain regions of subjects that were affected by Alzheimer’s Disease and synaptic measurements in schizophrenia.
In this webinar, Dr. Agenor Limon will present recent research integrating functional metrics with large anatomical, transcriptomic, and proteomic datasets to evaluate the relationship between synaptic E/I ratio and behavioral abnormalities across postmortem intervals and brain banks.
Key Topics Include:
- Understand the global synaptic excitation to inhibition ratio between excitatory and inhibitory synaptic receptors determined from reactivated frozen human brain tissue
- Understand the relationship between electrophysiological metrics of receptor function and multi-omic data in neurodegenerative disorders
- Understand the role of deviations of the excitation to inhibition ratio with clinical presentation in Alzheimer’s disease
Sensorimotor Network Development During Early Postnatal Life in the Awake and...InsideScientific
In the last decades, electrophysiological and imaging-based approaches provided significant new insights into the mechanisms of neuronal development. Nevertheless, many important questions remain unanswered. How does the fine control of a motor output develop? How does sensorimotor integration in the early and subsequent phases of brain development shape behavior? How does sensorimotor development evolve in awake and sleeping states? What role do myoclonic twitches play in this process?
Answering these questions requires performing high-precision tests in the brain of non-anesthetized animals across sleep and wake during the early stages of their postnatal development. Such tests require head-fixation apparatus suitable for neonatal and juvenile rodents. The Mobile HomeCage combines a stable head-fixation with an air-lifted cage that closely resembles laboratory rodents’ natural habitat – an optimal platform for studying early postnatal brain development.
In this webinar, Dr. Cavaccini (Prof. Karayannis’s lab at the Brain Research Institute, University of Zurich) and Dr. Dooley (Prof. Blumberg’s lab at the University of Iowa), share their insights into the development of rodent sensorimotor neuronal circuits during early postnatal life. They elucidate the cortical and subcortical mechanisms involved in the development of sensorimotor circuitry during wakefulness (in a mouse model) and REM sleep (in a rat model).
Key Takeaways
Dr. Anna Cavaccini:
- Anatomical and functional changes occur at the striatal level before and after the onset of different sensory modalities
- Locomotor activity changes throughout early development, and it correlates with striatal function
- Sensory information coming from whiskers affects locomotion and striatal function before and after the onset of different sensory modalities
Dr. James Dooley:
- Myoclonic twitches in REM sleep continue to trigger cortical and thalamic activity beyond the early postnatal period
- Twitch-related thalamic activity is spatiotemporally refined by the third postnatal week
- Motor thalamus activity reflects an internal model of movement produced by twitches and is dependent on the cerebellar output
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
Impact of percutaneous coronary intervention on the levels of interleukin-6 and C-reactive protein in the coronary circulation of subjects with coronary artery disease
An inhibitory pull–push circuit in frontal cortexTaruna Ikrar
Push–pull is a canonical computation of excitatory cortical circuits. By contrast, we identify a pull–push inhibitory circuit in frontal cortex that originates in vasoactive intestinal polypeptide (VIP)-expressing interneurons. During arousal, VIP cells rapidly and directly inhibit pyramidal neurons; VIP cells also indirectly excite these pyramidal neurons via parallel disinhibition. Thus, arousal exerts a feedback pull–push influence on excitatory neurons—an inversion of the canonical push–pull of feedforward input.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Earlysensoryexperienceinstructsthematurationofneuralcircuitry in the cortex1,2. This has been studied extensively in the primary visualcortex,inwhichlossofvisiontooneeyepermanentlydegrades corticalresponsivenesstothateye3,4,aphenomenonknownasocular dominance plasticity (ODP). Cortical inhibition mediates this process4–6,butthepreciseroleofspecificclassesofinhibitoryneurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediatelydropbyhalfwhenvisionisrestrictedtooneeye,butgradually return to normal over the followingtwenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation resultsfromarapid,althoughtransient,reductioninthefiringrates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turncanbeattributedtoadecreaseinlocalexcitatorycircuitinput onto PV interneurons.This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODPandappearstobenecessaryforsubsequentshiftsinexcitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. Thesefindingsdefinethemicrocircuitchangesinitiatingcompetitive
plasticityduringcriticalperiodsofcorticaldevelopment.Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
Pten and eph b4 regulate the establishment of perisomatic inhibition in mouse...Taruna Ikrar
Perisomatic inhibition of pyramidal neurons is established by fast-spiking, parvalbuminexpressing interneurons (PV cells). Failure to assemble adequate perisomatic inhibition is thought to underlie the aetiology of neurological dysfunction in seizures, autism spectrum disorders and schizophrenia. Here we show that in mouse visual cortex, strong perisomatic inhibition does not develop if PV cells lack a single copy of Pten. PTEN signalling appears to drive the assembly of perisomatic inhibition in an experience-dependent manner by suppressing the expression of EphB4; PVcells hemizygous for Pten show an B2-fold increase in expression of EphB4, and over-expression of EphB4 in adult PV cells causes a dismantling of perisomatic inhibition. These findings implicate a molecular disinhibitory mechanism driving the establishment of perisomatic inhibition whereby visual experience enhances Pten signalling, resulting in the suppression of EphB4 expression; this relieves a native synaptic repulsion between PV cells and pyramidal neurons, thereby promoting the assembly of perisomatic inhibition.
A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the “critical period,” when the circuitry of
primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during
the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A
disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal
nogo-66-receptor1(ngr1/rtn4r) is required to close the critical period.Herewecombinedmousegenetics, electrophysiology,andcircuitmapping
with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1.We demonstrate that ngr1
mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of
intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a “lower PV network configuration” in both
critical-period wild-type miceandadult ngr1/mice.Wepropose that ngr1 limits disinhibition to close the critical period forODplasticityand
that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.
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 presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.
The field of neuroscience is undergoing significant change. The two main cell families that make up the brain, neurons and glial cells, each concealed a hybrid brain cell that fell somewhere in the middle.
Research projects on protein expression and immunostaining. An unknown protein may be localized by double labeling the tissue with a marker whose expression is known. Graduate student research project for a seminar on optical methods in molecular biology. Students were introduced to the imaging core which is equipped with confocal microscopes. The instructors aided with utilization of the computer software, switching between different objectives, and the choice of materials.
Similar to Ikrar Et Al (Cell Type Specific Regulation Of Cortical Excitability Through The Allatostatin Receptor System) Frontiers Journal (20)
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Ikrar Et Al (Cell Type Specific Regulation Of Cortical Excitability Through The Allatostatin Receptor System) Frontiers Journal
1. ORIGINAL RESEARCH ARTICLE
NEURAL CIRCUITS published: 20 January 2012
doi: 10.3389/fncir.2012.00002
Cell-type specific regulation of cortical excitability through
the allatostatin receptor system
Taruna Ikrar 1† , Yulin Shi 1† , Tomoko Velasquez 3† , Martyn Goulding 3† and Xiangmin Xu 1,2*†
1
Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA, USA
2
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, USA
3
Molecular Neurobiology Laboratories, Salk Institute for Biological Studies, San Diego, CA, USA
Edited by: Recent technical advances enable the regulation of neuronal circuit activity with high
Jessica Cardin, Yale University spatial and temporal resolution through genetic delivery of molecular activation or
School of Medicine, USA
inactivation systems. Among them, the allatostatin receptor (AlstR)/ligand system has
Reviewed by:
been developed for selective and quickly reversible silencing of mammalian neurons.
Bing Zhang, University of
Oklahoma, USA However, targeted AlstR-mediated inactivation of specific neuronal types, particularly
David J. Margolis, University of diverse types of inhibitory interneurons, remains to be established. In the present study,
Zurich, Switzerland we achieved Cre-directed expression of AlstRs to excitatory and inhibitory cell-types in
*Correspondence: the cortex, and found that the AlstR-mediated inactivation was specific and robust at
Xiangmin Xu, Department of
single-cell and neuronal population levels. Bath application of the allatostatin peptide
Anatomy and Neurobiology, School
of Medicine, University of California, markedly reduced spiking activity of AlstR-expressing excitatory and inhibitory neurons
Irvine, CA 92697-1275, USA. in response to intrasomatic current injections and laser photostimulation via glutamate
e-mail: xiangmin.xu@uci.edu uncaging, but control neurons without AlstR expression were not affected. As for the
† Author Contributions:
cortical network activity, the peptide application constrained photostimulation-evoked
Experiments were conducted by
Taruna Ikrar and Yulin Shi;
excitatory activity propagation detected by fast voltage-sensitive dye (VSD) imaging
Xiangmin Xu, Yulin Shi, and Taruna of the slices expressing AlstRs selectively in excitatory neurons, while it augmented
Ikrar performed data analysis; excitatory activity in those slices with inhibitory neurons expressing AlstRs. In addition,
Tomoko Velasquez and Martyn AlstR-mediated inactivation effectively suppressed pharmacologically induced seizure
Goulding provided the AlstR mouse;
Xiangmin Xu wrote the paper.
activity in the slices targeting AlstRs to excitatory neurons. Taken together, our work
demonstrated that the genetic delivery of AlstRs can be used for regulation of cortical
excitability in a cell-type specific manner, and suggested that the AlstR system can be
potentially used for fast seizure control.
Keywords: genetic inactivation, electrophysiology, photostimulation, voltage-sensitive dye imaging
INTRODUCTION protein-coupled inward-rectifying K+ (GIRK) channels in AlstR-
In the last decade, there has been considerable progress in the expressing neurons. The AlstR/AL-mediated inactivation is esti-
development and use of molecular and genetic methods for mated to occur within seconds to minutes, depending on the
studying the central nervous system (Luo et al., 2008). Genetic peptide delivery speed (Lechner et al., 2002; Tan et al., 2006).
strategies for activating or inactivating selected neurons have Endogenous expression of GIRK channels is common to the great
opened up new possibilities for understanding neuronal cir- majority of mature neurons in the mammalian brain (Karschin
cuitry with high spatial and temporal resolution (Lechner et al., et al., 1996), such that cotransfection with GIRK channel sub-
2002; Boyden et al., 2005; Karpova et al., 2005; Tan et al., units is unnecessary to effectively inactivate neurons using the
2006; Armbruster et al., 2007; Lerchner et al., 2007; Chow AlstR/AL system (Lerchner et al., 2007; Wehr et al., 2009). Because
et al., 2010). Along with other newly developed molecular tech- mammals do not express the AL peptide or its receptor, pep-
niques, the allatostatin receptor (AlstR) inactivation system can tide application should specifically inactivate only the neurons
be a potent tool for regulating neocortical circuit activity with expressing the AlstR transgene.
a high degree of specificity and effectiveness. The AlstR and Selective AlstR expression and its neuronal inactivation in
its ligand peptide, allatostatin (AL), are found in insect brains, the brains of mice, ferrets, and monkeys has been demonstrated
not native to mammalian neuronal tissue (Birgul et al., 1999). via viral infection or using transgenic approaches (Gosgnach
When expressed in mammalian neurons through genetic meth- et al., 2006; Tan et al., 2006, 2008; Wehr et al., 2009). However,
ods (Lechner et al., 2002; Gosgnach et al., 2006; Tan et al., targeted AlstR-mediated inactivation of specific neuronal types,
2006, 2008; Wehr et al., 2009), the receptor system can been particularly diverse types of inhibitory interneurons in the neo-
used for silencing of neuronal activity as the application of the cortex, remains to be established. Given that there are multiple
exogenous ligand quickly increases K+ channel conductance and cell-types and each type is likely to have a unique role in cor-
keeps membrane potentials from depolarizing above threshold tical circuits, in the present study, we aimed to express the
through G protein-coupled AlstRs, which gate endogenous G AlstR system to excitatory or inhibitory cell-types for cell-type
Frontiers in Neural Circuits www.frontiersin.org January 2012 | Volume 6 | Article 2 | 1
2. Ikrar et al. AlstR-mediated regulation of cortical excitability
selective regulation of cortical excitability through a double trans- To prepare living brain slices, the double transgenic mouse
genic mouse approach. The transgenic mouse line conditionally pups (postnatal day 16–21) or control pups (AlstR or Cre
expressing AlstR following Cre recombination (see below) was mouse pups) were deeply anesthetized with pentobarbital
cross-bred with an Emx1-Cre mouse line (expressing Cre recom- sodium (>100 mg/kg, i.p.) and rapidly decapitated, and their
binase in most excitatory neurons of the cortex) (Guo et al., brains removed. For electrophysiological experiments, primary
2000; Kuhlman and Huang, 2008) or a Dlx5/6-Cre mouse line somatosensory (S1) cortical slices of 400 µm thick were cut
(expressing Cre in forebrain GABAergic neurons) for selective in sucrose-containing artificial cerebrospinal fluid (ACSF) (in
delivery of AlstRs to either excitatory or inhibitory cell-types in mM: 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH2 PO4 ,
the brain. 4 MgCl2 , 0.5 CaCl2 , and 24 NaHCO3 ). Slices were first incu-
We combined electrophysiology, laser scanning photostimu- bated in sucrose-containing ACSF for 30 minutes–one hour
lation, and fast voltage-sensitive dye (VSD) imaging to examine at 32◦ C, and then transferred to recording ACSF (in mM:
AlstR-mediated regulation of cortical excitability in living brain 126 NaCl, 2.5 KCl, 26 NaHCO3 , 2 CaCl2 , 2 MgCl2 , 1.25
slice preparations, and addressed the following questions: (a) NaH2 PO4 , and 10 glucose). For VSD imaging experiments, cor-
How effective is the Cre-mediated, selective expression of AlstRs? tical slices were cut in ACSF with a broad-spectrum excita-
(b) Is the AlstR-mediated inactivation specific or robust at single- tory amino acid antagonist kynurenic acid (0.2 mM). The ACSF
cell and neuronal population levels? (c) Can the AlstR system with kynurenic acid was found to better preserve slice health-
be used to suppress seizure activity when targeting AlstR expres- iness for dye staining than the sucrose-containing ACSF. After
sion to excitatory neurons? Our experiments demonstrated that the initial incubation at 32◦ C, slices were transferred for dye
the AlstR system can be effectively used for regulation of corti- staining at room temperature for one hour in the ACSF and
cal excitability in a cell-type specific manner, and suggested that 0.1 mg/ml of the absorption VSD, NK3630 (Nippon Kankoh-
the AlstR system can be developed for potential translational Shikiso Kenkyusho Co. Ltd., Japan) and then maintained in the
applications in seizure control. recording ACSF before use. Throughout the cutting, incubation,
staining, and recording, the solutions were continuously supplied
MATERIALS AND METHODS with 95% O2 –5% CO2 .
SLICE PREPARATION
All animals were handled and experiments were conducted in ELECTROPHYSIOLOGY, LASER SCANNING PHOTOSTIMULATION
accordance with procedures approved by the Institutional Animal AND VOLTAGE-SENSITIVE DYE IMAGING
Care and Use Committee at the University of California, Irvine. Our overall system of electrophysiological recordings, photostim-
In this study, we used a newer AlstR mouse line conditionally ulation, and imaging was described previously (Xu et al., 2010;
expressing AlstR and green fluorescent protein (GFP) following Xu, 2011). The solution was fed into the slice recording cham-
Cre recombination. This AlstR mouse line (R26-AlstR) was cre- ber through a pressure driven flow system with pressurized 95%
ated by using the same targeting vector of transgene as the AlstR O2 –5% CO2 with a perfusion flow rate of about 2 ml/minute.
192 strain (Gosgnach et al., 2006), but the vector was inserted To perform whole-cell recording, individual neurons were visu-
at the Rosa26 locus to drive more stable expression. Specifically, alized at high magnification (60x objective), and were patched
the R26-AlstR mouse was first generated using a conditional with glass electrodes of 4–6 M resistance that were filled with
double-stop cassette system that has been described previously an internal solution containing (in mM) 126 K-gluconate, 4 KCl,
(Kim et al., 2009). The AlstR gene followed by an IRES-EGFP 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na, and 10 phosphocreatine
cassette was placed downstream of the CAG promoter and FSF- (pH 7.2, 300–305 mOsm). The internal solution also contained
LSL double-stop cassette. The Cre-dependent R26-AlstR mouse 0.1% biocytin for cell labeling and morphological identification.
line was then produced by removing the FSF stop cassette by Once stable whole-cell recordings were achieved with good access
crossing the original double-stop AlstR mouse with the mouse resistance (usually <20 M ), basic electrophysiological proper-
line of ACTB-FLPe transgene (Rodriguez et al., 2000). To achieve ties were examined through hyperpolarizing and depolarizing
Cre-directed expression of AlstRs, the R26-AlstR mouse line was current injections. We focused on analyzing neuronal input resis-
cross-bred with an Emx1-Cre mouse line (Guo et al., 2000) or a tance, spiking thresholds, and spike numbers at different steps of
Dlx5/6-Cre mouse line (Monory et al., 2006). Emx1 is a mouse depolarizing current pulses in control ACSF, in the presence of
homologue of the Drosophila homeobox gene empty spiracles AL, and after washout of AL. To examine AlstR-mediated inac-
and its expression is largely restricted to excitatory neurons in tivation, the ligand AL was added into the recording solution.
the developing and adult cerebral cortex and hippocampus (Guo Although nanomolar concentrations of AL could inactivated iso-
et al., 2000; Gorski et al., 2002; Kuhlman and Huang, 2008); lated cultured AlstR-expressing neurons (Lechner et al., 2002),
the Emx1-Cre mouse line was created with the Cre gene placed 1 µM AL (unless specified otherwise) were used for facilitating
directly downstream of the Emx1 promoter (Guo et al., 2000). the ligand penetration deep into the slices and increasing tem-
Dlx is a family of homeodomain transcription factors which are poral speeds of the ligand infusion. The AL application for 15
related to the Drosophila distal-less (Dll) gene (Panganiban and minutes was estimated to produce full AlstR effects, while the
Rubenstein, 2002); in the Dlx5/6 Cre mouse, expression of Cre washout of 20 minutes was considered to remove the added AL
is directed to virtually all forebrain GABAergic neurons during from the recording solution.
embryonic development by the I56i and I56ii enhancers from the During laser scanning photostimulation experiments, the
zebrafish dlx5a/dlx6a intergenic region (Monory et al., 2006). microscope objective was switched from 60x to 4x. Stock solution
Frontiers in Neural Circuits www.frontiersin.org January 2012 | Volume 6 | Article 2 | 2
3. Ikrar et al. AlstR-mediated regulation of cortical excitability
of MNI-caged-l-glutamate (Tocris Bioscience, Ellisville, MO) was pixel’s amplitude (equivalent to the detectable signal level in
added to 20 ml of ACSF for a concentration of 0.2 mM caged glu- the original VSD maps of I/I%). VSD images were smoothed
tamate. The cortical slice image was acquired by a high resolution by convolution with a Gaussian spatial filter (kernel size: five
digital CCD camera, which in turn was used for guiding and pixels; SD (σ ): one pixel), and a Gaussian temporal filter (ker-
registering photostimulation sites. A laser unit (DPSS Lasers, nel size: three frames; δ: one frame). In the present study,
Santa Clara, CA) was used to generate 355 nm UV laser for single-trial VSD signals were of sufficiently high amplitudes and
glutamate uncaging. Short pulses of laser flashes (1 ms, 20 mW) could be discerned from background noise; no averaging over
were controlled by using an electro-optical modulator and a multiple trials was used for data presentation unless specified.
mechanical shutter. The laser beam formed uncaging spots, each To examine AlstR-mediated effects on photostimulation-evoked
approximating a Gaussian profile with a width of ∼100 µm VSD responses, the total numbers of activated pixels and response
laterally at the focal plane. During the experiments examining amplitudes across the 10 frames around the peak VSD activa-
neuronal excitation profiles, photostimulation was applied to tion (e.g., at 55 ms after photostimulation) were measured for
8 × 8 patterned sites (with an inter-site space of 50 µm2 ) centered comparison across control, AL application, and washout. Images
at the recorded neurons in a non-raster, non-random sequence were displayed and analyzed using custom-made MATLAB
to avoid revisiting the vicinity of recently stimulated sites, while programs.
current-clamp recordings measured membrane potential depo-
larization and spiking activity. The neuronal excitation profile STATISTICAL ANALYSIS
was simply assessed by the number of spiking locations to the For statistical comparisons across more than two groups, we used
photostimulation of 8 × 8 sites. Laser scanning photostimula- the Kruskal–Wallis test (non-parametric One-Way ANOVA) and
tion permits rapid evaluation of multiple stimulation locations the Mann–Whitney U-test for group comparisons. Alpha lev-
with no physical damage to the tissue under our experimental els of p ≤ 0.05 were considered significant. All the values were
conditions. presented as mean ± SE.
During VSD imaging experiments, a 705 nm light trans-
illuminated brain slices and voltage-dependent changes in the RESULTS
light absorbance of the dye were captured by the MiCAM02 SELECTIVE EXPRESSION OF AlstRs AND CHARACTERIZATION OF
fast imaging system (SciMedia USA Ltd, Costa Mesa, CA). The AlstR-MEDIATED NEURONAL INACTIVATION
illumination was supplied by optically filtering white light pro- In the present study, we used Cre-directed expression of the
duced by an Olympus tungsten-halogen lamp up to 100 W of AlstR transgene to excitatory and inhibitory cortical neurons
power. Optical recording of VSD signals was performed under by crossing the R26-AlstR mouse line conditionally expressing
the 2x objective with a sampling rate of 2.2 or 4.4 ms per AlstR and GFP following Cre recombination (Gosgnach et al.,
frame (frame resolution 88 (w) × 60 (h) pixels). The field of view 2006) with the Emx1-Cre mouse line (Guo et al., 2000) or
covered the area of 2.56 × 2.14 mm2 with a spatial resolution the Dlx5/6-Cre mouse line (Monory et al., 2006). We con-
of 29.2 × 35.8 µm2 /pixel. For the imaging of photostimulation- firmed the specificity and efficiency of these Cre mouse lines in
evoked excitatory activity with and without the AL application, achieving cell-type specific gene expression. As demonstrated
VSD imaging of evoked activity was triggered and synchronized in Figures 1A–F, through the use of a tdTomato Cre reporter
with each laser photostimulation at specified cortical sites. For line (Madisen et al., 2010), the Emx1-Cre mouse expresses Cre
each trial, the VSD imaging duration was 2000 frames including in a majority of excitatory pyramidal neurons of the cortex
500 baseline frames with an inter-trial interval of 12–14 seconds. (Guo et al., 2000; Gorski et al., 2002) (Figures 1A–C), while
To image pharmacologically induced spontaneous seizure-like the Dlx5/6 mouse expresses Cre specifically in inhibitory
activity, ACSF with 10 µM bicuculline were infused into the slices interneurons (Monory et al., 2006; Chao et al., 2010)
for 15 minutes before VSD imaging; the imaging experiments (Figures 1D–F).
consisted of seven sessions each at three perfusion conditions: Emx1-cre: AlstR mice expressed AlstRs with GFP in cortical
bicuculline only, bicuculline + AL, and bicuculline only again in excitatory neurons, which was verified through GABA immunos-
ACSF. Each session had nine consecutive trials conducted with taining (Figures 1G,H) and electrophysiological recordings (see
three minutes of off-illumination intervals between sessions. The below). Immunochemical examinations and living slice obser-
number of seizure occurrences per session was measured under vations indicated that about 50% of excitatory cortical neu-
different conditions. rons express AlstRs, detected by their strong GFP appearance.
VSD signals were originally measured by the percent change Similarly, AlstR expression in inhibitory cortical neurons of
in pixel light intensity [ I/I%; the % change in the intensity Dlx5/6-Cre: AlstR mice was strong, as the native fluorescence
( I) at each pixel relative to the initial intensity (I)]. In addition, enabled direct visualization and targeted electrophysiological
the mean and standard deviation (SD) of the baseline activ- recordings of the labeled neurons.
ity of each pixel across the 50 frames before the spontaneous We started to test whether AlstR expression through the
activity onset or preceding photostimulation was calculated, and transgenic mouse approach led to functional inactivation of
VSD signal amplitudes were then expressed as SD multiples neuronal activity with single-cell electrophysiology. As illus-
above the mean baseline signal for display and quantification. trated in Figures 2A–D, consistent with GIRK activation, bath
The activated pixel was empirically defined as the pixel with the application of AL significantly decreased neuronal excitabil-
amplitude ≥ 1 SD above the baseline mean of the corresponding ity of AlstR-expressing excitatory cells in response to somatic
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4. Ikrar et al. AlstR-mediated regulation of cortical excitability
FIGURE 1 | Use of Cre-driver lines for targeted expression of allatostatin exposure times for Emx1-Cre: tdTomato and Dlx5/6-Cre: tdTomato
receptors (AlstRs) to excitatory or inhibitory cell-types. (A–C) Data fluorescent images were at an order of difference due to an extremely
images from primary somatosensory (S1) cortex of a double transgenic high-level of fluorescence in Emx1-Cre: tdTomato slices. (F) Confocal
mouse obtained by crossing the Emx1-Cre mouse line (Guo et al., 2000) to a microscopy of tdTomato-expressing neurons (presumptive inhibitory neurons)
Rosa-CAG-LSL -tdTomato Cre reporter line (Madisen et al., 2010). (A) and (B) under higher magnification. Note the morphological differences of
are lower power images of a living cortical slice in bright field and tdTomato-expressing cells in (C) and (F). (G–I) AlstR-expressing neurons,
epi-fluorescent mode. (C) Confocal microscopy of tdTomato-expressing resulting from crossing of the Emx1-Cre mouse line to the AlstR mouse
neurons (presumptive excitatory neurons) under higher magnification. line, are not immunopositive for GABA. In (G), the AlstR-expressing neurons
The white arrows point to the dark areas which may represent the also express GFP (H) is the same field of view of the slice with GABA
.
somata of inhibitory cortical neurons. (D–F) Data images from S1 cortex immunostaining. (I) The overlay of the (G) and (H), with the arrowheads
of a double transgenic mouse by crossing the Dlx5/6–cre mouse line pointing to the GFP-AlstR-expressing neurons that are not GABA
(Monory et al., 2006) to the tdTomato Cre reporter line. The camera immunopositive.
current injections in Emx1-Cre: AlstR mouse slices. Similar AlstR-expressing (AlstR+) neurons (Figures 2B,D), and inhib-
to previous findings (Lechner et al., 2002; Wehr et al., ited action potential firing by the decreased spike numbers
2009), bath application of AL hyperpolarized resting membrane elicited across a range of depolarizing current injections
potentials, markedly reduced membrane input resistance of (Figures 2B,C). Control neurons without AlstR expression were
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5. Ikrar et al. AlstR-mediated regulation of cortical excitability
FIGURE 2 | Excitatory neuronal inactivation through the AlstR system. neurons (N = 6 cells) in the presence of AL, respectively. (E) Illustration
(A) One recorded excitatory neuron expressing GFP and allatostatin of the use of laser scanning photostimulation to assess evoked neuronal
receptors in a Emx1-Cre: AlstR cortical slice. AlstR-expressing neurons were excitability with the slice image superimposed with photostimulation
targeted based upon their GFP fluorescence (see the arrowheads), and sites (8 × 8 sites, cyan ∗ , 50 µm2 apart). The small red circle indicates
identified post hoc as pyramidal cells based upon their morphology revealed the recorded cell location. (F) Group data (N = 5 cells) of AlstR-mediated
by biocytin staining. (B) The AL application hyperpolarized the cell’s resting suppression of photostimulation-evoked excitability measured by the
membrane potential, reduced the cell’s membrane input resistance, and number of spiking locations. (G–I) Photostimulation-evoked activity
suppressed action potential firing by intrasomatic current injections. rmp: of the recorded neuron in (E) during control, AL application, and
resting membrane potential. The average hyperpolarization of excitatory washout, respectively. The AL application totally abolished
neuronal resting membrane potentials was −2.45 ± 0.78 mV (N = 6 cells). photostimulation-evoked spiking activity. In (D) and (F), ∗ , ∗∗ indicate
(C,D) Group data of significantly decreased intrinsic membrane statistical significance between control and AL application of p < 0.05 and
excitability and input resistance in AlstR-expressing (AlstR+) excitatory <0.005, respectively.
not affected (N = 10). Then we further characterized AlstR- we recorded from individual neurons and photostimulation
mediated inhibition of evoked excitability by laser scanning was applied across multiple sites. Our data established that
photostimulation. Compared to the examination of intrinsic the AlstR-mediated inactivation was sufficiently strong and
membrane excitability through current injections, the measure- it essentially abolished the photostimulation-evoked spiking
ment of photostimulation-evoked neuronal excitability via gluta- activity of the recorded AlstR-expressing excitatory neurons
mate uncaging is more physiologically meaningful as the evoked (Figures 2F–I).
excitability depends on the structure and membrane character- As for inhibitory cortical neurons, we showed that the
istics of the recorded neurons, and reflects the combination of AlstR system produced robust inhibition on major subtypes
intrinsic neuronal excitability and local synaptic input. Caged of inhibitory cortical neurons through examinations of AlstR-
glutamate was present in the bath solution, and only turned expressing inhibitory neurons in Dlx5/6-Cre: AlstR mouse slices.
active through focal UV photolysis. As shown in Figure 2E, The electrophysiological phenotype of the inhibitory neurons
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6. Ikrar et al. AlstR-mediated regulation of cortical excitability
examined was fast-spiking, adapting-spiking, low-threshold spik- AlstR-MEDIATED EFFECTS ON NEURONAL POPULATION ACTIVITY
ing, or late-spiking; the cells had morphologies resembling basket After the examination of AlstR-mediated effects at the level
cells, Martinotti cells, bitufted/bipolar cells, or neurogliaform of individual neurons, we went further to characterize the
cells. Compared to excitatory neurons, bath application of AL had AlstR-mediated inhibition on neuronal population activity with
a much larger hyperpolarizing effect on the resting membrane our newly developed functional imaging technique. This is
potentials of AlstR-expressing inhibitory neurons (Figures 3A,B). important because cortical circuits ultimately operate at the neu-
AL application consistently inhibited all the inhibitory neu- ronal population level. Optical imaging with fast VSD was used
rons tested by decreasing their intrinsic excitability to current to examine photostimulation-evoked population activity and to
injections (Figure 3C) and photostimulation-evoked excitability evaluate AlstR-mediated inhibition on cortical network activ-
(Figures 3D–L). It should be noted that the level of AlstR- ity. The VSD imaging can monitor neuronal activity in a large
mediated inhibition varied with cell-types. For example, the spik- area simultaneously (Xu et al., 2010; Xu, 2011), with changes in
ing activity of AlstR+, fast-spiking cells tended to be suppressed optical absorption closely correlating with neuronal membrane
completely while AlstR+, low-threshold spiking cells had remain- depolarization. The brain slices were stained with VSD and per-
ing spikes in the presence of AL (Figures 3E–L). However, the fused with ACSF containing caged glutamate. Then local cortical
overall effect of inhibition was substantial across interneuronal regions were stimulated with laser photostimulation, and circuit
subtypes (Figures 3C,D). activation and output directly visualized by fast VSD imaging. For
FIGURE 3 | Robust AlstR-mediated inhibition of diverse subtypes of (N = 9) of AlstR-mediated suppression of photostimulation-evoked
inhibitory neurons. (A) shows one recorded inhibitory neuron expressing excitability measured by the number of spiking locations. (E–H) and (I–L)
GFP and allatostatin receptors in the Dlx5/6-Cre: AlstR cortical slice. This cell shows photostimulation-evoked activity of the recorded low-threshold-spiking
exhibited a low-threshold-spiking phenotype with bitufted morphology. (B) neuron and a fast-spiking inhibitory neuron during control, AL application, and
and (C) shows the AL application caused a significant decrease in intrinsic washout, respectively. The AL application substantially suppressed
neuronal excitability of the examined AlstR-expressing (AlstR+) inhibitory photostimulation-evoked spiking activity of AlstR-expressing inhibitory
neurons (N = 12). The average hyperpolarization of inhibitory neuronal resting neurons. In (D), ∗∗ indicates statistical significance of p < 0.005 between
membrane potentials was −11.4 ± 0.94 mV. (D) shows the group data control and AL application.
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7. Ikrar et al. AlstR-mediated regulation of cortical excitability
the purpose of visual display, in Figure 4 and subsequent figures, cortical neurons expressed AlstRs in these slices, the AlstR-
the color scale codes the strength of excitatory activity (reflect- mediated inhibition of excitatory population activity was quite
ing membrane potential depolarization of neuronal ensembles) effective. The AL effect was reversible with the peptide washout
with warmer colors indicating greater excitation. In addition, the (Figures 4C1,C2). In comparison, AL application did not have
time course of VSD signal (in the percent change of pixel intensity effects on photostimulation-evoked population activity in the
[ I/I%]) from the specified region of interest (ROI) is shown cortical slices without AlstR expression (N = 4). The reduction
with the image data. of VSD activation with the AL application for Emx1-Cre:
AlstR-mediated inhibition of excitatory neuronal popula- AlstR slices, compared to control, is summarized in Figure 6A.
tion activity was found to be effective across different cortical On average, the total activation size during AL application
sites (Figure 4). In Emx1-Cre: AlstR mouse slices, under con- was 18% ± 2.0% of the control condition; the total response
trol conditions, photostimulation in local cortical regions initi- amplitude during AL application was 15.8% ± 1.8% relative
ated excitation around 5 ms after laser exposure, which quickly to control.
propagated to functionally connected cortical regions within Conversely, we found that AlstR-mediated inactivation of
50 ms in a columnar manner. Stimulation in the middle cor- GABAergic neurons augmented excitatory population activity.
tical layer (layer 4 or layer 5a) caused excitatory activity to Photostimulation-evoked response in the presence of AL
spread vertically to layers 2/3, 5, and 6 (Figures 4A1,A2 and was dramatically increased in Dlx5/6-Cre: AlstR mouse slices
5A1,A2). Although photostimulation-evoked excitatory activity (Figures 5A1,A2 and B1,B2), and the effect was reversed with
occurred in the presence of the AL peptide, evoked responses the AL washout (Figures 5C1,C2). In agreement with the previ-
of the AlstR-expressing brain slice was clearly reduced and con- ous finding that S1 barrels are functionally independent cortical
strained (Figures 4B1,B2). Given that about half of excitatory columns (Laaris et al., 2000; Petersen and Sakmann, 2001), in
FIGURE 4 | AlstR-mediated inhibition of excitatory neuronal population image frame in (A1). The arrowhead point the downward signal deflection
activity in an Emx1-Cre: AlstR mouse slice. (A1) Time series data of VSD caused by the laser excitation artifact. (B1) Time series of network activity in
imaging of photostimulation-evoked activity in S1 cortex in normal ACSF The . the same slice in the presence of the AL peptide, with excitatory response
color scale codes VSD signal amplitude expressed as standard deviation (SD) being clearly reduced compared to control. (B2) The time course of VSD
multiples above the mean baseline. The site of photostimulation between signal (in the percent change of pixel intensity [ I/I%]) from the ROI
layers 4 and 5a can be identified by the laser excitation artifact (purple) in the indicated by the small rectangle in the first image frame in (B1). (C1) Time
first frame, which is also indicated by the white arrowhead. Color-coded series of network activity in the same slice after the washout of the AL
excitatory activity is superimposed on the background slice image. (A2) The peptide. (C2) The time course of VSD signal (in the percent change of pixel
time course of VSD signal (in the percent change of pixel intensity [ I/I%]) intensity [ I/I%]) from the ROI indicated by the small rectangle in the first
from the region of interest (ROI) indicated by the small rectangle in the first image frame in (C1).
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8. Ikrar et al. AlstR-mediated regulation of cortical excitability
normal ACSF, the lateral extent of photostimulation response in across the whole slice lasting for about 200–300 ms per event
S1 layer 4 appeared to be limited to the cortical column defined (Figures 7A1–A4 and B1,B2). After stable occurrences of the
structurally by the barrel (Figure 5A1). Yet, the importance of seizure activity across seven imaging sessions (126 seconds per
inhibitory neuronal regulation of cortical excitatory informa- session), AL peptide was co-applied with bicuculline and seizure
tion flow is evident, as inhibition of GABAergic interneurons occurrences continued to be monitored by VSD imaging. As illus-
via the AlstR system caused the spread of excitation laterally in trated in Figures 7C,D, the tendency of reduced seizure occur-
layer 4 as well as in wide regions of upper and deeper layers rences was clearly visible during the AL application. Note that
(Figure 5B1). Across the slices, the increase of VSD activation the average durations or peak amplitudes of individual events
in the AL presence, compared to control, was 500.5% ± 171% during control and the AL infusion did not differ. Finally, the
and 621.2% ± 272.6% for the total activation size and response removal of AL from bath perfusion increased seizure occurrences,
amplitude, respectively (Figure 6B). which could get back to the normal control level. For the Emx1-
Cre: AlstR mouse slices tested, the average number of seizure
THE USE OF AlstR SYSTEM FOR SUPPRESSING SEIZURE ACTIVITY occurrences per minute was 1.09 ± 0.14, 0.41 ± 0.12, 0.68 ± 0.38
Given that targeted expression of AlstRs in excitatory neurons can for bicuculline-containing ACSF, co-application of bicuculline
strongly reduce cortical excitability with the ligand present, we and AL, and bicuculline-containing ACSF again, respectively. The
were interested in investigating whether the AlstR system could co-application of AL with bicuculline reduced the occurrence
be used for controlling seizure activity that is characterized by of bicuculline-induced seizure activity significantly (Figure 7E).
abnormal, excessive, or synchronous excitatory neuronal activity Thus, these data suggest that the AlstR system can be used to
in the brain. We performed the experiments in single Emx1- effectively suppress seizure activity.
cre: AlstR slices sequentially. First, bath application of bicu-
culline induced spontaneous seizure-like activity in the slices. DISCUSSION
The epileptiform activity detected by VSD imaging, occurred In this study, we used a Cre-dependent transgenic mouse
in a small region and evolved into large propagating activity approach to effectively express AlstRs to specific types of cortical
FIGURE 5 | AlstR-mediated inhibition of GABAergic neurons increases respectively. The site of photostimulation is in cortical layer 4. (A2–C2) The
excitatory population activity in a Dlx5/6-Cre: AlstR mouse slice. time course of VSD signal (in the percent change of pixel intensity [ I/I%])
(A1–C1) Time series data of VSD imaging of photostimulation-evoked activity from the ROI indicated by the small rectangle in the first image frame in
in S1 cortex, in normal ACSF in the presence and after washout of AL,
, (A1–C1), respectively.
Frontiers in Neural Circuits www.frontiersin.org January 2012 | Volume 6 | Article 2 | 8
9. Ikrar et al. AlstR-mediated regulation of cortical excitability
or RASSLs) (Conklin et al., 2008; Alexander et al., 2009). For
example, muscarinic acetylcholine receptors are engineered to
generate a family of GPCRs that are activated solely by a pharma-
cologically inert drug-like compound (clozapine-N-oxide). The
Gi-coupled designer receptor (hM4D) demonstrates its ability
to induce membrane hyperpolarization and neuronal silencing
in vitro and in vivo (Armbruster et al., 2007; Ferguson et al.,
2011). The designed drug ligands can be administered in the
periphery and cross the blood-brain barrier (BBB) to access
receptors in deep brain structures and/or widely distributed neu-
ronal populations. However, continued improvements are needed
for the use of RASSLs, as the engineered receptors may be acti-
vated by non-selective, pharmacologically active small molecules.
Furthermore, in some tissues RASSLs exhibit high levels of con-
stitutive signaling in the absence of designed small-molecule
agonists (Alexander et al., 2009), while the AlstR does not display
evidence of basal signaling. Although the AL peptide is unlikely
to cross the BBB in the current form and needs to be directly
infused into brain tissue, future translational studies using the AL
system will necessitate that AlstR ligands be designed with a capa-
bility of crossing the BBB. Systemic administration would likely
result in some loss of temporal control, but it may still be suffi-
cient to suppress excitatory activity of AlstR-expressing neurons.
Please note that while the techniques for making BBB-permeable
peptide analogs are established (Bickel et al., 2001; Egleton and
Davis, 2005), the AL analogs for realistic human therapy will have
to be developed toward this end.
A major focus of this work was to examine if the AlstR sys-
tem can regulate the activity of diverse types of inhibitory cortical
FIGURE 6 | Quantification of photostimulation-evoked responses in interneurons. Overall, inhibitory neurons can be inactivated, but
Emx1-Cre: AlstR and Dlx5/6-Cre: AlstR mouse slices. (A) Bar graph compared to excitatory neurons, the degree of AlstR-mediated
indicating the average activation size and response amplitude in the inhibition depended on interneuronal subtypes, which should
presence of AL for Emx1-Cre: AlstR slices (N = 4), relative to normal ACSF
control. (B) Bar graph indicating the average activation size and response
be related to the factors including intrinsic neuronal excitabil-
amplitude in the presence of AL for Dlx5/6-Cre: AlstR slices (N = 4), ity, AlstR expression levels, and endogenous GIRK densities.
relative to normal ACSF control. Given the availability of new interneuron-specific Cre driver lines
developed by a NIH Neuroscience Blueprint Project in the lab-
oratory of Josh Huang (available from the Jackson Laboratory)
neurons. The experiments with single-cell electrophysiology, laser (Taniguchi et al., 2011), AlstR expression can be targeted to
scanning photostimulation, and VSD imaging established that specific subsets of inhibitory cortical neurons for selectively
the AlstR-mediated inactivation of both excitatory and inhibitory suppressing their activity for future functional and behavioral
neurons was specific and robust at single-cell and population studies.
levels. Our data also indicate that the AlstR system has good In addition to the transgenic mouse approach, viral vectors can
potential to be used as a molecular anticonvulsant for fast seizure also be used for AlstR delivery in defined cell-types. Genetically
control via cell-type specific expression. modified adeno-associated viruses (AAV) and lentiviruses have
As further demonstrated in this study, the AlstR/AL system has been used for gene therapy in humans, enabling correcting cer-
the required merits and applicable features as a potent molecular tain genetic disorders in clinical trials (Davidson and Breakefield,
tool for regulation of cortical excitability. Optical inactivation of 2003; Kootstra and Verma, 2003). The viral vectors can yield sta-
neurons can be effectively achieved by using the halorhodopsin ble gene expression for months or years without apparent toxicity.
or Arch system, which activates chloride or proton pumps Compared with the transgenic approach, major advantages of
and offers high temporal precision (Han and Boyden, 2007; Chow viral transfection include restricted expression in defined cortical
et al., 2010). Although no ligand is needed, prolonged optical regions, stronger AlstR expression levels with high-tier viruses,
silencing over the course of several minutes causes photo-toxicity and the application to other species that transgenic approaches
and neuronal damage. In addition, optical illumination may are impractical.
interfere with functional imaging studies. One existing approach The AlstR approach represents an important advance for
similar to the use of the non-native AlstR system is to genetically genetic manipulation of neuronal activity and can be valuable
engineer G-protein-coupled receptors (GPCRs) of mammalian for many basic applications [see further discussion in (Callaway,
neurons (e.g., Receptors Activated Solely by Synthetic Ligands, 2005; Tan et al., 2006)]. We believe that the AlstR system can also
Frontiers in Neural Circuits www.frontiersin.org January 2012 | Volume 6 | Article 2 | 9
10. Ikrar et al. AlstR-mediated regulation of cortical excitability
FIGURE 7 | The AlstR system suppresses the occurrence of in the presence of bicuculline, bicuculline and AL, and bicuculline again for
bicuculline-induced spontaneous seizure-like activity. (A1–A4) Time two different example slices. Each imaging session consisted of nine
series data of VSD imaging of bicuculline-induced spontaneous seizure-like consecutive trials of sampling 8.8 seconds per trial (with a 14 seconds
activity in a Emx1-Cre: AlstR mouse slice. Specific time from the estimated inter-trial interval); the number of seizure events was measured for each
seizure onset is labeled on the image. The color scale codes VSD signal session. A interval of 15–20 minutes was allowed for the AL infusion and
amplitude expressed as SD multiples above the mean baseline. (B1, B2) washout before the next condition continued. (E) The data summary (N = 5
The time course of VSD signal (in the percent change of pixel intensity slices) showing that the co-application of AL with bicuculline (BIC) reduced
[ I/I%]) from the two ROIs (1 and 2) marked in A1 is plotted, respectively. the occurrence of bicuculline-induced seizure activity in Emx1-Cre: AlstR
Note that ROI 1 is close to the seizure initiation site. (C, D) The plots of mouse slices. ∗ Indicates statistical significance of p < 0.01 between
seizure occurrences per imaging session (126 seconds per session) BIC and BIC + AL.
Frontiers in Neural Circuits www.frontiersin.org January 2012 | Volume 6 | Article 2 | 10
11. Ikrar et al. AlstR-mediated regulation of cortical excitability
be potentially used in translational applications, for example, AlstR system, this genetic inactivation system can be potentially
in seizure control and epilepsy treatment. Despite the progress developed as a molecular anticonvulsant with few side effects.
in antiepileptic drug development and surgical management, in
about 30–40% of cases epileptic seizures do not respond well to ACKNOWLEDGMENTS
the conventional therapies. We should capitalize on the develop- This work was funded by the US National Institutes of Health
ment and use of new molecular and genetic tools to counteract grants DA023700 and DA023700S1 to Xiangmin Xu. We thank
seizure activity. Based upon the specificity and effectiveness of the Nicholas Olivas for helpful comments on the manuscript.
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