A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Hovis, Kenneth R.; Padmanabhan, Krishnan; Urban, Nathaniel N.

doi:10.1016/j.jneumeth.2010.05.017

Cited by 19 publications

(22 citation statements)

References 41 publications

(48 reference statements)

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“…The tip of the electrode was placed in the center of a GFP-positive glomerulus. The electroporation protocol consisted of 1200 pulses, (25msec duration, 1–2μA), at 2 Hz were given by an SIU box controlled by TTL pulses from the ITC-18 data acquisition board as described in (Hovis et al, 2010). This typically resulted in fluorescent dye loading of 6–10 mitral cell bodies and their dendrites per slice.…”

Section: Methodsmentioning

confidence: 99%

Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb

Hovis¹,

Ramnath²,

Dahlen³

et al. 2012

Journal of Neuroscience

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The mammalian accessory olfactory system is specialized for the detection of chemicals that identify kin and conspecifics. Vomeronasal sensory neurons (VSNs), residing in the vomeronasal organ, project axons to the accessory olfactory bulb (AOB) where they form synapses with principle neurons, known as mitral cells. The organization of this projection is quite precise and is believed to be essential for appropriate function of this system. However, how this precise connectivity is established is unknown. We show here that in mice the vomeronasal duct is open at birth, allowing external chemical stimuli access to sensory neurons, and that these sensory neurons are capable of releasing neurotransmitter to downstream neurons as early as the first post-natal day. Using major histocompatibility complex class I (MHC-1) peptides to activate a selective subset of VSNs during the first few post-natal days of development, we show that increased activity results in exuberant VSN axonal projections and a delay in axonal coalescence into well-defined glomeruli in the AOB. Finally, we show that mitral cell dendritic refinement occurs just after the coalescence of pre-synaptic axons. Such a mechanism may allow the formation of precise connectivity with specific glomeruli that receive input from sensory neurons expressing the same receptor type.

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Section: Methodsmentioning

confidence: 99%

Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb

Hovis¹,

Ramnath²,

Dahlen³

et al. 2012

Journal of Neuroscience

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“…In addition the demonstrated technique can also be used in combination with calcium sensitive dextrans or injected membrane-permeable calcium dyes to get functional information about the labeled neuron and/or the connected circuitry 7,19 . The availability of a wide range of fluorophores coupled to dextrans permits labeling of multiple individual cells or populations with different colors.…”

Section: Representative Resultsmentioning

confidence: 99%

“…Electroporation is a well established method to introduce charged macromolecules, like dextran-coupled dyes and DNA, into cells 6,7 . The cell membrane is permeabilized by application of short voltage pulses and the molecules are electrophoretically delivered into the cytosol 8 .…”

Section: Introductionmentioning

confidence: 99%

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology <em>In Vivo</em>

Hassenklöver

Manzini

2014

JoVE

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Citation: Hassenklöver, T., Manzini, I. The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo. J. Vis. Exp. (92), e52143, doi:10.3791/52143 (2014). AbstractThe olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

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“…In addition, single-cell electroporation has been used to load neurons with dyes and indicators. Loading can be done within minutes, and neurons can be studied for a few hours after electroporation (40)(41)(42). What then are the advantages of our technique, which is based on a very low sampling density?…”

Section: Discussionmentioning

confidence: 99%

Time-lapse electrical recordings of single neurons from the mouse neocortex

Cohen

Koffman

Meiri

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The ability of the brain to adapt to environmental demands implies that neurons can change throughout life. The extent to which single neurons actually change remains largely unstudied, however. To evaluate how functional properties of single neurons change over time, we devised a way to perform in vivo time-lapse electrophysiological recordings from the exact same neuron. We monitored the contralateral and ipsilateral sensory-evoked spiking activity of individual L2/3 neurons from the somatosensory cortex of mice. At the end of the first recording session, we electroporated the neuron with a DNA plasmid to drive GFP expression. Then, 2 wk later, we visually guided a recording electrode in vivo to the GFP-expressing neuron for the second time. We found that contralateral and ipsilateral evoked responses (i.e., probability to respond, latency, and preference), and spontaneous activity of individual L2/3 pyramidal neurons are stable under control conditions, but that this stability could be rapidly disrupted. Contralateral whisker deprivation induced robust changes in sensoryevoked response profiles of single neurons. Our experiments provide a framework for studying the stability and plasticity of single neurons over long time scales using electrophysiology.electroporation | two-photon imaging I t is well established that the morphology and response properties of neurons are highly dynamic during development. Immature cortical neurons are particularly amenable to change during specific stages of development, when spontaneous activity and sensory experience shape their morphology and physiology (1-4). Once formed and stabilized, neurons are thought to maintain some aspects of this plasticity during adulthood, albeit at a lower level (5-7); however, the extent to which single-neuron physiology changes or remains stable in the adult mammalian brain is poorly documented.Limitations to the study of single-neuron plasticity stem from technical difficulties as well as from the inherent heterogeneity of single neurons in a circuit. Fine changes, like those that underlie functional plasticity, can go undetected due to data averaging and homeostatic events that may cancel out each other. As a result, plasticity is made experimentally more tractable by studying populations after gross manipulations like sensory deprivation or injury. For example, receptive fields of neurons across adult rodent cortices are known to shift in response to different paradigms of sensory input manipulation (8-14). Whether and how other forms of plasticity, such as learning and memory, mark their signature on single neurons is more difficult to deduce from gross manipulation studies. One way to overcome some of these difficulties is through experiments based on time-lapse imaging and electrophysiology (14-18). However, the ability to measure fine temporal physiological events of identifiable single neurons over long time scales remains limited, especially with electrophysiology.To establish a method to follow the electrophysiology of single neuron...

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A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Cited by 19 publications

References 41 publications

Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb

Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology <em>In Vivo</em>

Time-lapse electrical recordings of single neurons from the mouse neocortex

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