Retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo. 2013

Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
Department of Biology, Washington University in St. Louis, USA.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular(1), juxtacellular(2) or loose-patch(3) iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology(4-6). However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons(7), but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult(8-11). The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS. The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals(12). We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes(13). However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods(11, 14). Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

UI MeSH Term Description Entries
D008636 Mesencephalon The middle of the three primitive cerebral vesicles of the embryonic brain. Without further subdivision, midbrain develops into a short, constricted portion connecting the PONS and the DIENCEPHALON. Midbrain contains two major parts, the dorsal TECTUM MESENCEPHALI and the ventral TEGMENTUM MESENCEPHALI, housing components of auditory, visual, and other sensorimoter systems. Midbrain,Mesencephalons,Midbrains
D008856 Microscopy, Fluorescence Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye. Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D009474 Neurons The basic cellular units of nervous tissue. Each neuron consists of a body, an axon, and dendrites. Their purpose is to receive, conduct, and transmit impulses in the NERVOUS SYSTEM. Nerve Cells,Cell, Nerve,Cells, Nerve,Nerve Cell,Neuron
D004555 Electric Fish Fishes which generate an electric discharge. The voltage of the discharge varies from weak to strong in various groups of fish. The ELECTRIC ORGAN and electroplax are of prime interest in this group. They occur in more than one family. Mormyrid,Mormyridae,Elephantfish,Elephantfishes,Fish, Electric,Mormyrids
D004594 Electrophysiology The study of the generation and behavior of electrical charges in living organisms particularly the nervous system and the effects of electricity on living organisms.
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001369 Axons Nerve fibers that are capable of rapidly conducting impulses away from the neuron cell body. Axon

Related Publications

Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
April 1984, Brain research bulletin,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
January 2011, Frontiers in neural circuits,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
November 2014, Journal of visualized experiments : JoVE,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
May 1984, Neuroscience letters,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
September 2019, Journal of neuroscience methods,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
November 2002, Genesis (New York, N.Y. : 2000),
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
March 2023, STAR protocols,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
October 2023, Nature protocols,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
February 1990, Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology,
Ariel M Lyons-Warren, and Tsunehiko Kohashi, and Steven Mennerick, and Bruce A Carlson
July 1973, IEEE transactions on bio-medical engineering,
Copied contents to your clipboard!