How To Put Together A Setup For In Vivo Electrophysiology Recordings In Anhestetized Animals

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From Art to Engineering? The Rising of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging

• Oliver Griesbeck

From Fine art to Engineering science? The Ascent of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging

• David Kleinfeld,
• Oliver Griesbeck

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• Published: October xi, 2005
• https://doi.org/10.1371/journal.pbio.0030355

Figures

Commendation: Kleinfeld D, Griesbeck O (2005) From Fine art to Technology? The Rise of In Vivo Mammalian Electrophysiology via Genetically Targeted Labeling and Nonlinear Imaging. PLoS Biol 3(10): e355. https://doi.org/x.1371/journal.pbio.0030355

Published: Oct eleven, 2005

Copyright: © 2005 Kleinfeld and Griesbeck. This is an open up-admission article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abbreviations: BAC, bacterial artificial chromosome; TPLSM, two-photon light amplification by stimulated emission of radiation scanning microscopy; XFP, fluorescent protein

For close to half a century, neurophysiologists have been able to tape electrical signals from the millions of individual neurons that compose fifty-fifty the smallest mammalian brain. Despite this long history, which has led to meaning strides toward agreement how neuronal activity translates into brain function, much of the way electrophysiological data is gathered is more of an art form than a science. Reproducibility, a cornerstone of scientific progress, hasn't always been forthcoming when recording from private neurons in the brain, largely because of the improbability or uncertainty that different investigators make their measurements from the same neurons or even the same select subpopulations of neurons. How does one put together the information well-nigh activity in single neurons that are recorded past different investigators in dissimilar ways? Farther nevertheless, how does i combine this information with cognition of the underlying circuitry to make sense of the firing patterns that underlie normal brain function? Progress volition come largely from the ability to reproducibly tape voltages, equally well every bit other variables that ascertain physiological function, from identified neuronal prison cell types. The ability to record from the same subpopulation of cells on a routine footing is the singular means to validate measurements across different laboratories and move electrophysiology beyond its electric current, largely anecdotal status.

Past definition, measurements from identified neuronal cell types depend on a ways to visualize the cells in question. For uncomplicated neuronal circuits in invertebrates, in which the function of a cell is often well correlated with its physical location inside a ganglion, unproblematic light microscopy imaging is adequate to uniquely identify a neuron. Similarly, in a mammalian brain slice, gross architectonic features tin can be discerned from the visual texture of the tissue, while individual neuronal boundaries may exist identified with optical techniques that minimize the interfering furnishings of scattered lite. For the case of recording from brains in living mammals, the technical challenges that must be surmounted to record from identified cells are far greater. Antidromic activation of projection neurons, a heroic approach, provides selectivity in some instances [1]. Yet, at nowadays, much of in vivo recording is performed blind, in the sense that jail cell morphology and phenotype are confirmed only from post hoc histology.

What advances prevarication alee to accelerate the qualitative nature of mammalian in vivo recording? In detail, can electrophysiology approach the level of precision and reproducibility that ane associates, for example, with biochemistry or molecular biology? The confluence of 3 avenues of technical advance—one in imaging, one in labeling, and one in behavioral training—propose that in vivo electrical and optical recording from identified neuronal phenotypes in the central nervous organization of awake behaving mice should soon be a mutual reality. That's the practiced news. Simply before we become likewise enthusiastic, information technology is important to realize that the major stumbling block in electrophysiology has yet to exist solved. Electrophysiology remains a labor-intensive art class. Data gathering involves many manually controlled processes that require an extended and constant level of vigilance. This is to be assorted with molecular biology, where standard tools and high levels of automation brand conquering relatively cheap in terms of time and expense, and thus shift the focus to conceptual synthesis. Fourth dimension will tell if the technical advances described below advance not just the reliability of electrophysiology simply further serve as a tipping point for its transition from an art to an engineering process.

Labeling of Specific Neuronal Phenotypes—Mus musculus as a "Uncomplicated" Nervous Organization

The age of transgenic animals and fluorescence labeling drives forward with ever greater carelessness. Neurobiology is one of the great beneficiaries of the evolution of a rainbow spectrum of fluorescent proteins (XFPs) [2–4], in the sense that transgenic expression of these proteins reveals the iii-dimensional outlines of individual living neurons with minimal cytosolic perturbation. For the electrophysiologist, this portends the engineering science of mice in which divers subclasses of neurons express a fluorescence label. While this technique in mammals is not quite at the level of precision it has in invertebrates, where one tin often identify individual neurons in vivo, such mice offer the possibility of allowing researchers to return to the same phenotypically defined neurons inside a given brain region. A demonstration of the power of this arroyo is a well appreciated series of transgenic mice labeled via nonhomologous incorporation of an expression cassette (a short sequence of Dna) that codes for the pan-neuronally expressed Thy-i promoter, a selected XFP, and ribosome bounden [v]. The type of expression varies significantly from line to line as a issue of strong positional and context sensitivity of the Thy-1 expression cassette when integrated into the genome. The resultant mosaic labeling is valuable for sure studies, but more importantly, information technology serves to illustrate that considerable "artistic" elements are currently at work in labeling the encephalon.

What are the essential difficulties in reproducibility and predictability in the generation of mice with labeled neurons? The utilise of expression cassettes in mammals suffers from the difficulty of identifying fundamental regulatory elements, such as enhancers or silencers, that are necessary for the right expression of a transgene [6]. A related source of variability is that expression of the label is influenced by the DNA sequences that flank the inserted DNA, yet the site of integration into the genome differs between transgenic animals. These difficulties are diminished through the use of bacterial artificial chromosomes (BACs) [vii,viii], which incorporate the entire transcription unit and large pieces of sequence 5′ and 3′ of it (Figure 1A). Although this approach is not perfect and can yet miss out on important regulatory elements in some cases (Figure 1B), in the ideal example the BAC includes all necessary elements to limited a reporter gene in the correct fashion.

Effigy 1. Schematic for the Production of a Modified BAC for the Targeted Expression of an XFP or an XFP-Based Reporter in Mice

(A) A library of suitable BAC clones is scanned using bioinformatics and an appropriate clone, encoding a suitable cell-type-specific transcriptional unit with ample flanking regions, is selected. Note that but a few of the many possible enhancer and silencer regions are drawn. An exon that lies downstream of the ATC starting time sequence is selected to be replaced by the XFP/reporter sequence by homologous recombination (exon 2 in this case), and a shuttle vector that codes for the label together with flanking regions around the exon ("a" and "b") is synthetic. The enzyme RecA is used to interchange the sequence for the exon and the label to course a modified BAC clone that codes for the label. The modified clone is injected into a mouse oocyte, where the dominant incorporation into the host Deoxyribonucleic acid occurs through nonhomologous recombination.

(B) Many factors influence the phenotype of a given transgenic mouse, and thus the same clone may result in a number of lines with slightly different properties. The insert shows the XFP expression pattern for a line based on a BAC clone that contains the transcriptional unit of a glycine transporter.

(Paradigm: Jean-Marc Fritschy and Hanns-Ulrich Zeilhofer)

https://doi.org/10.1371/periodical.pbio.0030355.g001

Methods to incorporate reporter genes into BAC constructs are relatively straightforward (Figure 1A) and have led to an virtually industrial-scale effort to generate and characterize a collection of mice with defined labeled neurons for further anatomical and physiological assay [9]. Recent examples of transgenic mouse engineering based on BAC clones demonstrate the accurate labeling of neurons containing the neurotransmitter glycine in the spinal cord, brainstem, and cerebellum (Figure 1B) [10], and the labeling of neurons expressing both parvalbumin and GABA throughout neocortex [xi]. Other examples used clones with the factor for glutamic acrid dehydrogenase (GAD-27) to select for all GABAergic neurons, but observed expression in but the parvalbumin-positive subpopulation [12,13]. It is to be expected that the precision of molecular biological science volition further evolve to produce mice with ever increasing specificity of subtype labeling.

In Vivo Visually Guided Recording of Labeled Cortical Neurons—Laser Jocks Turned Neuroscientists

Making mice with fluorescent neurons is just the first step; the 2d requires the means to visualize the axons and dendrites of these neurons, which tin can be less than a micrometer in thickness. In vivo ii-photon laser scanning microscopy (TPLSM) [14,fifteen] provides a unique means to prototype fluorescently labeled neurons that lie beneath the surface of the brain [16]. When used in conjunction with transgenic mice that are labeled past the expression of a fluorescent protein, TPLSM provides the necessary visualization to target a fine glass electrode to the membrane surface of ane'due south neuron of pick [17] (Figure 2). TPLSM can paradigm deep into scattering encephalon tissue, in backlog of 500 μm under normal conditions [18] and down to i,000 μm nether special circumstances [19]. Although technical challenges—such as increasing the rate at which images are scanned and compensating for optical aberrations—1 can, in principle, image and thus target neurons throughout almost the entire depth of mouse cortex.

Figure 2. Targeted Electric Recording of Transgenically Labeled Inhibitory Interneurons in Mouse Cortex

(A) The 2-photon laser scanning microscope is shown schematically. The disquisitional features are the use of split fluorophores, one for the label (GFP in this instance) and some other to mark the intracellular fluid of electrode (Alexa in this example) that have overlapping excitation spectra and different emission spectra (meet [B]). The intracellular voltage shows a trace obtained under whole-cell patch of the response to vibrissa stimulation. Alexa, Alexa 594 dye; fs laser, titanium:sapphire fashion-locked light amplification by stimulated emission of radiation with 100 fs output pulse width; GFP, greenish fluorescent protein; PMT, photomultiplier tube.

(B and C) Emission spectra and fluorescent images from the GFP and Alexa channels. Confirmation of whole-jail cell patch is achieved past injecting Alexa into the GFP-filled prison cell, as illustrated in the overlay.

(Images: Troy Margrie)

https://doi.org/x.1371/journal.pbio.0030355.g002

Biomolecular Reporters and Drivers of Land Variables—Proteins every bit Spies and Membrane Provocateurs

Apart from their role as phenomenally good, noninvasive labels of neuronal structure, XFPs take become the footing for a series of sensors of physiological variables and events, such as membrane-potential fluctuations and intracellular messenger dynamics [20]. Genetically encoded, these sensors are generated inside cells, exercise not require cofactors, and do not leak out of cells even during prolonged studies. These sensors will do good considerably from the increasing accuracy of neuronal labeling via modified BAC clones (run into Figure 1). Indicators of synaptic release [21–23] or intracellular [Ca^two+] dynamics [24–28] might initially exist the nigh appealing. While bug, such as bespeak strength and response kinetics, accept still to be sorted out, recent work on transgenic mice that express these and other probes proved the feasibility of the arroyo (Table i). An exquisite example is the expression of the pH indicator synaptopHluorin in olfactory sensory neurons of the mouse, which allowed for the in vivo imaging of patterns of activation in the olfactory bulb after odorant stimulation [21]. To the extent that optical microscopy is able to resolve their blueprint of expression, XFP-based molecular probes offering a means to read out activity non just from a few but ideally from whole populations of identified neurons.

The complement to optical-based probes of neuronal land variables is optical-based perturbation mediated past intrinsic chromophores. The ability to perturb the land of neuronal activation plays two essential roles in systems identification. The first is to make up one's mind the effect of a depolarizing perturbation in neighboring as well as downstream cells. The challenge has been met, in non-mammalian systems, through the use of cloned photoreceptor complexes [29] and photolabile organic cages that release agonists of excitatory neurotransmission onto cloned channels that are expressed in defined phenotypes [30]. The second role is the inactivation of neuronal pathways every bit a means to open feedback loops and determine the management of indicate flow. For example, a modified M⁺ channel in which photoisomerization drives the reversible transition between closed and conducting states has been demonstrated in vitro [31]. I clear challenge is the functional incorporation of these and related photograph-activated agents in defined mammalian cells.

Targeted Recording from the Awake Rodent—Molecular Biology Meets Consciousness

Immobilization is by and large necessary for virtually forms of recording. Needless to say, immobilization achieved by anesthesia blatantly disrupts neural function, and the whole notion of attentive-based activation besides as motor output per se is lost. This problem is avoided with primates through the use of "caput-fixed" animals that are trained to sit quietly while they perceive the world through arrays of projectors and tactile pads. The aforementioned form of constraint can be brought to studies with rodents through the utilize of head-fixed preparations [32], which has proved to be of disquisitional importance for the study of behavioral [33] and electrophysiological [34] aspects of whisking. This strategy has as well provided a ways to record both optically and electrically from private neurons that are labeled with organic [Ca^two+] indicators (Figure 3A; J. Waters and F. Helmchen, unpublished data), and information technology is anticipated that recording and perturbation from cells labeled via viral transfection will soon be forthcoming [35]. The nigh-term challenge is to record from awake head-fixed mice.

Figure three. Prospects for Recording from Awake but Caput-Restrained Animals

(A) Photograph of a trained rat that is awake and head-restrained, ready for imaging of organic [Ca²⁺]-sensitive dyes. All aspects of the recording procedure demonstrated in primates are expected hold for mice besides. (Image: Jack Waters)

(B) Photograph and set-up of visual virtual reality for rodents. In this instance, the rat is trunk-fixed, and tin rotate on an axis, but is not head-fixed. The visual earth of the beast is controlled past projected images, and reward is administered through a food tube. (Images: Hansjuergen Dahmen)

https://doi.org/10.1371/journal.pbio.0030355.g003

A final issue concerns the extent of behavior that may be expected with head-fixed animals, specially every bit a large block of research concerns spatial tasks and hippocampal role. Both primate electrophysiological studies [36] and human psychophysical studies [37] have avant-garde with the utilize of virtual reality. Recently, the aforementioned level of sophistication has been brought to bear on rodent studies [38] (Effigy 3B), where body-fixed rats are constrained to walk on a near frictionless ball while they observe a virtual visual world. This advance already provides a means to record from rats when the tether, such as that for a head-mounted scanner [39], is too short for use with animals in mazes. In the best of worlds, this advance is a stepping rock to recording from head-stock-still mice as they respond to novel environments.

Putting It All Together

The tools are there to perform targeted electric and optical-based ion recording, and stimulation, of identified neuronal phenotypes in mice. Nonlinear microscopy, while all the same a tool of the aficionado, is approaching maturity [40]. The blueprint of endogenous molecular sensors of cell part, while in early days, has attained a set of heuristics and material successes (Table 1). This suggests that signaling and circuitry in the mammalian nervous organisation may exist addressed in a reliable and logical, if painstaking, way. Other contempo work, involving methods to automate histology at the synaptic [41] and cellular [42] levels, will assist place physiological measurements in the framework of detailed architectonics. The greatest challenges for in vivo electrophysiology appear to lie primarily in the areas of molecular biology and behavior. Gene expression through the use of BACs has been successfully targeted to only a few neuronal subtypes and so far, still must be pushed to all jail cell types. This highlights a need for better phenotyping of neurons, both by conventional histochemistry and by microarray analysis of gene expression, and a ameliorate understanding of the transcription factor logic that defines expression. Automatic ways for shaping animal behavior demand to be advanced [43]. Critically, while the bias that "mice cannot be trained" is pervasive, at that place has been little concerted effort to breed and train calm mice that could be the background for transgenesis. Behavioral issues bated, it is a expert bet that a mixture of genetics and optics will play a ascendant role in delimiting the algorithms of brain function.

Acknowledgments

The ideas in this essay originated from presentations and discussions at the Imaging Neurons and Neural Activeness: New Methods, New Results biannual briefing and the Imaging Construction and Function in the Nervous System annual summer schoolhouse, both held at Common cold Spring Harbor Laboratory in 2005. Nosotros give thanks Eve Marder and Ofer Tchernichovski for boosted discussions and Ed Callaway and Beth Friedman for critical reading of the essay.

Note Added in Proof

A contempo report demonstrates viral incorporation of a photo-activated cation channel into mammalian neurons and the use of this channel to gate spiking [54].

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