High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo
- University of California, Berkeley, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- National Institute on Drug Abuse, United States
- Johns Hopkins University School of Medicine, United States
Abstract
Optical imaging has become a powerful tool for studying brains in vivo. The opacity of adult brains makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus in vivo.
Data availability
Almost all data needed to evaluate the conclusions in the paper are present in the paper or the supplementary materials; Raw image data for Figs. 2, 4 & 9 are available from Dryad, 10.5061/dryad.pr4t978
Article and author information
Author details
Joshua Tate Dudman
Funding
Howard Hughes Medical Institute
- Guanghan Meng
- Yajie Liang
- Wan-chen Jiang
- Rongwen Lu
- Joshua Tate Dudman
- Na Ji
National Institute of Neurological Disorders and Stroke
- Guanghan Meng
- Na Ji
National Institute on Drug Abuse
- Sarah Sarsfield
- Yeka Aponte
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were conducted according to the United States National Institutes of Health guidelines for animal research. Procedures and protocols were approved by the Institutional Animal Care and Use Committee at Janelia Research Campus, Howard Hughes Medical Institute (protocol number: 16-147)
Copyright
© 2019, Meng et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo
eLife 8:e40805.
https://doi.org/10.7554/eLife.40805
Further reading
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At many vertebrate synapses, presynaptic functions are tuned by expression of different Cav2 channels. Most invertebrate genomes contain only one Cav2 gene. The Drosophila Cav2 homolog, cacophony (cac), induces synaptic vesicle release at presynaptic active zones (AZs). We hypothesize that Drosophila cac functional diversity is enhanced by two mutually exclusive exon pairs that are not conserved in vertebrates, one in the voltage sensor and one in the loop binding Caβ and Gβγ subunits. We find that alternative splicing in the voltage sensor affects channel activation voltage. Only the isoform with the higher activation voltage localizes to AZs at the glutamatergic Drosophila larval neuromuscular junction and is imperative for normal synapse function. By contrast, alternative splicing at the other alternative exon pair tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number affects short-term plasticity, which is rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
