Ultrafast optogenetic stimulation of the auditory pathway by targeting‐optimized Chronos
2018; Springer Nature; Volume: 37; Issue: 24 Linguagem: Inglês
10.15252/embj.201899649
ISSN1460-2075
AutoresDaniel Keppeler, Ricardo Martins Merino, David López de la Morena, Burak Bali, Antoine Huet, Anna Gehrt, Christian Wrobel, Swati Subramanian, Tobias Dombrowski, Fred Wolf, Vladan Rankovic, Andreas Neef, Tobias Moser,
Tópico(s)Light effects on plants
ResumoResource5 November 2018free access Transparent process Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos Daniel Keppeler orcid.org/0000-0002-2638-8731 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Ricardo Martins Merino Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author David Lopez de la Morena orcid.org/0000-0003-0835-2732 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Search for more papers by this author Burak Bali Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Restorative Cochlear Genomics Group, German Primate Center, Göttingen, Germany Search for more papers by this author Antoine Tarquin Huet Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Search for more papers by this author Anna Gehrt Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Christian Wrobel Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Swati Subramanian Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Tobias Dombrowski Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Fred Wolf Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Bernstein Center for Computational Neuroscience, Göttingen, Germany Campus Institute for Dynamics of Biological Networks, Göttingen, Germany Search for more papers by this author Vladan Rankovic Corresponding Author [email protected] orcid.org/0000-0003-0285-5232 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Restorative Cochlear Genomics Group, German Primate Center, Göttingen, Germany Search for more papers by this author Andreas Neef Corresponding Author [email protected] orcid.org/0000-0003-4445-7478 Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Campus Institute for Dynamics of Biological Networks, Göttingen, Germany Search for more papers by this author Tobias Moser Corresponding Author [email protected] orcid.org/0000-0001-7145-0533 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Daniel Keppeler orcid.org/0000-0002-2638-8731 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Ricardo Martins Merino Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author David Lopez de la Morena orcid.org/0000-0003-0835-2732 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Search for more papers by this author Burak Bali Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Restorative Cochlear Genomics Group, German Primate Center, Göttingen, Germany Search for more papers by this author Antoine Tarquin Huet Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Search for more papers by this author Anna Gehrt Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Christian Wrobel Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Swati Subramanian Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Tobias Dombrowski Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Fred Wolf Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Bernstein Center for Computational Neuroscience, Göttingen, Germany Campus Institute for Dynamics of Biological Networks, Göttingen, Germany Search for more papers by this author Vladan Rankovic Corresponding Author [email protected] orcid.org/0000-0003-0285-5232 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Restorative Cochlear Genomics Group, German Primate Center, Göttingen, Germany Search for more papers by this author Andreas Neef Corresponding Author [email protected] orcid.org/0000-0003-4445-7478 Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany Campus Institute for Dynamics of Biological Networks, Göttingen, Germany Search for more papers by this author Tobias Moser Corresponding Author [email protected] orcid.org/0000-0001-7145-0533 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany Max Planck Institute for Experimental Medicine, Göttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Author Information Daniel Keppeler1,2,‡, Ricardo Martins Merino2,3,4,5,‡, David Lopez de la Morena1,2,6,‡, Burak Bali1,2,7,‡, Antoine Tarquin Huet1,3,6,‡, Anna Gehrt1,8, Christian Wrobel1,8, Swati Subramanian1,2, Tobias Dombrowski1,†, Fred Wolf4,5,8,9,10, Vladan Rankovic *,1,7, Andreas Neef *,3,4,10 and Tobias Moser *,1,2,3,5,6,8,11 1Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany 2Göttingen Graduate School for Neurosciences and Molecular Biosciences, University of Göttingen, Göttingen, Germany 3Biophysics of Neural Computation Group, Bernstein Center for Computational Neuroscience Göttingen, Göttingen, Germany 4Neurophysics Group, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany 5Max Planck Institute for Experimental Medicine, Göttingen, Germany 6Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, Göttingen, Germany 7Restorative Cochlear Genomics Group, German Primate Center, Göttingen, Germany 8Collaborative Research Center 889, University of Göttingen, Göttingen, Germany 9Bernstein Center for Computational Neuroscience, Göttingen, Germany 10Campus Institute for Dynamics of Biological Networks, Göttingen, Germany 11Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany †Present address: Department of Otorhinolaryngology, Head and Neck Surgery, St. Elisabeth Hospital, Ruhr University Bochum, Bochum, Germany ‡These authors contributed equally to this work ‡These authors contributed equally to this work *Corresponding author. Tel: +49 551 3851 209; E-mail: [email protected] *Corresponding author. Tel: +49 551 3961 107; E-mail: [email protected] *Corresponding author. Tel: +49 551 3963 070; E-mail: [email protected] EMBO J (2018)37:e99649https://doi.org/10.15252/embj.201899649 See also: E Ronzitti et al (December 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Optogenetic tools, providing non-invasive control over selected cells, have the potential to revolutionize sensory prostheses for humans. Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in cochlear implants. However, most channelrhodopsins do not support the high temporal fidelity pertinent to auditory coding because they require milliseconds to close after light-off. Here, we biophysically characterized the fast channelrhodopsin Chronos and revealed a deactivation time constant of less than a millisecond at body temperature. In order to enhance neural expression, we improved its trafficking to the plasma membrane (Chronos-ES/TS). Following efficient transduction of SGNs using early postnatal injection of the adeno-associated virus AAV-PHP.B into the mouse cochlea, fiber-based optical stimulation elicited optical auditory brainstem responses (oABR) with minimal latencies of 1 ms, thresholds of 5 μJ and 100 μs per pulse, and sizable amplitudes even at 1,000 Hz of stimulation. Recordings from single SGNs demonstrated good temporal precision of light-evoked spiking. In conclusion, efficient virus-mediated expression of targeting-optimized Chronos-ES/TS achieves ultrafast optogenetic control of neurons. Synopsis Here we biophysically characterized and molecularly improved the fast gating blue-light activated channelrhodopsin Chronos. Employing the potent viral vector AAV-PHP.B we postnatally expressed the improved Chronos in cochlear neurons and achieved ultrafast neural control. Biophysical characterization of the fast channelrhodopsin Chronos revealed a deactivation time constant of less than a millisecond at body temperature. Molecular engineering of Chronos via adding trafficking sequences enhanced plasma membrane abundance of the opsin and use of postnatal AAV-PHP.B carrying Chronos into the mouse cochlea enabled efficient expression in spiral ganglion neurons. Chronos enabled synchronized optically driven firing in spiral ganglion neurons for stimulation rates of up to hundreds of Hz as required for future optical cochlear implants. Introduction Since the discovery of channelrhodopsins (ChRs, Nagel et al, 2002, 2003) and the application of these light-gated ion channels for controlling excitable cells (Boyden et al, 2005), the concept of optogenetics has revolutionized the life sciences (Adamantidis et al, 2015; Kim et al, 2017). Application of optogenetics to restore sensory function in the immune-privileged eye and the ear is thought to have a fair chance of clinical translation (Sahel & Roska, 2013; Jeschke & Moser, 2015). Indeed, AAV-mediated optogenetics for vision restoration has recently entered a first clinical trial in a dose-finding effort (RST-001 Phase I/II Trial for Advanced Retinitis Pigmentosa—Full Text View—ClinicalTrials.gov). While neural coding of visual information can likely be achieved with ChRs that deactivate within several milliseconds (Busskamp et al, 2012), faster ChRs are required for sound coding in spiral ganglion neurons of the ear (SGNs) that spike at hundreds of Hz with sub-millisecond precision (Jeschke & Moser, 2015). If such ultrafast optogenetic control of neural activity was available, it would serve auditory research and could fuel the development of future optical cochlear implants (oCIs). Clinically, this is highly relevant as approximately 466 million people\xF6over 5% of the world's population\xF6suffer from a disabling hearing impairment (WHO, 2018) and we are still lacking causal therapies for the most common form, sensorineural hearing impairment. Consequences are impaired communication, often social isolation, depression, and reduction in professional capabilities. As of today, partial restoration of auditory function by hearing aids and electrical CIs (eCI) represents the options of choice for rehabilitation in sensorineural hearing impairment, which results from cochlear dysfunction or degeneration. The eCI bypasses dysfunctional or lost cochlear hair cells via direct electric stimulation of SGNs and, with most of the approximately 500,000 users achieving open speech comprehension, is considered the most successful neuroprosthesis (Zeng, 2017; Lenarz, 2018). Nonetheless, there is an urgent need for further improvement of the CI. The biggest bottleneck of the eCI is the poor spectral resolution of coding that arises from the widespread of current around each electrode contact (Kral et al, 1998). Using light for stimulation in oCI is one of the present developments to improve spectral coding by CIs, as light can be better spatially confined than electric current (e.g., Richter et al, 2011; Hernandez et al, 2014). One of the implementations used optogenetic stimulation of SGNs for a first proof-of-principle study on activation of the auditory pathway up to the midbrain (inferior colliculus, IC), demonstrating a lower spread of cochlear excitation for fiber-based oCI than for monopolar eCI (Hernandez et al, 2014). However, the temporal fidelity of ChR2-mediated optogenetic control of SGN firing seemed limited; auditory brainstem responses broke down even below 100 Hz of stimulation. Higher temporal fidelity of optogenetic SGN stimulation might be achieved when using faster ChRs such as Chronos (Klapoetke et al, 2014), the newly engineered Chronos mutant ChroME (Mardinly et al, 2018), or fast Chrimson mutants (Mager et al, 2018). Therefore, characterizing and optimizing fast ChRs are of great importance for fast-spiking neurons in the auditory system, but also in the somatosensory system, cerebellum, and a wide range of inhibitory circuits. Here, we targeted Chronos, the fastest ChR reported so far, and first dissected its gating by patch-clamp recordings of photocurrents. Toward its application for optogenetic stimulation of mouse SGNs, we optimized Chronos, the viral vector and virus injection approach for achieving high plasma membrane expression. As described for another opsin (Gradinaru et al, 2010), we appended sequences for improved exit from the endoplasmic reticulum (ES) (Ma et al, 2001; Stockklausner et al, 2001) and trafficking to the plasma membrane (TS) (Hofherr et al, 2005) to Chronos (Chronos-ES/TS) and performed postnatal injections of AAV-PHP.B serotype (Deverman et al, 2016), which drove highly efficient Chronos-ES/TS expression. We demonstrate by recordings of oABR and single SGN firing that Chronos-ES/TS enables ultrafast stimulation of the auditory pathway. Results Chronos undergoes sub-millisecond on/off transitions at 36°C The kinetic properties of ChRs are best studied in cells with little background conductance to characterize the light-induced conductance in isolation. To this end, we used human embryonic kidney cells 293T (HEK-293T cells) expressing Chronos or ChR2 (Materials and Methods). We clamped the membrane voltage to −60 mV where any light-induced change in the conductance is then linearly reflected in a change of the pipette current. First, we compared gating kinetics of ChR2 and Chronos at a low light intensity of 0.27 mW mm−2, far below the half-maximal activation. At 22°C, we found activation and deactivation time constants (mean ± SEM) of τact = 4.9 ± 0.3 ms, τdeact = 9.4 ± 1.0 ms (n = 6) for ChR2 and τact = 1.5 ± 0.1 ms, τdeact = 3.0 ± 0.2 ms (n = 21) for Chronos (Fig 1A). When increasing the temperature to 36°C, activation and deactivation accelerated, with τact = 0.58 ± 0.02 ms and τdeact = 0.76 ± 0.05 ms (n = 6), Chronos reached the sub-millisecond range, while ChR2 gating kinetics, τact = 2.3 ± 0.1 ms, τdeact = 3.0 ± 0.3 ms (n = 6 and 5, respectively), at 36°C were comparable to the values achieved by Chronos at 22°C. Hence, already at such a low light intensity, Chronos, but not ChR2, activation and deactivation should permit signal transmission with a bandwidth of several hundred Hertz. Probing the frequency bandwidth directly by applying light chirps, Chronos confers a much higher bandwidth compared to ChR2 (Fig 1B). The gain of chirp responses could be very well characterized by a single cutoff frequency of 24 Hz and 86 Hz for ChR2 and Chronos, respectively, at 22°C and 63 Hz and 150 Hz at 36°C (Fig 1C). Figure 1. Chronos mediates fast light-driven currents One-second-long light pulses (LED centered 480 nm, 0.27 mW/mm2) elicit current responses in HEK-293T cells expressing ChR2 and Chronos, tested at 22 and 36°C. Right panels show activation and deactivation at higher time resolution. A quasi-periodic chirp stimulus is used to directly probe the bandwidth of Chronos- and ChR2-mediated photocurrents in HEK-293T cells. Top: stimulus, middle: full response, bottom: sections from the beginning, middle, and end of the response. Note the substantially larger frequency range over which Chronos currents follow the light stimulus. At 36°C, this range is extended even further. Analysis of the chirp responses of HEK-293T cells as in (B). The current amplitude modulation is plotted against the stimulus frequency. The smooth lines represent fits to the magnitude of the transfer function of a single pole filter abs ((1+ if/fcut)−1). Download figure Download PowerPoint Improving the plasma membrane expression of Chronos The above biophysical characterization had indicated Chronos as a strong candidate for optogenetic stimulation of SGNs with the required high temporal fidelity, provided sufficient plasma membrane expression can be achieved. Recent studies have shown that adding ER export and trafficking signals, isolated from a vertebrate inward rectifier potassium channel, to the cytoplasmic C terminus of opsins promote their plasma membrane expression (Gradinaru et al, 2010). Hence, we added these sequences, here nick-named ES (Export Signal) and TS (Trafficking Signal), sandwiching EYFP, to Chronos (Chronos-ES/TS, Fig 2A). We first compared expression of Chronos-ES/TS and Chronos in HEK-293T cells transfected with the respective pAAV plasmid by immunocytochemistry. Using confocal and stimulated emission depletion (STED) microscopy, we found a preferentially peripheral, likely plasmalemmal, localization of Chronos-ES/TS with some intracellular signal most likely arising from the Golgi, while the original Chronos construct was more diffusely distributed throughout the extra-nuclear intracellular space (Fig 2B–D). We quantified the cellular distribution of the opsin by line profile analysis (Fig 2C) and found a significantly greater peripheral than central immunofluorescence for Chronos-ES/TS when compared to the original Chronos construct (Fig 2C and D). The larger variance of the ratio of membrane and intracellular fluorescence for Chronos-ES/TS (Fig 2D) is likely explained by dividing by the relatively low, yet varying intracellular abundance of the opsin. In summary, the data suggest improved trafficking to the plasma membrane of Chronos-ES/TS. Figure 2. Optimizing membrane expression of Chronos by adding ER-exit and trafficking signals: HEK-293T cells pAAV vector used in the study Chronos with a trafficking signal (TS), EYFP and ER export signal (ES) Chronos-ES/TS (upper) or containing the original Chronos-EGFP (Klapoetke et al, 2014; lower). In each, expression was driven by the human synapsin promoter (hSyn) and enhanced by the Woodchuck hepatitis virus posttranslational regulatory element (WPRE) and bovine growth hormone (bGH) polyadenylation signal (bGH poly A) sequences. ITR: inverted terminal repeats. Confocal and STED section of representative HEK-293T cells transfected with Chronos-ES/TS (upper) and Chronos (lower) and immunolabeled for FP: membranous labeling is more obvious for Chronos-ES/TS. Scale bars: 10, 5 and 2 μm for left, middle, and right panels. Peak-normalized line profiles (7.5 μm) centered on the estimated membrane of HEK-293T cells expressing Chronos-ES/TS (blue) or Chronos (green) as in (B): mean ± SEM. Chronos-ES/TS-expressing cells showed a clear peripheral, likely membrane peak, which is missing in Chronos-expressing cells. Right panels show exemplary line profile placements (yellow). One line per cell was placed perpendicular and centered to cell edge, aiming at sufficient intracellular coverage and avoiding fluorescent aggregates (arrowheads). N corresponds to analyzed cells (1 sample/cell). Box and whisker plot ratio of maximal membrane and maximal cytoplasmic fluorescence of immunolabeled HEK-293T cells expressing Chronos-ES/TS or Chronos: Mann–Whitney U-test showed significantly higher ratio in Chronos-ES/TS cells demonstrating an improved membrane expression of Chronos-ES/TS (P-value = 4.4e-4). The horizontal line within the box indicates the median, boundaries of the box indicate the 0.25- and 0.75-percentile, and the whiskers indicate the highest and lowest values of the results. Squares: individual data points. For details on membranous and cytoplasmic area, see Materials and Methods section. Download figure Download PowerPoint Similar findings were also obtained in hippocampal neurons in culture transduced by AAV2/6 or AAV-PHP.B (Fig 3). This indicates that neurons, too, struggle to traffic Chronos to the plasma membrane and that this can be alleviated when adding the ES and TS signals, at least when tested in culture. Figure 3. Improved membrane expression of optimized Chronos-ES/TS in hippocampal neurons Hippocampal neurons infected at DIV 10 with two different versions of Chronos show very distinct expression patterns. Neurons infected with Chronos-ES/TS (upper panels) either using AAV2/6 (left) or PHP.B virus (middle) show very specific plasma membrane expression in somatic regions and proximal dendrites. Infection of neurons with Chronos (lower panels) either using AAV2/6 (left) or AAV-PHP.B (middle) showed more intracellular opsin abundance. Right panels show exemplary line profile placement (yellow). Scale bar: 50 μm applies to all panels. Peak-normalized line profiles (1.5 μm) centered on the outer cell edge of AAV-PHP.B transduced hippocampal proximal dendrites expressing Chronos-ES/TS (blue) or Chronos (green) as in (A): mean ± SEM. Chronos-ES/TS-expressing cells showed a clear peripheral, likely plasmalemmal peak, which is missing in Chronos-expressing cells. Box and whisker plot of the ratio of maximal membrane and maximal intracellular fluorescence of immunolabeled hippocampal neurons expressing Chronos-ES/TS or Chronos: Mann–Whitney U-test showed significantly higher ratio in Chronos-ES/TS cells demonstrating an improved membrane expression of Chronos-ES/TS (P-value = 4.2e-8). The horizontal line within the box indicates the median, boundaries of the box indicate the 0.25- and 0.75-percentile, and the whiskers indicate the highest and lowest values of the results. Squares: individual data points. For details on membranous and cytoplasmic area, see Materials and Methods section. Download figure Download PowerPoint AAV-mediated expression of Chronos and Chronos-ES/TS in mouse SGNs Next, we turned to expression of Chronos in mouse SGNs in vivo and, once more, compared the original Chronos and Chronos-ES/TS. We aimed to establish efficient AAV-mediated transduction of SGNs and employed the human synapsin promoter (hSyn, Fig 2A) that had turned out to drive efficient and specific SGN expression (Hernandez et al, 2014). We first followed our previous protocol using transuterine injections of AAV2/6 into the otocyst of mouse embryos at embryonic day 11.5 (Fig 4A, upper). In most of the cases, the expression of Chronos-EGFP was absent or sparse (Fig 4B, left and middle). Exceptionally we saw high expression levels (Fig 4B right). As before (Hernandez et al, 2014), the expression, if any, was largely limited to the SGNs of the basal cochlear turn and was never seen in inner hair cells (Fig 4B right, inset). Figure 4. Establishing efficient expression of Chronos in SGNs: use of Chronos-ES/TS, potent AAV-PHP.B, and postnatal mode of AAV injection Upper panel: schematic representation of the viral injection into the embryonic otocyst (left: black cylinder marks the light guide used to trans-illuminate the embryo in the uterus after mobilization from the abdominal cavity, green: micropipette filled with fast green-colored AAV suspension). Middle panel: schematic representation of AAV injection into the postnatal cochlea via the round window (RW). Lower panel: surgical situs of a p7 mouse with retroauricular incision, graphical aid encircles the injection site). Inset shows ex vivo cochlea just after AAV injection via RW. Scale bar: 2 mm. Maximum projection of confocal images of immunolabeled mid-modiolar cochlear cryosections (exemplary sections of basal turn) of embryonically AAV2/6-Chronos-injected mice collected at 4 weeks of age. EYFP (green) marks transduced SGNs, calretinin (magenta) was used as generic marker of SGNs, scale bar: 50 μm. In the inset, color code for EYFP channel was changed to fire (EYFP). Left panel: most common, non-expressing example, inset shows zoom of negative SGNs. Middle panel: occasional, sparsely expressing example, inset: one out of two positive SGNs. Right panel: rare, highly expressing example, inset: negative inner hair cell (calretinin in gray), exclusive localization of EYFP in the SGN boutons and fibers. Postnatally AAV-PHP.B-Chronos-ES/TS-injected mouse (see (B) except where stated differently). EYFP (green) marks transduced SGNs, parvalbumin (magenta) was used as generic marker of SGNs, scale bar: 50 μm. High transduction rate, good membrane expression. In inset, color code for the green channel was changed to fire for better visualization. Similar to (B). Postnatally AAV-PHP.B-Chronos-injected mouse (see (C) except where stated differently). Substantial SGN transduction, poor membrane expression. Line profile analysis of FP immunofluorescence across the membrane of SGN somata. Traces were centered at the transi
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