Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.
Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.
The peripheral nervous system (PNS) connects the central nervous system (CNS) to its distal targets, permitting the relay of efferent signals to exert motor control and afferent signals to provide sensory feedback. Using the multitude of advances in mouse genetics, scientists have developed different mouse models to investigate many diseases/syndromes afflicting the PNS1,2,3. As most neurodegenerative pathologies are multifactorial with cell autonomous and non-autonomous contributions4,5, untangling cell-/neuron-specific pathologies can provide crucial, novel insights into disease mechanisms.
To this end, the development of bacterial artificial chromosome (BAC)-transgenic mice6 has enabled selective endogenous expression of fluorescent proteins in targeted subsets of neurons. For example, BAC-transgenic mice are available, which express green fluorescent protein (GFP) in cholinergic7 or glycinergic neurons8, or a variant red fluorescent protein (tdTomato) in parvalbumin-positive neurons9. Alternatively, selective neuronal expression of fluorescent proteins can be achieved via Cre-loxP technology10. For instance, mouse strains expressing Cre-recombinase in subsets of neurons (e.g., choline acetyltransferase (ChAT)-Cre) can be bred with mice that express a fluorescent protein (e.g., tdTomato or GFP) from a constitutive locus (e.g., Gt(ROSA)26Sor) under the control of a transcriptional repressor flanked by loxP sites11 (e.g., generating mice that express tdTomato only in cholinergic neurons). Indeed, using Cre-loxP recombination, transgenic mice have been generated that express yellow fluorescent protein in axons of the descending corticospinal tract12.
In addition, recent advances in CRISPR/Cas9 gene editing, such as ORANGE, enable the fluorescent tagging of multiple endogenous neuronal proteins, with expression achievable at nanoscale resolution13. Moreover, in combination with Cre-expressing mouse strains, ORANGE-CAKE can be used to tag multiple endogenous proteins in individual neurons13. Alternatively, viral-mediated neuronal tracing also allows the labeling of neuronal subsets and can be achieved with targeted combinations of viral serotypes and/or cell-specific promoters14,15,16,17.
Further to the neuronal labeling methods, mouse lines have also been engineered to express reporter proteins targeting specific organelles, such as mitochondria expressing cyan fluorescent protein (Mito.CFP)18 or autophagosomes expressing GFP (LC3.GFP)19. Moreover, mouse lines have been engineered to assess calcium dynamics specifically in neurons (e.g., Thy1.GCaMP)20,21. Altogether, with the advancement of such models, novel experimental applications enable scientists to ask more precise biological and pathological questions about the CNS and PNS.
The main role of peripheral motor nerves is to transmit electrical signals to skeletal muscle to elicit movement. In addition, and occurring over longer time-scales, neurochemical and physiological messages in the form of diverse organelles (e.g., mitochondria, endolysosomes, signaling endosomes) traverse the cytoskeletal network in a uni- or bi-directional manner to help maintain neuronal homeostasis22,23,24. Impairments in axonal transport have disastrous consequences for neuronal health and are linked to many neurodevelopmental and neurodegenerative diseases25. At the molecular level, impairments in axonal transport can disrupt physiological events regulating synaptic signaling and plasticity, gene transcription, and local translation throughout the axon26,27. While there is a multitude of tools to study these events in cultured cells/neurons28,29, assessing axonal transport dynamics and axonal-linked biological events in vivo are required to confirm key insights into physiological and pathological processes30.
Over the years, the Schiavo Laboratory has optimized protocols to ask diverse questions about axonal transport31,32,33,34,35,36. These experiments have expanded from the discovery that a fluorescently labeled atoxic fragment of tetanus neurotoxin (HCT) is internalized into axon terminals in skeletal muscle through interactions with nidogens and polysialogangliosides37. Once internalized, HCT is retrogradely transported in Rab7-positive, neurotrophin-containing signaling endosomes that are destined for the cell bodies of motor and sensory neurons38,39,40,41. In parallel, advances in imaging technology have enabled the real-time analysis of peripheral nerve bundles and individual axons in live, anesthetized mice30. The first foray into assessing in vivo axonal transport dynamics in pathology revealed presymptomatic impairments in the transport of signaling endosomes and mitochondria in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS)35. Importantly, these defects are unlikely to simply represent secondary consequences of neurodegeneration, given the finding that motor neuron loss can occur in the absence of axonal transport perturbations in a mouse model of Kennedy's disease42 and a heterozygous mutant FUS model of ALS43. Such axonal transport deficits can be remedied in ALS mice using inhibitors of specific kinases33 or growth factor receptors34. Moreover, treating neurons with a specific histone deacetylase blocker alters mitochondrial transport in vivo36. Most recently, we report that the BDNF-dependent modulation of axonal transport is dysregulated in distinct motor neuron subtypes in ALS mice44.
By using an ever-expanding toolkit for assessing axonal transport dynamics28,29, this video protocol outlines several applications that will permit further insights into different biological and pathological scenarios. First, transgenic mice that selectively express fluorescent proteins in cholinergic neurons (i.e., motor neurons) are used to discriminate between motor and sensory axons both in vivo and ex vivo. Fluorescently labeled HCT is then loaded into signaling endosomes in three transgenic lines to differentiate axonal transport dynamics in distinct peripheral neurons. The next experimental protocol details a multiplex fluorescence approach to assess mitochondrial transport specifically in motor neurons by breeding ChAT.tdTomato mice with Mito-CFP mice. Finally, instructions are provided on how to concurrently image mitochondria and signaling endosomes within the same axon in vivo.
This protocol details steps to assess in vivo axonal transport of signaling endosomes and mitochondria in intact axons of the mouse sciatic nerve. Indeed, an experimental setup is provided that enables users to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice expressing fluorescent reporter proteins selectively expressed in motor neurons; 2) assess in vivo axonal transport of signaling endosomes specifically in motor neuron axons using three different transgenic mice; 3) investigate in vivo axonal transport of mitochondria specifically in motor neuron axons; and 4) concurrently assess in vivo transport dynamics of signaling endosomes and mitochondria within the same axon. This approach has vast potential for investigating axonal transport in basal conditions and can be used to assess pathological perturbations in different diseases affecting peripheral motor and sensory nerves.
Using previous experimental paradigms as a foundation31,32, here we have detailed novel, robust ways to differentiate axonal transport occurring in motor versus sensory neurons using transgenic reporter mice. Using the Mito.CFP mouse, this approach has been further developed to assess in vivo mitochondrial transport by avoiding intrasciatic nerve injections of TMRE36. This circumvents possible neural damage and perturbations in axonal transport caused by the intranerve injection of the probe. Furthermore, this protocol allows the visualization of axonal transport of multiple organelles in motor axons innervating muscles with distinct physiological properties (e.g., fast-twitch fatigable muscles vs. slow-twitch fatigue-resistant muscles). As such, signaling endosome and/or mitochondrial axonal transport dynamics can be assessed in different subsets of α-motor neurons44. Moreover, the axonal transport of those organelles in pathological settings can also be assessed through crossbreeding with mouse models of different neurodegenerative diseases1,2,3.
The axonal transport toolkit is continuously expanding28,29, and ex vivo protocols have been developed to assess transport dynamics using cultured mouse ventral horn explants49 or excised mouse nerve-muscle preparations50. Furthermore, the development of protocols to assess axonal transport in induced human pluripotent stem cell (hiPSC)-derived cortical51 neurons or hiPSC-derived spinal motor neurons52 has enabled investigation of human neurons with disease-causing mutations. Such cutting-edge protocols in mouse tissue and human cells can provide critical insights into neuronal function, facilitate novel pathomechanistic discovery in neurodegenerative disease models, and be used to test therapeutic molecules and strategies.
Several critical steps need to be followed for the successful implementation of these techniques, and some important notes have been provided in the protocol section. The major requirements for intravital imaging are the inverted confocal microscope with customized stage insert and the equipment to maintain anesthesia and optimum temperature. Indeed, a specialized mobile anesthetic system is needed for 1) induction of anesthesia, 2) dissection/tissue processing (i.e., exposing the sciatic nerve), and 3) maintaining anesthesia during intravital imaging (as previously detailed in 31,32). Especially when using higher magnification objectives (e.g., 40x or 63x), the depth of anesthesia can impact the image quality, as deeper anesthesia induces large 'gaspy' breaths that result in frequent shifts in focus. Such large movements will undoubtedly impact post imaging transport analyses (e.g., tracking cargoes using the Fiji plugins TrackMate53 or KymoAnalyzer54) as the breathing movements produce artifacts in time-lapse videos that can render them unsuitable for automated tracking or require more time-consuming assessment. Moreover, we have also observed imaging artifacts caused by pulsating arteries within the sciatic nerve, which can only be resolved by choosing a different imaging region. The microscope must be equipped with an environmental chamber capable of maintaining constant body temperature, as temperature and pH influence axonal transport55. Furthermore, the application of analgesics post surgery should be avoided, as they can alter transport dynamics56. If the experimental design is longitudinal and requires repeated imaging (e.g.,57), the dissection protocols need to be appropriately adjusted to be minimally invasive and may require additional ethical/licence approval.
Certain experimental considerations need to be kept in mind. First, most of the protocols detailed herein involve the use of transgenic mice that possess fluorescent reporter proteins in mitochondria or motor neuron axons. Each of these mouse lines should be bred and imaged as hemi-/heterozygote. The exceptions, however, are the ChAT.Cre and Rosa26.tdTomato mouse lines that can be separately maintained as homozygotes, with the resulting hemizygote offspring enabling tdTomato expression in cholinergic neurons after Cre-loxP recombination. When cross-breeding transgenic hemi-/heterozygote mice (e.g., Mito.CFP) with other transgenic hemi-/heterozygote mice (e.g., ChAT.eGFP), one needs to carefully consider the breeding strategy, as obtaining the desired numbers of double-mutant progeny can be time-consuming. Moreover, when breeding the F1 generation of ChAT.Cre and Rosa26.tdTomato mice (i.e., ChAT.tdTomato) with additional transgenic strains (e.g., Mito.CFP), one should expect even fewer mice carrying the desired triple transgenes. In addition, one must also consider the potential fluorophore overlap when breeding two-reporter mice with nearby wavelength properties (e.g., Mito-CFP-excitation: 435 nm, emission: 485 nm, bred with ChAT.eGFP-excitation: 488 nm, emission: 510 nm), although it may be possible to overcome this problem with spectral unmixing58.
This technique has some limitations to be considered. In this work and our previous protocols31,32, we have shown how several genetically encoded markers and different staining methods can be used to label and track distinct organelles in vivo. However, not all probes are suitable for this experimental approach. We assessed injections into either TA or soleus muscle of cholera toxin beta subunit (CTB)-488 (0.5-1.5 µg/µL ~4 h before imaging), a probe routinely used to label motor neuron cell bodies in in vivo retrograde tracer experiments59,60. However, when injected alone or co-injected with HCT-555, the CTB-488 labeling was poor despite using concentrations similar to those used for successful retrograde motor neuron tracing. Thus, we conclude that, despite CTB being an excellent in vitro marker of signaling endosomes in neuronal cultures61, HCT remains the gold-standard probe to identify signaling endosomes in vivo in sciatic nerve axons.
Using different routes, we also tested probes routinely used for labeling lysosomes, such as LysoTracker green DND-26, and markers of active lysosomal hydrolases, such as BODIPY-FL-pepstatin A for Cathepsin D62 and Magic Red for Cathepsin B, but with no success. We tried intramuscular delivery of BODIPY-FL-pepstatin A (2.5 µg into the TA ~4 h before imaging), as well as intrasciatic nerve injection of 2 µL of LysoTracker (10 µM), BODIPY-FL-pepstatin A (10 µM) or Magic Red (1/10) 30-60 min before imaging. Despite these probes highlighting the nerve, we were unable to find clearly labeled organelles. The probes accumulated around axons, likely being retained by Schwann cells. Hence, the unsuccessful labeling of lysosomes may be due to deficient probe delivery into neurons, although the existence of more suitable concentrations cannot be ruled out. Given that TMRE labeling works under similar conditions (i.e., intrasciatic nerve injections), the labeling intensity may be dye-dependent and must be tested for each marker independently. However, we conclude that targeting lysosomes in vivo with these probes is not feasible at the concentrations stated above.
Methods of anesthesia can alter distinct physiological readouts (e.g., cochlea function63 and cortical electrophysiology64); however, whether anesthesia influences in vivo axonal transport in the sciatic nerve is currently unknown. Given the reduced neuromuscular activity under isoflurane-induced anesthesia, it is possible that transport kinetics differ compared to the wakeful state. However, the only in vivo study that has directly investigated this revealed that transport of dense core vesicles in thalamocortical projections does not differ between anesthetized and awake mice65. Furthermore, because distinctions in transport between wild-type and disease model mice are detectable under anesthesia35,43, it is clear that isoflurane exposure does not prevent the identification of perturbances in signaling endosome or mitochondrial trafficking.
This protocol has other potential applications, which have been described below. Breeding of the transgenic mice outlined in this protocol (e.g., Mito.CFP, ChAT.eGFP) with neurodegenerative disease mouse models1,2,3 will enable neuron subtype- and/or cargo-specific investigations. Moreover, recently developed mouse Cre lines66 would also permit the visualization of fluorescent reporter proteins in distinct sensory axon populations. For example, Rosa26.tdTomato mice can be crossed with a neuropeptide Y receptor-2-expressing (Npy2r).Cre mouse to enable tdTomato fluorescence in myelinated A-fiber nociceptors67. Furthermore, temporal control can also be achieved by using inducible Cre systems (e.g., tamoxifen)68. Another potential application relies on the availability of transgenic mice expressing fluorescent reporter proteins in Schwann cells. Indeed, S100-GFP69 and PLP-GFP70 mice enable in vivo and/or in situ imaging of Schwann cells and have been at the forefront of research involved in Schwann cell migration during peripheral nerve regeneration.
In addition to these applications and complementing the Mito.CFP mouse is the availability of several transgenic mouse lines that express fluorescent proteins in distinct organelles, such as mitochondria and autophagosomes. For example, investigating in vivo mitochondrial transport might be possible with the mito::mKate2 mouse71 or the photoconvertible mitoDendra mouse57. Moreover, in vivo mitophagosome transport may be possible using the pH-sensitive mito-Keima mouse72 and the mito-QC mouse73 for mitophagy analyses. Furthermore, the lysosomal labeling difficulties we encountered may be overcome by using mice expressing LAMP1-GFP, with the caveat that LAMP1 is also present in endocytic organelles distinct from lysosomes74.
In summary, we have provided novel ways to assess in vivo axonal transport of several organelles in specific peripheral nerve axons from diverse transgenic mice. The concurrent imaging of different organelles will be particularly important, given recent findings of axonal interactions and co-trafficking of organelles such as mitochondria and endosomes75,76. We believe that the presented methods will be useful for improving understanding of the basal physiology of axons in vivo and untangling important pathomechanisms driving neurodegeneration of peripheral nerves.
The authors have nothing to disclose.
We would like to thank Robert M. Brownstone (Queen Square Institute of Neurology, University College London) for sharing the ChAT-eGFP, ChAT.Cre and Rosa26.tdTomato mice, and Pietro Fratta (Queen Square Institute of Neurology, University College London) for sharing the HB9.GFP mouse. We would like to thank Elena R. Rhymes, Charlotte J.P. Kremers, and Qiuhan Lang (Queen Square Institute of Neurology, University College London) for critical reading of the manuscript. This work was supported by a Junior Non-Clinical Fellowship from the Motor Neuron Disease Association (UK) (Tosolini/Oct20/973-799) (APT), the Wellcome Trust Senior Investigator Awards (107116/Z/15/Z and 223022/Z/21/Z) (GS), a UK Dementia Research Institute Foundation award (GS); and a Medical Research Council Career Development Award (MR/S006990/1) (JNS).
0.2 mL PCR tube | |||
70% (v/v) ethanol in distilled water | |||
AlexaFlour555 C2 maleimide | ThermoFisher Scientific | A-20346 | Can also use AlexaFlour-488 or -647 Maleimide |
B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J | Jackson Laboratory | 7909 | Rosa26.tdTomato mice |
B6.Cg-Tg(Hlxb9-GFP)1Tmj/J mice | Jackson Laboratory | 5029 | HB9.GFP mice |
B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice | Jackson Laboratory | 7967 | Mito.CFP mice |
B6;129S6-Chattm2(cre)Lowl/J mice | Jackson Laboratory | 6410 | ChAT.Cre mice |
Computer with microscope control and image acquisition software | Zeiss | Zen | |
Cotton swab | |||
Desktop centrifuge | |||
Dissecting microscope | |||
Eye lubricant | |||
Fine curved forceps | Dumont | ||
Fine straight forceps | Dumont | ||
Glass coverslip (22 x 64 mm, thickness no. 1) | |||
Graduated, glass micropipette with microliter markings and plunger | Drummond Scientific | 5-000-1001-X10 | |
Hair clippers | |||
Hamilton microliter syringe (701 N, volume 10 μL, needle size 26 s G, bevel tip, needle L 51 mm) | Merck | 20779 | |
HcT-441 | N/A | N/A | See Restani et al., 2012 for more details |
Heating pad | |||
Immersion oil for fluorescent imaging at 37 °C | |||
Inverted confocal microscope with environmental chamber | Zeiss | LSM 780 | Most inverted confocals should be adaptable |
Isoflurane | |||
Isoflurane vaporizer/anesthesia machine with induction cham-ber and mask stabilizer | |||
Magic tape | invisible tape | ||
Micropipette puller | |||
Parafilm | Parafilm | ||
Phosphate-buffered saline (PBS): 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4–HCl, pH 7.4 | |||
Recombinant human brain-derived neurotrophic factor (BDNF) | Peprotech | 450-02 | BDNF that can be used to co-inject with HcT-555 |
Saline | |||
Scalpel blade | Dumont | ||
Small spring scissors | Dumont | ||
Surgery/operating microscope | |||
Surgical drape | |||
Surgical suture | |||
Surgical tape | |||
Tg(Chat-EGFP) GH293Gsat/Mmucd mice | MMRRC | 000296-UCD | ChAT.eGFP |
Vortex mixer |