Neural labeling and manipulation by neonatal intraventricular viral injection in mice

This step-by-step protocol provides a fast and easy technique to label and/or genetically manipulate neural cells, achieved by intraventricular injection of viral vectors into neonatal mice under ultrasound guidance. Successful injection of ad-eno-associated viral vectors (AAV) induces neural transduction as fast as 3 days post injection (dpi) in both the central and peripheral nervous systems. Virally driven expression persists until early adulthood. The same setup enables injec-tion of other viral vectors as well as intramuscular injection. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) and Brill et al. (2016)

Niwaki Instead of Random Forests: Targeted Serial Sectioning Scanning Electron Microscopy With Reimaging Capabilities for Exploring Central Nervous System Cell Biology and Pathology

Ultrastructural analysis of discrete neurobiological structures by volume scanning electron microscopy (SEM) often constitutes a “needle-in-the-haystack” problem and therefore relies on sophisticated search strategies. The appropriate SEM approach for a given relocation task not only depends on the desired final image quality but also on the complexity and required accuracy of the screening process. Block-face SEM techniques like Focused Ion Beam or serial block-face SEM are “one-shot” imaging runs by nature and, thus, require precise relocation prior to acquisition. In contrast, “multi-shot” approaches conserve the sectioned tissue through the collection of serial sections onto solid support and allow reimaging. These tissue libraries generated by Array Tomography or Automated Tape Collecting Ultramicrotomy can be screened at low resolution to target high resolution SEM. This is particularly useful if a structure of interest is rare or has been predetermined by correlated light microscopy, which can assign molecular, dynamic and functional information to an ultrastructure. As such approaches require bridging mm to nm scales, they rely on tissue trimming at different stages of sample processing. Relocation is facilitated by endogenous or exogenous landmarks that are visible by several imaging modalities, combined with appropriate registration strategies that allow overlaying images of various sources. Here, we discuss the opportunities of using multi-shot serial sectioning SEM approaches, as well as suitable trimming and registration techniques, to slim down the high-resolution imaging volume to the actual structure of interest and hence facilitate ambitious targeted volume SEM projects

Nerve-independent formation of a topologically complex postsynaptic apparatus

As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)–rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity

Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo

We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resolution. By combining various technical improvements, we tracked individual mitochondria with unprecedented spatiotemporal resolution over distances of >100 mu m. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resolution in a living organism offers a new approach to elucidating their function in its complete physiological context

The specific frequencies of ultra-compact dwarf galaxies

We aim at quantifying the specific frequency of UCDs in a range of environments and at relating this to the frequency of globular clusters (GCs) and potential progenitor dwarf galaxies. Are the frequencies of UCDs consistent with being the bright tail of the GC luminosity function (GCLF)? We propose a definition for the specific frequency of UCDs, S_{N,UCD}=N_{UCD}*10^{0.4*(M_{V,host}-M_{V,0})}*c_{w}. The parameter M_{V,0} is the zeropoint of the definition, chosen such that the specific frequency of UCDs is the same as those of globular clusters, S_{N,GC}, if UCDs follow a simple extrapolation of the GCLF. The parameter c_{w} is a correction term for the GCLF width sigma. We apply our definition of S_{N,UCD} to results of spectroscopic UCD searches in the Fornax, Hydra and Centaurus galaxy clusters, two Hickson Compact Groups, and the Local Group. This includes a large database of 180 confirmed UCDs in Fornax. We find that the specific frequencies derived for UCDs match those of GCs very well, to within 10-50%. The ratio {S_{N,UCD}}/{S_{N,GC}} is 1.00 +- 0.44 for the four environments Fornax, Hydra, Centaurus, and Local Group, which have S_{N,GC} values. This good match also holds for individual giant galaxies in Fornax and in the Fornax intracluster-space. The error ranges of the derived UCD specific frequencies in the various environments then imply that not more than 50% of UCDs were formed from dwarf galaxies. We show that such a scenario would require >90% of primordial dwarfs in galaxy cluster centers (<100 kpc) to have been stripped of their stars. We conclude that the number counts of UCDs are fully consistent with them being the bright tail of the GC population. From a statistical point of view there is no need to invoke an additional formation channel.Comment: 11 pages, 6 figures, A&A accepted. Press release http://www.aanda.org/index.php?option=com_content&task=view&id=788&Itemid=27

A search for massive UCDs in the Centaurus Galaxy Cluster

We recently initiated a search for ultra-compact dwarf galaxies (UCDs) in the Centaurus galaxy cluster (Mieske et al. 2007), resulting in the discovery of 27 compact objects with -12.2<M_V<-10.9 mag. Our overall survey completeness was 15-20% within 120 kpc projected clustercentric distance. In order to better constrain the luminosity distribution of the brightest UCDs in Centaurus, we continue our search by substantially improving our survey completeness specifically in the regime M_V<-12 mag (V_0<21.3 mag). Using VIMOS at the VLT, we obtain low-resolution spectra of 400 compact objects with 19.3<V_0<21.3 mag (-14<M_V<-12 mag at the Centaurus distance) in the central 25' of the Centaurus cluster, which corresponds to a projected radius of ~150 kpc. Our survey yields complete area coverage within ~120 kpc. For 94% of the sources included in the masks we successfully measure a redshift. Due to incompleteness in the slit assignment, our final completeness in the area surveyed is 52%. Among our targets we find three new UCDs in the magnitude range -12.2<M_V<-12 mag, hence at the faint limit of our survey. One of them is covered by archival HST WFPC2 imaging, yielding a size estimate of r_h <= 8-9 pc. At 95% confidence we can reject the hypothesis that in the area surveyed there are more than 2 massive UCDs with M_V<-12.2 mag and r_eff <=70 pc. Our survey hence confirms the extreme rareness of massive UCDs. We find that the radial distributions of Centaurus and Fornax UCDs with respect to their host clusters' centers agree within the 2 sigma level.Comment: 9 pages, 7 figures, accepted as Research Note for A&

Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization

Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated alpha-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated alpha-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating alpha-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function

Remodeling of Axonal Connections Contributes to Recovery in an Animal Model of Multiple Sclerosis

In multiple sclerosis (MS), inflammation in the central nervous system (CNS) leads to damage of axons and myelin. Early during the clinical course, patients can compensate this damage, but little is known about the changes that underlie this improvement of neurological function. To study axonal changes that may contribute to recovery, we made use of an animal model of MS, which allows us to target inflammatory lesions to the corticospinal tract (CST), a major descending motor pathway. We demonstrate that axons remodel at multiple levels in response to a single neuroinflammatory lesion as follows: (a) surrounding the lesion, local interneurons show regenerative sprouting; (b) above the lesion, descending CST axons extend new collaterals that establish a “detour” circuit to the lumbar target area, whereas below the lesion, spared CST axons increase their terminal branching; and (c) in the motor cortex, the distribution of projection neurons is remodeled, and new neurons are recruited to the cortical motor pool. Behavioral tests directly show the importance of these changes for recovery. This paper provides evidence for a highly plastic response of the motor system to a single neuroinflammatory lesion. This framework will help to understand the endogenous repair capacity of the CNS and to develop therapeutic strategies to support it

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated a-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated a-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating a-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.This work was supported by: FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciéncia, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028336 (PTDC/MED-NEU/28336/2017); Norte-01-0145-FEDER-000008 – Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through FEDER; and Thompson Family Foundation (TFFI) award, RO1AG050658 (NIH/National Institute on Aging) and R21NS120076 (NIH/NINDS) awards to FB, and a PRIN-2017FJC3-004 (MIUR) to MEP. TM is supported by the Deutsche Forshcunggemeinschaft (EXC 2145 SyNergy – ID 390857198; TRR 274/1 2020 – ID 408885537). JE is a FCT fellow (SFRH/BD/116343/2016). MAL is an FCT Investigator (IF/00902/2015)