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Background Despite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons. Results To gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NAD+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system. Conclusions Our data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain.

Background The development of neural circuits within the embryonic cerebral cortex relies on the timely production of neurons, their positioning within the embryonic cerebral cortex as well as their terminal differentiation and dendritic spine connectivity. The RhoA GTPases Rnd2 and Rnd3 are important for neurogenesis and cell migration within the embryonic cortex (Nat Commun 4:1635, 2013), and we recently identified the BTB/POZ domain-containing Adaptor for Cul3-mediated RhoA Degradation family member Bacurd2 (also known as Tnfaip1) as an interacting partner to Rnd2 for the migration of embryonic mouse cortical neurons (Neural Dev 10:9, 2015). Findings We have extended this work and report that Bacurd1/Kctd13 and Bacurd2/Tnfaip1 are interacting partners to Rnd2 and Rnd3 in vitro. Given that these genes are expressed during cortical development, we performed a series of in utero electroporation studies in mice and found that disruptions to Bacurd1/Kctd13 or Bacurd2/Tnfaip1 expression impair the long-term positioning of E14.5-born cortical neurons within the postnatal (P17) mouse cerebral cortex. We also find that forced expression of Bacurd1/Kctd13 and Bacurd2/Tnfaip1 alters the branching and dendritic spine properties of layer II/III projection neurons. Conclusions We identify Bacurd1/Kctd13 and Bacurd2/Tnfaip1 as interacting partners to Rnd proteins which influence the development of cortical neurons. Their neurodevelopmental functions are likely to be relevant to human brain development and disease. Electronic supplementary material The online version of this article (doi:10.1186/s13064-016-0062-1) contains supplementary material, which is available to authorized users.

In vivo two-photon (2P) imaging enables neural circuitry to be repeatedly visualized in both normal conditions and following trauma. This protocol describes how laser-mediated neuronal microlesions can be created in the cerebral cortex using an ultrafast laser without causing a significant inflammatory reaction or compromising the blood-brain barrier. Furthermore, directives are provided for the acute and chronic in vivo imaging of the lesion site, as well as for post-hoc analysis of the lesion site in fixed tissue, which can be correlated with the live imaging phase.

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline.

Purpose : Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnositcs and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. Methods : Fluorescence-activated cell sorting (FAC) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The nanobody was then labeled with 99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of 99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU 145) overexpression of EGFR. Results : FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of 99mTc-8B6 Nanobody is relatively fast (half-life, 1.5h) and mainly via the kidneys. At 3h postinjection, total kidney accumulation is high(46.6+0.9%IA) compared to total liver uptake (18.9+0.6IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+0.5%IA/cm3) of 99mTc-8B6 Nanobody compared to DU145 xenografts (1.8+0.3%IA/cm3, pConclusion : The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with 99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, (99m)Tc-7C12 and (99m)Tc-7D12.Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g).Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-7C12, but both showed short plasma half-lives (10 min).The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, (99m)Tc-7C12 was selected for further studies.