A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing

Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases.Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing,making these vectors powerful candidates for the dissection of synaptic inputs.Although several attenuated rabies viral vectors have been developed,their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors.To overcome these limitations,we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus.Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably:(1)N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro,and the efficiency was six times higher than that of rAAV9-Retro;(2)the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG;(3)CVS-N2c-ΔG could delivery modified genes for neural activity monitoring,and the time window during which this was maintained was 3 weeks;and(4)CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination.These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Protective effects of gonadal hormones on spinal motoneurons following spinal cord injury

Spinal cord injury（SCI） results in lesions that destroy tissue and disrupt spinal tracts, producing deficits in locomotor and autonomic function. The majority of treatment strategies after SCI have concentrated on the damaged spinal cord, for example working to reduce lesion size or spread, or encouraging regrowth of severed descending axonal projections through the lesion, hoping to re-establish synaptic connectivity with caudal targets. In our work, we have focused on a novel target for treatment after SCI, surviving spinal motoneurons and their target musculature, with the hope of developing effective treatments to preserve or restore lost function following SCI. We previously demonstrated that motoneurons, and the muscles they innervate, show pronounced atrophy after SCI. Importantly, SCI-induced atrophy of motoneuron dendrites can be attenuated by treatment with gonadal hormones, testosterone and its active metabolites, estradiol and dihydrotestosterone. Similarly, SCI-induced reductions in muscle fiber cross-sectional areas can be prevented by treatment with androgens. Together, these findings suggest that regressive changes in motoneuron and muscle morphology seen after SCI can be ameliorated by treatment with gonadal hormones, further supporting a role for steroid hormones as neurotherapeutic agents in the injured nervous system.

Protective Effect of Paeoniflorin against Optic Nerve Crush

In order to evaluate the efficacy of traditional paeonia extract paeoniflorin against optic nerve crush, 16 Brown Norway rats were divided into two groups at random, with 8 rats in each group. In paeoniaflorin-treated group, 2 mg paeoniaflorin （total volum： 1 mL） was injected into rat＇s peritoneum one time a day for a period of 8 days. Rats in untreated group were given a single dose of vehicle. The optic nerve was crushed by a special forceps for 30 s in the left eye and a sham procedure was performed in the right eye on the 2nd day after the first injection. The retrograde fluorogold labeling of ganglion cells was conducted 5 days after optic nerve crush. The whole retina was flat-mounted thereafter. The ganglion cells that survived the crush were counted under fluorescent microscope by using an automatic counting software. As compared with the contralateral eye, the survival rate of ganglion cells in the left eye increased from 40.22% to 64.53% with a significant difference found between them （t=2.55, P=0.023）. The results suggested that the paeonia extract paeoniflorin possessed a protective effect against optic nerve crush.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically,both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment.However,in the clinical treatment of facial nerve defects,allogeneic donors are limited.In this experiment,we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect.Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group.In the autograft group,the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve,and in the xenogeneic acellular nerve graft group,this was done using a xenogeneic acellular nerve graft with trunk-branches.Examinations of facial symmetry,nerve-muscle electrophysiology,retrograde transport of labeled neuronal tracers,and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement,and that they could actively close their eyelids completely.The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups.Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey.Fluoro Gold-labeled neurons could be detected in the facial nuclei on the injured side.Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves.A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope.Taken together,our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys,with an effect similar to that of autologous nerve transplantation.Thus,a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available.The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No.2 People's Hospital of Nanjing Medical University,China(approval No.2018-D-1)on March 15,2018.

Femoral nerve regeneration and its accuracy under different injury mechanisms

Surgical accuracy has greatly improved with the advent of microsurgical techniques. However, complete functional recovery after peripheral nerve injury has not been achieved to date. The mechanisms hindering accurate regeneration of damaged axons after peripheral nerve injury are in urgent need of exploration. The present study was designed to explore the mechanisms of peripheral nerve regeneration after different types of injury. Femoral nerves of rats were injured by crushing or freezing. At 2, 3, 6, and 12 weeks after injury, axons were retrogradely labeled using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate（Dil） and True Blue, and motor and sensory axons that had regenerated at the site of injury were counted. The number and percentage of Dil-labeled neurons in the anterior horn of the spinal cord increased over time. No significant differences were found in the number of labeled neurons between the freeze and crush injury groups at any time point. Our results confirmed that the accuracy of peripheral nerve regeneration increased with time, after both crush and freeze injury, and indicated that axonal regeneration accuracy was still satisfactory after freezing, despite the prolonged damage.

Magnesium acetyltaurate prevents retinal damage and visual impairment in rats through suppression of NMDA-induced upregulation of NF-κB,p53 and AP-1(c-Jun/c-Fos)

Magnesium acetyltaurate(MgAT)has been shown to have a protective effect against N-methyl-D-aspartate(NMDA)-induced retinal cell apoptosis.The current study investigated the involvement of nuclear factor kappa-B(NF-κB),p53 and AP-1 family members(c-Jun/c-Fos)in neuroprotection by MgAT against NMDA-induced retinal damage.In this study,Sprague-Dawley rats were randomized to undergo intravitreal injection of vehicle,NMDA or MgAT as pre-treatment to NMDA.Seven days after injections,retinal ganglion cells survival was detected using retrograde labelling with fluorogold and BRN3A immunostaining.Functional outcome of retinal damage was assessed using electroretinography,and the mechanisms underlying antiapoptotic effect of MgAT were investigated through assessment of retinal gene expression of NF-κB,p53 and AP-1 family members(c-Jun/c-Fos)using reverse transcription-polymerase chain reaction.Retinal phospho-NF-κB,phospho-p53 and AP-1 levels were evaluated using western blot assay.Rat visual functions were evaluated using visual object recognition tests.Both retrograde labelling and BRN3A immunostaining revealed a significant increase in the number of retinal ganglion cells in rats receiving intravitreal injection of MgAT compared with the rats receiving intravitreal injection of NMDA.Electroretinography indicated that pre-treatment with MgAT partially preserved the functional activity of NMDA-exposed retinas.MgAT abolished NMDA-induced increase of retinal phospho-NF-κB,phospho-p53 and AP-1 expression and suppressed NMDA-induced transcriptional activity of NF-κB,p53 and AP-1 family members(c-Jun/c-Fos).Visual object recognition tests showed that MgAT reduced difficulties in recognizing the visual cues(i.e.objects with different shapes)after NMDA exposure,suggesting that visual functions of rats were relatively preserved by pre-treatment with MgAT.In conclusion,pre-treatment with MgAT prevents NMDA induced retinal injury by inhibiting NMDA-induced neuronal apoptosis via downregulation of transcriptional activity of NF-κB,p53 and AP-1-mediated c-Jun/c-Fos.The experiments were approved by the Animal Ethics Committee of Universiti Teknologi MARA(UiTM),Malaysia,UiTM CARE No 118/2015 on December 4,2015 and UiTM CARE No 220/7/2017 on December 8,2017 and Ethics Committee of Belgorod State National Research University,Russia,No 02/20 on January 10,2020.

Advances in genomic study of cortical projection neurons

The mammalian neocortex gives rise to perception and initiates voluntary motor responses.The cortical laminae are comprised of six distinct cellular layers of local circuit neurons and projection neurons.To explore molecular identities of the distinct cortical projection neurons,discovery-orientated genomic approaches have been adopted.Microarray analysis of dissected cortical tissues has been applied to identify cortical layer markers.Early neuronal cells were sorted by FACS from GFP-labeled embryonic brains for gene expression profiling.Laser capture microdissection of retrograde-labeled pro-jection neurons,when coupled with optimal RNA amplification technology,has become a valuable strategy for neuronal isolation and gene expression analysis in differentiated neurons.RNA sequencing technology is promising not only for the determination of gene expression,but also for discovery of posttranscriptional modifications of the complex neural system.There is no doubt that advances in genomic studies are opening up novel research avenues for our understanding of the cortical neuronal functions.