Integrated neural tracing and in-situ barcoded sequencing reveals the logic of SCN efferent circuits in regulating circadian behaviors

The circadian clock coordinates rhythms in numerous physiological processes to maintain organismal homeostasis. Since the suprachiasmatic nucleus(SCN) is widely accepted as the circadian pacemaker, it is critical to understand the neural mechanisms by which rhythmic information is transferred from the SCN to peripheral clocks. Here, we present the first comprehensive map of SCN efferent connections and suggest a molecular logic underlying these projections. The SCN projects broadly to most major regions of the brain, rather than solely to the hypothalamus and thalamus. The efferent projections from different subtypes of SCN neurons vary in distance and intensity, and blocking synaptic transmission of these circuits affects circadian rhythms in locomotion and feeding to different extents. We also developed a barcoding system to integrate retrograde tracing with in-situ sequencing, allowing us to link circuit anatomy and spatial patterns of gene expression. Analyses using this system revealed that brain regions functioning downstream of the SCN receive input from multiple neuropeptidergic cell types within the SCN, and that individual SCN neurons generally project to a single downstream brain region.This map of SCN efferent connections provides a critical foundation for future investigations into the neural circuits underlying SCNmediated rhythms in physiology. Further, our new barcoded tracing method provides a tool for revealing the molecular logic of neuronal circuits within heterogeneous brain regions.

Morphological characteristics of the striatal neural pathway by biotinylated dextran amine tracing in rats

Using neural pathway tracing and immunohistochemical technique, the striato-direct pathway （BDA3 kDa injected into the rat lateral globus pallidus） and striato-indirect pathway （BDA3 kDa injected into the substantia nigra pars reticulata） neurons were specifically labeled, and then subjected to double-labeled immunohistochemistry for mu-OPIOID Receptor （specifically-labeled striatal patch compartment）, D1, and D2, respectively. The experimental findings showed that there are no statistically significant differences in the soma diameter and the number of primary dendrites between the striato-direct （substantia nigra pars reticularis） and indirect （globus pallidum externum） neurons labeled retrograde by BDA3 kDa. In addition, these two kinds of projection neurons revealed no obvious coexistence. This evidence indicates that as a highly sensitive neural pathway tracer, BDA could yield reliably and exquisitely detailed labeling of target neurons and synaptic structures. The variance of the morphologic structures and the localization of neurons were not statistically significant between the striato-substantia nigra pars reticularis and the globus pallidum externum projection neurons. Mesencephalic and thalamic neurons correlated with striatal neurons in morphology. Especially the latter which make typical excitatory synaptic contacts with striato-direct and -indirect neurons. Thus, this evidence suggests that thalamic neurons may extensively excite striatal neurons.

MEMOIR: A Novel System for Neural Lineage Tracing

Memory by Engineered Mutagenesis with Optical In situ Readout（MEMOIR）is a novel strategy for lineage tracing that combines Cas9/g RNA and sequential multiplexed single-molecule RNA fluorescence hybridization（seqFISH）[1],which was created by Cai Long et al.at the California Institute of Technology[2].In MEMOIR,dynamic cellular event histories are recorded,then read out in single cells using seq FISH.Here,we introduce the

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

End-to-side neurorrhaphy repairs peripheral nerve injury：sensory nerve induces motor nerve regeneration

End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve.It involves suturing the distal stump of the disconnected nerve（recipient nerve） to the side of the intimate adjacent nerve（donor nerve）.However,the motor-sensory specificity after end-to-side neurorrhaphy remains unclear.This study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves after end-to-side neurorrhaphy.Thirty rats were randomized into three groups：（1） end-to-side neurorrhaphy using the ulnar nerve（mixed sensory and motor） as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve;（2） the sham group：ulnar nerve and cutaneous antebrachii medialis nerve were just exposed;and（3） the transected nerve group：cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied.At 5 months,acetylcholinesterase staining results showed that 34% ± 16% of the myelinated axons were stained in the end-to-side group,and none of the myelinated axons were stained in either the sham or transected nerve groups.Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%.In contrast,no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment.These results confirmed that motor neuron regeneration occurred after cutaneous nerve end-to-side neurorrhaphy.

Illuminating Neural Circuits in Alzheimer’s Disease

Alzheimer’s disease(AD)is the most common neurodegenerative disorder and there is currently no cure.Neural circuit dysfunction is the fundamental mechanism underlying the learning and memory deficits in patients with AD.Therefore,it is important to understand the structural features and mechanisms underlying the deregulated circuits during AD progression,by which new tools for intervention can be developed.Here,we briefly summarize the most recently established cutting-edge experimental approaches and key techniques that enable neural circuit tracing and manipulation of their activity.We also discuss the advantages and limitations of these approaches.Finally,we review the applications of these techniques in the discovery of circuit mechanisms underlyingβ-amyloid and tau pathologies during AD progression,and as well as the strategies for targeted AD treatments.

HSV-1 H129-Derived Anterograde Neural Circuit Tracers:Improvements, Production, and Applications

Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1(HSV-1) strain H129(H129), and have been successfully applied to map diverse neural circuits.Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

The Expression of Calcitonin Gene-related Peptide on the Neurons Associated Zusanli(ST 36)in Rats

Objective： To investigate the biochemical characteristic of the neurons associated Zusanli （ST 36） in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B （AF594-CTB） neural tracing and calcitonin gene-related peptide （CGRP） fluorescent immunohistochemical techniques. Methods： Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body. After 3 surviving days, the rat＇s spinal cord and dorsal root ganglia （DRGs） at lumbar segments were dissected following perfusion with 4% paraformaldehyde, cut into sections, and then stained with CGRP- fluorescent immunohistochemical method. Results： AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG, and the labeled motor neurons located in the dorsolateral and intermediate regions of lamina IX from L3-L5 segments with high concentration at L4. Meanwhile, CGRP- positive neural labeling distributed symmetrically on both sides of DRGs, anterior and dorsal horns of spinal cord. In the AF594-CTB labeled neurons, 37% sensory neurons and 100% motor neurons expressed CGRP- positive. Conclusion： These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns, and contained CGRP-expression.

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain.Two-photon Ca^(2+)imaging in vivo using a cranial window and specific neuronal labeling enables realtime,in situ,and long-term imaging of the living brain.Here,we constructed a recombinant rabies virus containing the Ca^(2+)indicator GCaMP6 s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6 s signal reliability.This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca^(2+)activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca^(2+)imaging.Notably,we were able to record single-spine spontaneous Ca2+activity in specific circuits.Distinct spontaneous Ca^(2+)dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits.Our method can be applied to monitor Ca^(2+)dynamics in specific input circuits in vivo,and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.