A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling

Background Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK. Methods Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties. Results H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer’s disease and control mouse brains. Conclusions gKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

An extended toolkit for production and use of RVdG-CVS-N2c rabies viral vectors uncovers hidden hippocampal connections

From the large collection of molecular tools used to investigate neuronal connectivity, envA-pseudotyped rabies viral vectors (RVdGenvA) uniquely enable cell-type specific, trans-synaptic retrograde labeling. However, widespread use of the powerful and flexible method is to date hindered by low-yield and cumbersome production pipelines. Here, we report the development of new cell lines, which significantly reduce production time while increasing viral titer and eliminating background contamination from native-coat particles. We further show that RVdGenvA-CVS-N2c vectors produced using this system retain their enhanced retrograde-trafficking when compared with SAD-B19 vectors, allowing us to uncover undescribed cortico-hippocampal connections and to monitor activity in a cortical microcircuit of behaving animals. Along with new suites of AAV and RVdG-CVS-N2c vectors, developed to enable retrograde labeling from a wide range of neuronal populations and tailored for diverse experimental requirements, we present here an optimal system for mapping, manipulating and imaging of neuronal circuits.

Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague–Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

Three-Dimensional Mapping of Retrograde Multi-Labeled Motor Neuron Columns in the Spinal Cord

The quantification and distribution characteristics of spinal motor neurons play important roles in the study of spinal cord and peripheral nerve injury and repair. In most research, the sole retrograde labeling of each nerve or muscle could not simultaneously obtain the distributions of different motor neuron subpopulations. Therefore, it did not allow mapping of spatial relationships of different motor neuron columns for disclosing the functional relationship of different nerve branches. Here, we combined the multiple retrograde labeling, optical clearing, and imaging for three-dimensional (3D) visualization of motor neurons of multiple brachial plexus branches. After screening fluorescent tracers by the labeling feasibility of motor neurons and fluorescence compatibility with optical clearing, we performed mapping and quantification of the motor neurons of ulnar, median, and radial nerves in the spinal cord, then disclosed the relative spatial distribution among different neuronal subpopulations. This work will provide valuable mapping data for the understanding of the functional relationships among brachial plexus branches, hopefully facilitating the study of regeneration of axons and remodeling of motor neurons in peripheral nerve repair.

Rostro-Caudal Specificity of Corticospinal Tract Projections in Mice

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III–V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

Connection “Stripes” in the Primate Insula

The insula has been classically divided into vast granular, dysgranular and agranular sectors. Over the years, several distinct studies proposed subdivisions of these sectors, with however no consensus. We recently proposed a cyto- and myelo-architectonic partition in which each sector contained sharply delimited areas (Evrard et al. 2014 J Comp Neurol 522: 64-97). Some of these areas were further divided into distinct subareas with obvious functional implications. Here, we examined the spatial relationship between architectonic boundaries and tract-tracing labeling in the insula in the macaque monkey. Injections of neuronal tracers in distinct areas of the prefrontal or anterior cingulate cortices produced heterogeneous and discontinuous patterns of anterograde and retrograde labeling in the insula. These patterns were made of sharply delimited patches forming anteroposterior stripes across consecutive coronal sections. While the overall pattern of labeling varied with the injection site, the patches systematically coincided with specific architectonic subareas, particularly in the dysgranular insula. This unequivocally validates our prior architectonic partition and strongly supports the idea of a refined modular Bauplan of the primate insula. This modular organization may underlie a serial stream of integration of interoception with ‘self-agency’ and ‘social’ activities across distinct insulo-prefrontal processing units that need to be explored.

Rostro-caudal specificity of corticospinal tract projections in mice

ABSTRACTRostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT-reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex, the rostral forebrain area (RFA), and a region in the lateral cortex near the barrel field. Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the RFA or lateral cortex. With BDA injections into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic-sacral levels. With BDA injections into the RFA and lateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae; Axons from lateral sensorimotor cortex terminated primarily in deep layers of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.SIGNIFICANCEMice are used extensively for studies of regeneration following spinal cord injury because of the ability to create genetic modifications to explore ways to enhance repair and enable axon regeneration. A particular focus has been the corticospinal tract (CST) because of its importance for voluntary motor function. Here, we document features of the rostro-caudal specificity of CST