A Cre-Dependent, Anterograde Transsynaptic Viral Tracer for Mapping Output Pathways of Genetically Marked Neurons

Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread trans-synaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde trans-synaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde trans-neuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knock-in mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons and anterograde-specific. These data provide proof-of-principle for a conditional, non-diluting anterograde trans-synaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations

Sympathetic neuroblasts undergo a developmental switch in trophic dependence

Sympathetic neurons require NGF for survival, but it is not known when these cells first become dependent on neurotrophic factors. We have examined in vitro mitotically active sympathetic neuroblasts immuno-isolated from different embryonic stages, and have correlated this functional data with the expression of neurotrophin receptor mRNAs in vivo. Cells from E14.5 ganglia are supported by neurotrophin-3 (NT-3) in a serum-free medium, but not by NGF; NT-3 acts as a bona fide survival factor for these cells and not simply as a mitogen. By birth, sympathetic neurons are well-supported by NGF, whereas NT-3 supports survival only weakly and at very high doses. This change in neurotrophin-responsiveness is correlated with a reciprocal switch in the expression of trkC and trkA mRNAs by sympathetic neuroblasts in vivo. These data suggest that neurotrophic factors may control neuronal number at earlier stages of development than previously anticipated. They also suggest that the acquisition of NGF-dependence may occur, at least in part, through the loss of receptors for these interim survival factors

Connectional architecture of a mouse hypothalamic circuit node controlling social behavior

Type 1 estrogen receptor-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl^(Esr1)) play a causal role in the control of social behaviors, including aggression. Here we use six different viral-genetic tracing methods to systematically map the connectional architecture of VMHvl^(Esr1) neurons. These data reveal a high level of input convergence and output divergence (“fan-in/fan-out”) from and to over 30 distinct brain regions, with a high degree (∼90%) of bidirectionality, including both direct as well as indirect feedback. Unbiased collateralization mapping experiments indicate that VMHvl^(Esr1) neurons project to multiple targets. However, we identify two anatomically distinct subpopulations with anterior vs. posterior biases in their collateralization targets. Nevertheless, these two subpopulations receive indistinguishable inputs. These studies suggest an overall system architecture in which an anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. This architecture differs from the “brain-inspired,” hierarchical feed-forward circuits used in certain types of artificial intelligence networks

Connectional architecture of a mouse hypothalamic circuit node controlling social behavior

Type 1 estrogen receptor-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl^(Esr1)) play a causal role in the control of social behaviors, including aggression. Here we use six different viral-genetic tracing methods to systematically map the connectional architecture of VMHvl^(Esr1) neurons. These data reveal a high level of input convergence and output divergence (“fan-in/fan-out”) from and to over 30 distinct brain regions, with a high degree (∼90%) of bidirectionality, including both direct as well as indirect feedback. Unbiased collateralization mapping experiments indicate that VMHvl^(Esr1) neurons project to multiple targets. However, we identify two anatomically distinct subpopulations with anterior vs. posterior biases in their collateralization targets. Nevertheless, these two subpopulations receive indistinguishable inputs. These studies suggest an overall system architecture in which an anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. This architecture differs from the “brain-inspired,” hierarchical feed-forward circuits used in certain types of artificial intelligence networks

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms—SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)—and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity

Postmigratory Neural Crest Cells Expressing c-RET Display Restricted Developmental and Proliferative Capacities

c-RET is an orphan receptor tyrosine kinase essential for enteric neurogenesis in mice and is involved in several human genetic disorders. RET is also one of the earliest surface markers expressed by postmigratory neural crest cells in the gut. We generated anti-RET monoclonal antibodies to isolate such cells. We find that RET+ cells are antigenically and functionally distinct from neural crest stem cells (NCSCs) characterized previously. Unlike NCSCs, which are RET− and MASH1−, most RET+ cells express MASH1. Moreover, unlike NCSCs, which are multipotent and have high proliferative capacity, many RET+ cells generate only neurons following a limited number of divisions. This behavior is observed even in the presence of glial growth factor, a polypeptide that suppresses neuronal and promotes glial differentiation by NCSCs. These data provide direct evidence for the existence of committed neuronal progenitor cells and support a model of neural crest lineage diversification by progressive restriction of developmental potential

MASH1 maintains competence for BMP2-induced neuronal differentiation in post-migratory neural crest cells

Background: The interplay between growth factors and transcription factors in vertebrate neurogenesis is poorly understood. MASH1 is a basic helix–loop–helix (bHLH) transcription factor that is essential for autonomic neurogenesis. Bone morphogenetic protein (BMP) 2, and its relative BMP4, have been shown to induce expression of MASH1 and to promote autonomic neuronal differentiation in neural crest stem cells. The relationship between expression of MASH1 and the neurogenic competence of neural crest cells has not been investigated, however. Results: We have examined the function of MASH1 in neurogenic competence using a population of immuno-isolated neural-crest-derived progenitor cells. Post-migratory neural crest cells isolated from fetal rat gut expressed Mash1, yet comprised a mixture of committed neuronal precursors and non-neuronal cells. The non-neuronal cells remained competent to differentiate to neurons, however, if challenged with BMP2. Such competence declines with time and is paralleled by a decline in Mash1 expression in the cells. Expression of endogenous Mash1 can be maintained by BMP2; in turn, constitutive expression of Mash1 from a retroviral vector maintains competence for neuronal differentiation in response to late addition of BMP2. Conclusions: These data suggest that MASH1 promotes competence for neurogenesis, in a manner similar to its homologs, the proneural genes achaete–scute in Drosophila. They also reveal an unexpected feedback interaction between BMP2 and MASH1 during neuronal differentiation. MASH1 may play multiple roles at successive stages of development within a neurogenic lineage, only one of which is revealed by a loss-of-function mutation

SOX10 Maintains Multipotency and Inhibits Neuronal Differentiation of Neural Crest Stem Cells

The mechanisms that establish and maintain the multipotency of stem cells are poorly understood. In neural crest stem cells (NCSCs), the HMG-box factor SOX10 preserves not only glial, but surprisingly, also neuronal potential from extinction by lineage commitment signals. The latter function is reflected in the requirement of SOX10 in vivo for induction of MASH1 and PHOX2B, two neurogenic transcription factors. Simultaneously, SOX10 inhibits or delays overt neuronal differentiation, both in vitro and in vivo. However, this activity requires a higher Sox10 gene dosage than does the maintenance of neurogenic potential. The opponent functions of SOX10 to maintain neural lineage potentials, while simultaneously serving to inhibit or delay neuronal differentiation, suggest that it functions in stem or progenitor cell maintenance, in addition to its established role in peripheral gliogenesis