Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity, or weak signals. We developed an anterograde viral system based on a live attenuated vaccine of yellow fever – YFV-17D. Replication-deficient or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.

Several large or mid-scale collections of Drosophila enhancer traps have been recently created to allow for genetic swapping of GAL4 coding sequences to versatile transcription activators or suppressors such as LexA, QF, split-GAL4 (GAL4-AD and GAL4-DBD), GAL80 and QS. Yet a systematic analysis of the feasibility and reproducibility of these tools is lacking. Here we focused on InSITE GAL4 drivers that specifically label different subpopulations of olfactory neurons, particularly local interneurons (LNs), and genetically swapped the GAL4 domain for LexA, GAL80 or QF at the same locus. We found that the major utility-limiting factor for these genetic swaps is that many do not fully reproduce the original GAL4 expression patterns. Different donors exhibit distinct efficacies for reproducing original GAL4 expression patterns. The successfully swapped lines reported here will serve as valuable reagents and expand the genetic toolkits of Drosophila olfactory circuit research.

The hippocampus and the striatum represent two major systems in the brain for learning, memory and navigation. Although they were traditionally considered as two parallel systems responsible for distinct types of learning or navigation, increasing evidence indicates a close synergetic or competitive interaction between them. Both the hippocampus and the striatum consist of multiple anatomical and functional domains. Besides the limited direct projection from the hippocampus to the ventral striatum, most of the functional interaction between them may be mediated by polysynaptic projections. Polysynaptic connectivity has been difficult to examine due to a lack of methods to continuously track the pathways in a controlled manner. Here we developed a novel approach for directed stepwise polysynaptic tracing by reconstituting a replication–deficient retrograde transneuronal virus – pseudorabies virus lacking gene IE180 (PRVΔIE. We minimized PRV neurotoxicity by temporally restricting viral replication; and enabled both anatomical tracing and functional analysis of the circuits. With these tools, we delineated a hippocampus striatum wiring diagram, which consists of pathways from specific functional domains in the hippocampus to corresponding domains in the striatum via distinct intermediate regions. This polysynaptic wring diagram provides a structural foundation for further elucidation of the interaction between the hippocampus and the stratum in multiple brain functions.

Although the hippocampus is generally considered a cognitive center for spatial representation, learning, and memory, increasing evidence supports its roles in regulating locomotion. However, the neuronal mechanisms of the hippocampal regulation of locomotion and exploratory behavior remain unclear. In this study, we found that the inhibitory hippocampal synaptic projection to the medial septum (MS) bi-directionally controls the locomotor speed of mice. The activation of the MS-projecting interneurons in the hippocampus or the activation of the hippocampus-originated inhibitory synaptic terminals in the MS decreased locomotion and exploratory behavior. On the other hand, the inhibition of the hippocampus-originated inhibitory synaptic terminals in the MS increased locomotion. Unlike the septal projecting interneurons, the activation of the hippocampal interneurons projecting to the retrosplenial cortex did not change animal locomotion. Therefore, this study reveals a specific long-range inhibitory synaptic output from the hippocampus to the medial septum in the regulation of animal locomotion.

Although the hippocampus is generally considered a cognitive center for spatial representation, learning and memory, increasing evidence supports its roles in regulation of locomotion. However, the neuronal mechanisms of hippocampal regulation of locomotion and exploratory behavior remain unclear. Here we found that the inhibitory hippocampo-septal projection bi–directionally controls locomotion speed of mice. Pharmacogenetic activation of these septum–projecting interneurons decreased locomotion and exploratory behavior. Similarly, activation of the hippocampus–originated inhibitory terminal in the medial septum reduced locomotion. On the other hand, inhibition of the hippocampus–originated inhibitory terminal increased locomotion. The locomotion-regulative roles were specific to the septal projecting interneurons as activation of hippocampal interneurons projecting to the retrosplenial cortex did not change animal locomotion. Therefore, this study reveals a specific long-range inhibitory output from the hippocampus in the regulation of animal locomotion.