The medial part of the olfactory tubercle (OT) is a brain structure located at the interface of the reward and olfactory system. It is closely related to pheromone-rewards, natural reinforcement, addiction and many other behaviors. However, the structure of the anatomic circuitry of the medial part of the OT is still unclear. In the present study, the medial part of the OT was found to be highly connected with a wide range of brain areas with the help of the pseudorabies virus tracing tool. In order to further investigate the detailed connections for specific neurons, another tracing tool – rabies virus was utilized for D1R-cre and D2R-cre mice. The D1R and D2R neurons in the medial part of the OT were both preferentially innervated by the olfactory areas, especially the piriform cortex, and both had similar direct input patterns. With the help of the adeno-associated virus labeling, it was found that the two subpopulations of neurons primarily innervate with the reward related brain regions, with slightly less axons projecting to the olfactory areas. Thus, the whole-brain input and output circuitry structures for specific types of neurons in the medial part of the OT were systematically investigated, and the results revealed many unique connecting features. This work could provide new insights for further study into the physiological functions of the medial part of the OT.

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The mammalian basal forebrain (BF), a heterogenous structure providing the primary cholinergic inputs to cortical and limbic structures, plays a crucial role in various physiological processes such as learning/memory and attention. Despite the involvement of the BF cholinergic neurons (BFCNs) in olfaction related memory has been reported, the underlying neural circuits remain poorly understood. Here, we combined viral trans-synaptic tracing systems and ChAT-cre transgenic mice to systematically reveal the relationship between the olfactory system and the different subsets of BFCNs. The retrograde adeno-associated virus and rabies virus (AAV-RV) tracing showed that different subregional BFCNs received diverse inputs from multiple olfactory cortices. The cholinergic neurons in medial and caudal horizontal diagonal band Broca (HDB), magnocellular preoptic area (MCPO) and ventral substantia innominate (SI; hereafter HMS complex, HMSc) received the inputs from the entire olfactory system such as the olfactory bulb (OB), anterior olfactory nucleus (AON), entorhinal cortex (ENT), basolateral amygdala and especially the piriform cortex (PC) and hippocampus (HIP); while medial septum (MS/DB) and a part of rostral HDB (hereafter MS/DB complex, MS/DBc), predominantly from HIP; and nucleus basalis Meynert (NBM) and dorsal SI (hereafter NBM complex, NBMc), mainly from the central amygdala. The anterograde vesicular stomatitis virus (VSV) tracing further validated that the major target of the OB to the BF is HMSc. To correlate these structural relations between the BFCNs and olfactory functions, the neurons activated in the BF during olfaction related task were mapped with c-fos immunostaining. It was found that some of the BFCNs were activated in go/no-go olfactory discrimination task, but with different activated patterns. Interestingly, the BFCNs in HMSc were more significantly activated than the other subregions. Therefore, our data have demonstrated that among the different subgroups of BFCNs, HMSc is more closely related to the olfactory system, both structurally and functionally. This work provides the evidence for distinct roles of different subsets of BFNCs in olfaction associated memory.

Background: Efficient viral vectors for mapping and manipulating long projection neuronal circuits are crucial in brain structural and functional studies. The glycoprotein gene-deleted SAD strain rabies virus pseudotyped with the N2C glycoprotein (SAD-RV(ΔG)-N2C(G)) shows high neuro-tropism in cell culture, but its in vivo retrograde infection efficiency and neuro-tropism have not been systematically characterized. Methods: SAD-RV(ΔG)-N2C(G) and two other broadly used retrograde tracers, SAD-RV(ΔG)-B19(G) and rAAV2-retro were respectively injected into the VTA or DG in C57BL/6 mice. The neuron numbers labeled across the whole brain regions were counted and analyzed by measuring the retrograde infection efficiencies and tropisms of these viral tools. The labeled neural types were analyzed using fluorescence immunohistochemistry or GAD67-GFP mice. Result: We found that SAD-RV (ΔG)-N2C (G) enhanced the infection efficiency of long-projecting neurons by ~ 10 times but with very similar neuro-tropism, compared with SAD-RV (ΔG)-B19(G). On the other hand, SAD-RV(ΔG)-N2C(G) showed comparable infection efficiency with rAAV2-retro, but had a more restricted diffusion range, and broader tropism to different types and regions of long-projecting neuronal populations. Conclusions: These results demonstrate that SAD-RV(ΔG)-N2C(G) can serve as an effective retrograde vector for studying neuronal circuits. Key words：Viral vector, N2C Glycoprotein, Neuronal circuits, Retrograde tracing Background In the central nervous system, distinct brain regions work corporately through particular circuit connections to process different and complex information [1-6]. Neuronal

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that has broad range of hosts. Stable JEV vector has not been reported yet. Here, we constructed a JEV-EGFP by inserting a fragment of C38 (the N-terminal 38 amino acids of capsid)-EGFP-FMDV2A into the junction between 5'UTR and the N-terminus of capsid gene. An adaptive nucleotide mutation T45G (location at the N-terminus of capsid gene), resulting in an amino acid change from asparagine to lysine (N15K), was identified by genome sequencing. It stabilized the vector and enlarged the virion. The stabilizing effect might be general because it is also stable when EGFP was replaced with another marker, SNAP. A model was proposed for this stabilization effect based on previously published and our data. This finding may be used to construct various JEV-based stable delivery systems for virological studies and neural circuit tracing.

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