Brain-wide map of projections from mice ventral subiculum

Neuroscience Letters 629 (2016) 171–179

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Research article

Brain-wide map of projections from mice ventral subiculum He Tang a,b , Gui-Sheng Wu a , Jing Xie a,b , Xiaobin He c , Ke Deng a , Huadong Wang c , Fuqiang Xu c,d,∗∗ , Huai-Rong Luo a,∗ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China b University of Chinese Academy of Sciences, Beijing 100039, China c State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China d Wuhan National Laboratory for Optoelectronics, Wuhan 430074, China

h i g h l i g h t s • Brain regions receive direct projections from ventral subiculum were identified. • Brain regions receive secondary projections from ventral subiculum were determined. • The transneuronal tracing of herpes simplex virus could be clearly identified.

a r t i c l e

i n f o

Article history: Received 26 April 2016 Received in revised form 6 July 2016 Accepted 11 July 2016 Available online 12 July 2016 Keywords: Hippocampal formation Ventral subiculum Herpes simplex virus Neural circuits Brain map

a b s t r a c t The hippocampal formation plays a critical role in episodic memory formation and spatial navigation. Within the hippocampus, the subiculum is considered to be a hub connecting the hippocampal formation to the remainder of the brain. There are functional differences between the dorsal and ventral part of subiculum, while the ventral subiculum (vSub) plays a role in anxiety, stress and emotion. In the present study, we examined the projection of the ventral subiculum to the whole brain in mice by using a modified herpes simplex virus 1 strain H129 with an inserted fluorescent protein gene. In our experiments, the modified H129 transits the primary-order, second-order, and third-order neuronal projections at 36–44, 52–60 and 68–76 h after inoculation in mice, respectively. Our data revealed that vSub directly projects to the medial entorhinal cortex, amygdalohippocampal area, anterodorsal thalamic nucleus, medial hypothalamus, supramammillary nucleus, medial septal nucleus and adjacent diagonal band, the connections between median raphe nucleus and interpeduncular nucleus in brain stem, while ventral prefrontal cortex, laterodorsal tegmental nucleus and locus coeruleus receives second-order projections from vSub. Our data would help further understanding the functional connections of vSub with other brain regions. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Extensive studies have been focused on the function and structure of hippocampal formation. The hippocampal formation plays a central role in episodic memory formation by integrating sensory

∗ Corresponding author at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, 134 Lanhei Road, Kunming, Yunnan 650201, China. ∗∗ Corresponding author at: Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, 30 West Xiaohongshan Road, Wuhan 430071, China. E-mail addresses: [email protected] (F. Xu), [email protected] (H.-R. Luo). http://dx.doi.org/10.1016/j.neulet.2016.07.014 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.

and emotional information into space and time. The hippocampal formation is composed of the dentate gyrus, areas CA3 and CA1, entorhinal cortex and subiculum (Sub). The subiculum lies between CA1 and the entorhinal cortex, and receives extensive projections from CA1 and other parts of the hippocampus as well as projects back to these regions [1,2] and adjacent portions of the ventral and rostral parts of the pre- and parasubiculum [3,4]. Except the hippocampal formation, the subiculum is reported to project to medial and lateral entorhinal cortex (MEC and LEC), septal nucleus, amygdala, the bed nuclei of the stria-terminalis (BST), the nucleus accumbens, frontal cortex, hypothalamus, and mammillary bodies [4–7]. Via these regions, the subiculum further projects to the medial preoptic area, medial orbital cortices, and the prefrontal and

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the prelimbic regions, possibly including infralimbic and anterior cingulate cortices [8,9]. Through this diverse interplay of connectivity, the subiculum serves as the hub for information processing between the hippocampus and the remaining parts of the brain [10]. It has been proposed that the dorsal and ventral components of the subiculum serve in discrete functional roles beyond just being the hub of the hippocampus. The dorsal component is important for the processing of space, movement and memory, while the ventral component is responsible for emotion and motivated behavior [11–13]. Development and application of neuronal tracers, such as Fluoro Gold (FG), cholera toxin B subunit (CTb), Phaseolus vulgarisleucoagglutinin (PHA-L) or biotinylated dextran amines (BDA) methods. These neuronal tracers could be taken up by the neurons and are transported across the synapse, staining the neuronal fibers efficiently. But injected tracer molecules could be diluted during transport along neuronal fibers [14,15], leading to sensitivity reduction after trans-synaptic transmission. To help address some of these concerns, neurotropic viruses have been developed as effective transneuronal tracers. These viruses replicate themselves in recipient neurons and produce strong transneuronal labeling [16,17]. The order of sequential projection of neurons can also be discerned based on knowledge of the life cycle of virus [18,19]. In this study, we used transgenic herpes simplex virus and immunohistochemical methods to map the whole-brain distribution patterns of vSub [16]. Our results showed that vSub directly projectes to the medial entorhinal cortex, amygdalohippocampal area, anterodorsal thalamic nucleus, medial hypothalamus, supramammillary nucleus, medial septal nucleus and adjacent diagonal band, the connections between median raphe nucleus and interpeduncular nucleus in brain stem, while ventral prefrontal cortex, laterodorsal tegmental nucleus and locus coeruleus receives second-order projections from vSub.

2. Materials and methods 2.1. Animals C57BL/6 male mice (6 weeks, 18–22 g) were purchased from Vital River Co., Ltd. (Beijing, China). The animals were housed at 4 per cage under pathogen free conditions with 12/12 h light/dark cycles, temperature of 22 ± 2 ◦ C, relative humidity 50–60%, with free access to food and water. They were habituated to the animal facility for one week before surgical procedure. The experiments were performed in strict accordance with the protocols approved by the Institutional Animal Care and Use Committee of Wuhan Institute of Physics and Mathematics and the Kunming Institute of Botany, Chinese Academy of Sciences.

2.2. Preparation of recombinant H129 virus The herpes simplex virus 1 strain H129 tagged by tdTomato (H129-TT) was used for multisynaptic anterograde tracing. H129TT is a multiple transsynaptic tracer that was kindly supplied by L. W. Enquist. It expresses Td-tomato driven by CAG promoter and was generated by propagating a Cre-dependent, anterograde viral tracer- H129TK-TT in cell-line 293T expressing Cre recombinase. The Cre recombinase removed the loxP-STOP-loxP element from the genome of H129TK-TT, and made H129-TT expressing Td-Tomato. The recombinant H129-TT was purified through 3 cycles of fluorescence plaque purification and further propagated in Vero cells (ATCC, Manassas, VA). The culture solution was concentrated at 4000 g for 10 min. The supernatant was collected, filtered with 0.45 ␮m filters (Millipore) and further concentrated 100-fold

through ultra-high speed centrifugation (Beckman). H129-TT was then assayed and stored in aliquots at −80 ◦ C. 2.3. Surgical procedure and viral injection The mice were anesthetized with pentobarbital (80 mg/kg) by intraperitoneal injection and then mounted in a stereotaxic holder. The skull was exposed and a small hole was drilled above the injection site. 150 nl of H129-tdTomato virus (6 × 109 pfu/ml) was injected into the vSub with coordinate AP/DV/ML: −4.10/−3.85/−2.5 mm using a pulled glass pipette at a speed of 30 nl/min (Nanoliter 2000, WPI, USA). After the injection, the pipette was left in place for 15 min and then was drawn out gently. Beginning at 28 h, three mice were sacrificed every 8 h, until 76 h. All operations with virus were conducted in a Level-2 Biosafety laboratory. 2.4. Histology and immunostaining After pentobarbital overdose, mice were intracardially perfused with 0.9% saline solution followed by 4% paraformaldehyde in PBS. Mouse brains were removed immediately and placed in 4% paraformaldehyde solution overnight for post fixation and then soaked in 30% sucrose solution for at least 48 h for cryoprotection. With a cryostat (Leica CM1950), the brains were sequentially cut into 40 ␮m thick coronal sections from the olfactory bulb to cerebellum (approximately from Bregma +4.50 mm to −6.40 mm). The sections were cleaned with PBS and mounted on chrome-gelatin subbed glass slides in sequence. To enhance the fluorescence intensity, the sections were blocked with 3% BSA in PBS-0.3% Triton X-100 for 1 h at 37 ◦ C and subsequently incubated with primary antibody (1:500, rabbit anti-DsRed;Takara Clontech, Cat# 632496, RRID: AB 10013483) for 20 h at 4 ◦ C. After washing, the sections were incubated with Cy3 goat anti-rabbit secondary antibody (1:300, Jackson ImmunoResearch, Cat# 111-165-144, RRID: AB 2338006) for 1 h at 37 ◦ C. Finally, brain sections were coverslipped with 70% DAPI-glycerol mounting medium. 2.5. Imaging and analysis The images of whole brain sections were acquired with a digital slide scanner (Nicon Eclipse Ti) and adjusted by NIS-Elements. The locations of the labeled neurons and outlines of the brain nuclei were manually defined according to the mouse brain atlas [20]. 3. Results Three animals were sacrificed at each time point of 24, 36, 44, 52, 60, 68 and 76 h after injection of virus. The injection sites were highly circumscribed within the ventral part of subiculum (Fig. 1) and the infected neurons could be observed clearly (Fig. 2). Animals killed at 24 h did not show the transport of the virus beyond the injection site (data not shown). The density and distribution of viral infection in animals sacrificed at 36 and 44 h after virus injection were the same, and were determined as the first-order projection sites. These sites ranged from Bregma +1.18 mm to −4.60 mm in the rostro-caudal axis (Fig. 3, Table 1). Animals sacrificed at 52 and 60 h after inoculation shared the same density and distribution of viral infection. The newly present infected regions after the firstorder projections were considered second-order projection sites, which ranged from Bregma +2.46 mm to −5.88 mm in the rostrocaudal axis (Fig. 3, Table 1). The regions infected during 68 and 76 h were identical and determined as third-order projection sites, which were distributed from Bregma +3.56 mm to −5.88 mm (Fig. 3, Table 1).

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Fig. 1. The viral injection site in ventral subiculum (vSub).

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The coronal section of Bregma −4.10 mm illustrates the injection site (arrowhead).

Fig. 2. The representative fluorescence micrographs showing the neurons infected by H129.

The neurons initially infected during 36–44 h were in the posteroventral dentate gyrus and medial entorhinal cortex of the hippocampal formation, indicating direct projection from vSub (Fig. 4a and d). The neurons infected during 52–60 h were distributed in the posterodorsal dentate gyrus, most of medial and lateral entorhinal cortex, and the ventral part of parasubiculum (Fig. 4b and e). These regions might receive the projections from vSub secondarily. After 68 h of viral injection, the infected regions in hippocampal formation were larger (Fig. 4c and f). In the amygdala and adjacent areas, the projections from vSub primarily appeared in the amygdalohippocampal area at 36–44 h (Fig. 4g). Subsequently the neurons receiving secondary projection from vSub were densely evident in amygdala and expanding to adjacent areas (Fig. 4h), including central, medial, lateral, basal and cortical amygdaloid nuclei, and the intra-amygdaloid division of bed nucleus of the striaterminalis (Fig. 4i).

In the diencephalon, the neurons receiving projections from vSub appeared in the hypothalamus, thalamus and septal nucleus. In the ventral part of the hypothalamus, neurons infected at 36–44 h after inoculation appeared in the medial part of supramammillary nucleus (Fig. 5a). At 52–60 h, the labeled neurons had increased in number within the supramammillary nucleus and were present in the dorsal and ventral border of mammillary nucleus (Fig. 5b). At 68–76 h after inoculation, the labeled neurons of mammillary nucleus were distributed to the lateral, ventral and central part of the mammillary nucleus (Fig. 5c). In the middle of hypothalamus, the neurons infected 36–44 h after virus injection were found sparsely scattered in the lateral and posterior hypothalamus (Fig. 5d). At 52–60 h after virus injection, the labeled neurons could be observed in most parts of the hypothalamus, including various hypothalamic nuclei and the preoptic nucleus (Fig. 5e).

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Fig. 3. A summary illustration for the distribution of projections from mice ventral subiculum. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) The distance indicated beneath the coronal section was its location relative to Bregma. The distribution of infected area during 36–44 h, 52–60 h, and 68–76 h after inoculation were indicated by light red, moderate red, and dark red, respectively.

Table 1 The distribution of projections from mice ventral subiculum. First order (36–44 h)

Second order (52–60 h)

Third order (68–76 h) Medial part of the dorsal tenia tecta

Septum

Medial septal nucleus; Diagonal band nucleus

Thalamus

Rostral part of the anterodorsal thalamic nucleus; Anterior part of the paraventricular, paratenial, paracentral, central medial and reuniens thalamic nucleus Ventromedial part of the lateral and posterior hypothalamus; Medial part of the supramammillary nucleus Amygdalohippocampal area

Dorsal peduncular cortex; Dorsal part of the dorsal tenia tecta; Medial and ventral orbital cortex Ventral and intermediate parts of the lateral septal nucleus; Accumbens nucleus Medial and lateral part of the anterior thalamus; Medial part of the posterior thalamus

Preoptic nucleus; Medial part of the medial and lateral hypothalamus; Dorsal and ventral border of the mammillary nucleus

Lateral and vental part of the hypothalamus; Lateral, ventral and central part of the mammillary nucleus Lateral part of the central, medial, lateral, basal and cortical amygdaloid nuclei

Anterior Part

Hypothalamus

Amygdala

Hippocampal Formation

Ventral part of the medial entorhinal cortex; Posteroventral dentate gyrus

Brain Stem

Connections between the median raphe nucleus and interpeduncular nucleus

Central part of the central, medial, lateral, basal and cortical amygdaloid nuclei; Intra-amygdaloid division of bed nucleus of the striaterminalis Dorsal part of the medial entorhinal cortex; Ventral part of the lateral entorhinal cortex; Posterodorsal dentate gyrus; Ventral part of parasubiculum Medial part of the raphe nucleus; Interpeduncular nucleus; Laterodorsal tegmental nucleus; Locus coeruleus

After 68–76 h, the neurons in the medial and ventral parts of the hypothalamus were densely labeled (Fig. 5f). In thalamus, as 36–44 h after inoculation, densely labeled neurons appeared in the rostral part of the anterodorsal thalamic nucleus but not the anteroventral and anteromedial thalamic nucleus (Fig. 5g). Sparsely labeled neurons could be observed in

Dorsal part of the lateral septal nucleus

Lateral part of the thalamus

Dorsal part of the lateral entorhinal cortex

Lateral part of the raphe nucleus

anterior paraventricular nucleus, paratenial nucleus, paracentral nucleus, and central medial nucleus of the rostral thalamus and the reuniens nucleus (Fig. 5g). By 52–60 h after inoculation, the infected neurons were much greater in number and were incorporated in various thalamic subdivisions (Fig. 5h and i).

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Fig. 4. Distribution of anterograde labeled neurons in the hippocampus, entorhinal cortex, and amygdala. Representative fluorescent micrographs show the distribution of anterograde labeled neurons in hippocampus, entorhinal cortex, and amygdala. Coronal section of Bregma −3.90 mm shows the distribution of labeled neurons in hippocampus at 36–44 h (a), 52–60 h (b), and 68–76 h (c) after inoculation. Coronal section of Bregma −4.48 mm shows the distribution of infected neurons in entorhinal cortex at 36–44 h (d), 52–60 h (e), and 68–76 h (f) after inoculation. Coronal section of Bregma −2.00 mm shows the distribution of infected neurons in amygdala at 36–44 h (g), 52–60 h (h), and 68–76 h (i) after inoculation. AHi: amygdalohippocampal area; BLA: basolateral amygdaloid nucleus, anterior part; BLP: basolateral amygdaloid nucleus, posterior part; BMP: basomedial amygdaloid nucleus, posterior part; LEnt: lateral entorhinal cortex; MEnt: medial entorhinal cortex; PaS: parasubiculum; PLCo: posterolateral cortical amygdaloid nucleus; PMCo: posteromedial cortical amygdaloid nucleus.

The septal nucleus and adjacent areas also receive primary projections from vSub evidenced by the neurons infected at 36–44 h, although few labeled neurons appeared in medial septal nucleus and the diagonal band nucleus (Fig. 5j). At 52–60 h after inoculation, the labeled neurons increased and spread to most of the medial septal nucleus and diagonal band nucleus, and ventral and intermediate parts of the lateral septal nucleus (Fig. 5k). At this period, the accumbens nucleus was also lightly labeled (Fig. 5k), suggesting secondary projection from vSub. At 68–76 h, the dorsal part of lateral septal nucleus was also labeled (Fig. 5l).

In the brain stem, a few labeled neurons in the connections of the median raphe nucleus and interpeduncular nucleus could be observed at 36–44 h after virus injection, indicating first-order projection from vSub (Fig. 6a). After that, the dense labeled region spread to the raphe nucleus (Fig. 7e and f) and almost the entire interpeduncular nucleus (Fig. 6b and c). No infected neurons in the prefrontal cortex could be found at 36–44 h after inoculation, indicating no direct projections from vSub are likely (Fig. 7a). At 52–60 h after injection, infected neurons could be found in the medial orbital cortex, ventral orbital

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Fig. 5. Distribution of anterograde labeled neurons in the diencephalon. Representative fluorescent micrographs show the distribution of anterograde labeled neurons in mammillary body, hypothalamus, thalamus, and septal nucleus. Coronal section of Bregma −3.00 mm shows the distribution of labeled neurons in mammillary body at 36–44 h (a), 52–60 h (b), and 68–76 h (c) after inoculation. Coronal section of Bregma −2.30 mm shows the distribution of infected neurons in hypothalamus at 36–44 h (d), 52–60 h (e), and 68–76 h (f) after inoculation. Coronal section of Bregma −0.40 mm shows the distribution of infected neurons in thalamus at 36–44 h (g), 52–60 h (h), and 68–76 h (i) after inoculation. Coronal section of Bregma +0.90 mm shows the distribution of labeled neurons in septal nucleus at 36–44 h (j), 52–60 h (k), and 68–76 h (l) after inoculation. Acb: accumbens nucleus; AD: anterodorsal thalamic nucleus; Arc: arcuate hypothalamic nucleus; HDB: nucleus of the horizontal limb of the diagonal band; LH: lateral hypothalamic area; LSV: ventral part of lateral septal nucleus; MM: medial mammillary nucleus; MS: medial septal nucleus; PH: posterior hypothalamic area; PT: paratenial thalamic nucleus; PVA: anterior part of paraventricular thalamic nucleus; SuM: supramammillary nucleus; VDB: nucleus of the vertical limb of the diagonal band.

cortex, the dorsal peduncular cortex and dorsal part of the dorsal tenia tecta, indicating second-order projection from vSub (Fig. 7b). Labeled neurons within these regions were further increased at 68–76 h after inoculation (Fig. 7c). Locus coeruleus and laterodorsal tegmental nucleus (Fig. 7g) also received no primary projections, but evidence of second-order projection from vSub was indicated by the labeled neurons appearing at 52–60 h (Fig. 7h). Over time, the density of infected neurons increased, especially in the locus coeruleus (Fig. 7i).

4. Discussion In this study, the infected neurons could be observed at about 28 h after inoculation, and was limited within the injection site. In previous studies, the H129 infected neurons were detected at 2 days after inoculation [21]. Therefore, the spread of H129 virus from the injection site to other regions occurs from 28 h to 2 days after inoculation [19]. The most recent studies indicate that a small fraction of H129 virus could be transported retrogradely through circuitry

[22]. But the retrograde transneuronal spread of H129 is temporally delayed and the anterograde projections labeled by H129 still have high reliability [23]. In the subicular-rhinal projections, there were subicular axonal distributed in the entorhinal, perirhinal and postrhinal cortices [24]. Our results showed that the ventral subiculum project directly to medial entorhinal cortex, followed by secondary or indirect projection to parasubiculum and lateral entorhinal cortex (Fig. 4). The medial entorhinal cortex is heavily involved in special memory and navigation [25]. The connection of ventral subiculum and medial entorhinal cortex might also play an important role in spatial localization and contextual recognition. In the amygdala and adjacent area, subicular axons were reported to appear in the amygdalohippocampal area and various amygdaloid nuclei, which included the central, medial, lateral, basal and cortical nuclei [9,26]. Our results in the mouse demonstrate that neurons infected 36–44 h after inoculation were mainly present in the amygdalohippocampal area (Fig. 4g), followed by expansion to other regions of amygdaloid nucleus and intraamyg-

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Fig. 6. Distribution of anterograde labeled neurons in the median raphe nucleus and interpeduncular nucleus. Representative fluorescent micrographs show the distribution of anterograde labeled neurons in median raphe nucleus and interpeduncular nucleus. Coronal section of Bregma −4.00 mm shows the distribution of labeled neurons in median raphe nucleus and interpeduncular nucleus at 36–44 h (a), 52–60 h (b), and 68–76 h (c) after inoculation. IP: interpeduncular nucleus; MnR: median raphe nucleus.

Fig. 7. Distribution of anterograde labeled neurons in prefrontal cortex, laterodorsal tegmental nucleus and locus coeruleus. Representative fluorescent micrographs show the distribution of anterograde labeled neurons in prefrontal cortex, laterodorsal tegmental nucleus and locus coeruleus. Coronal section of Bregma +2.10 mm shows the distribution of labeled neurons in prefrontal cortex and interpeduncular nucleus at 36–44 h (a), 52–60 h (b), and 68–76 h (c) after inoculation. Coronal section of Bregma −5.00 mm shows the distribution of infected neurons in laterodorsal tegmental nucleus at 36–44 h (d), 52–60 h (e), and 68–76 h (f) after inoculation. Coronal section of Bregma −5.70 mm shows the distribution of infected neurons in locus coeruleus at 36–44 h (g), 52–60 h (h), and 68–76 h (i) after inoculation. DP: dorsal peduncular cortex; DRC: caudal part dorsal raphe nucleus; DTT: dorsal tenia tecta; LC: locus coeruleus; LDTg: laterodorsal tegmental nucleus; MO: medial orbital cortex; VO: ventral orbital cortex.

daloid division of the bed nucleus of the striaterminalis (Fig. 4h and i). These data support that vSub directly projects to the amygdalohippocampal area, which might be the interface between vSub and the amygdala. The subiculum is reported to project to most regions or nuclei of the hypothalamus, such as the preoptic region, periventricular nucleus, paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus, posterior nucleus, and mammillary region [9,27]. We found only sparse infected neurons at 36–44 h after virus injection in most portions of the hypothalamus, indicating weak direct projection from vSub (Fig. 5d). Our results also showed that it is the supramammillary nucleus in the mammillary region that receive these primary projections from vSub (Fig. 5a–c). The subiculum

reciprocally connects with various thalamic nuclei, and the investigations of their functions have been largely focused on the anterior thalamic nucleus [28]. This seems appropriate, for our results reveal that the primary and densest projections from vSub to thalamus indeed appeared in anterodorsal thalamic nucleus (Fig. 5g). Previous studies revealed that the subiculum projected to the diagonal band, lateral and medial septal nuclei, and the accumbens nucleus, while the diagonal band and medial septal nucleus in turn project to the hippocampus [29,30]. Our results showed that the medial septal nucleus and diagonal band were the primary projection sites from vSub (Fig. 5j), while accumbens nucleus apparently received secondary projection from vSub (Fig. 5k).

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There is close functional cooperation between the hippocampus and the prefrontal cortex. Wide connections between the prefrontal cortex and subiculum, hippocampus, entorhinal cortex, piriform cortex, the amygdalohippocampal area and other regions has been reported [8,31]. In our study, the prefrontal cortex did not receive direct projection from vSub (Fig. 7a and b). The connection between prefrontal cortex and vSub might therefore occur through other areas that do receive direct projections from vSub, such as the amygdalohippocampal area or entorhinal cortex. In the brain stem, we observed rare but direct projections from vSub to the median raphe nucleus and interpeduncular nucleus (Fig. 6a). However, indirect projections from vSub to the raphe nucleus, interpeduncular nucleus, laterodorsal tegmental nucleus and locus coeruleus were widely evident (Fig. 7). vSub is the chief target of serotonergic projections from the raphe nucleus and noradrenergic neurons from the locus coeruleus [31,32]. Thus, the projections from vSub to the raphe nucleus and locus coeruleus likely serve a feedback role in regulation of emotional status. The conventional anterograde neural tracers, such as Phaseolus vulgaris leucoagglutinin (PHAL) and biotin-dextran amine (BDA), have revealed much useful information about ventral subicular projections [9,27]. Here we used a modified herpes simplex virus 1 strain H129 with fluorescent protein gene to reveal the ventral subicular neuron projections [16] while relying knowledge of the virus lifecycle to help us distinguish primary vs secondary/tertiary neuronal projections. In our experiments, the fluorescent protein labeled H129 reveals primary-order, second-order, and third-order neuron projections at time periods of 36–44, 48–52, 56–62 h, respectively. Our results showed that, vSub directly projects to the medial entorhinal cortex, amygdalohippocampal area, anterodorsal thalamic nucleus, medial hypothalamus, supramammillary nucleus, medial septal nucleus and adjacent diagonal band, the connections between median raphe nucleus and interpeduncular nucleus in brain stem. The ventral prefrontal cortex, laterodorsal tegmental nucleus and locus coeruleus receives second-order projections from vSub. Understanding the direct projection of vSub was crucial to obtain the fundamental functional role of vSub, our results provide the guide for study on the functional connections of vSub with other regions. Acknowledgements This work was partially supported by the National Natural Science Foundation of China (81370453) and Natural Science Foundation of Yunnan Province (2013FA045, 2014BC011 and 2015FB172). We thank David J. Anderson (California Institute of Technology) for providing herpes simplex virus. We also thank Sen Jin, Zhijian Zhang and Xutao Zhu (Wuhan Institute of Physics and Mathematics, CAS) for technical assistance.

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