Whole-brain mapping of afferent projections to the bed nucleus of the stria terminalis in tree shrews

Accepted Manuscript Whole-brain mapping of afferent projections to the bed nucleus of the stria terminalis in tree shrews Rong-Jun Ni, Peng-Hao Luo, Yu-Mian Shu, Ju-Tao Chen, Jiang-Ning Zhou PII: DOI: Reference:

S0306-4522(16)30322-0 http://dx.doi.org/10.1016/j.neuroscience.2016.07.017 NSC 17221

To appear in:

Neuroscience

Accepted Date:

11 July 2016

Please cite this article as: R-J. Ni, P-H. Luo, Y-M. Shu, J-T. Chen, J-N. Zhou, Whole-brain mapping of afferent projections to the bed nucleus of the stria terminalis in tree shrews, Neuroscience (2016), doi: http://dx.doi.org/ 10.1016/j.neuroscience.2016.07.017

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Whole-brain mapping of afferent projections to the bed nucleus of the stria terminalis in tree shrews Rong-Jun Ni1, Peng-Hao Luo1, Yu-Mian Shu1,2, Ju-Tao Chen1, Jiang-Ning Zhou1* 1 Chinese Academy of Science Key Laboratory of Brain Function and Diseases, School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui, P.R. China 2 School of Architecture and Civil Engineering, Chengdu University, Chengdu, Sichuan, P.R. China

Running Title: Inputs to bed nucleus of the stria terminalis Keywords: prefrontal cortex; amygdala; hypothalamus; hippocampus; ventral tegmental area; parabrachial nucleus.

* Correspondence to: Jiang-Ning Zhou, MD PhD School of Life Science, University of Science and Technology of China, Hefei 230027, Anhui, PR China Tel.: +86 -551-63607658; Fax: +86 -551-63600408 E-mail address: [email protected]

The number of words in the manuscript: 8815 The number of figure: 6

Abstract The bed nucleus of the stria terminalis (BST) plays an important role in integrating and relaying input information to other brain regions in response to stress. The cytoarchitecture of the BST in tree shrews (Tupaia belangeri chinensis) has been comprehensively described in our previous publications. However, the inputs to the BST have not been described in previous reports. The aim of the present study was to investigate the sources of afferent projections to the BST throughout the brain of tree shrews using the retrograde tracer Fluoro-Gold. The present results provide the first detailed whole-brain mapping of BST-projecting neurons in the tree shrew brain. The BST was densely innervated by the prefrontal cortex, entorhinal cortex, ventral subiculum, amygdala, ventral tegmental area, and parabrachial nucleus. Moreover, moderate projections to the BST originated from the medial preoptic area, supramammillary nucleus, paraventricular thalamic nucleus, pedunculopontine tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and nucleus of the solitary tract. Afferent projections to the BST are identified in the ventral pallidum, nucleus of the diagonal band, ventral posteromedial thalamic nucleus, posterior complex of the thalamus, interfascicular nucleus, retrorubral field, rhabdoid nucleus, intermediate reticular nucleus, and parvicellular reticular nucleus. In addition, the different densities of BST-projecting neurons in various regions were analyzed in the tree shrew brains. In summary, whole-brain mapping of direct inputs to the BST is delineated in tree shrews. These brain circuits are implicated in the regulation of numerous physiological and behavioral processes including stress, reward, food intake, and arousal.

Introduction The bed nucleus of the stria terminalis (BST), surrounding the anterior commissure and anterior to the hypothalamus, is sometimes referred to as a key part of the extended amygdala (de Olmos and Heimer, 1999, Oler et al., 2016). It is organized into several subdivisions (Franklin and Paxinos, 2007, Paxinos and Watson, 2007, Lebow and Chen, 2016). Different BST subdivisions are connected with different parts of the brain in rodents by anterograde and retrograde tracing methods (Dong et al., 2001, Dong and Swanson, 2003, 2004a, b, Wood and Swann, 2005, Dong and Swanson, 2006a, b). The afferent and efferent projections of the BST have been examined in non-human primates (deCampo and Fudge, 2013, Oler et al., 2016), rodents (Dong and Swanson, 2006b, Kudo et al., 2012), cat (Holstege et al., 1985), and pigeon (Berk, 1987, Atoji et al., 2006). These neural circuits indicate the possible role of the BST in addiction-related behaviors (Stamatakis et al., 2014), ingestive behaviors (Dong and Swanson, 2003, Naka et al., 2013), and stress response (Davis et al., 2010, Radley and Sawchenko, 2011). Tree shrews are day-active animals that live in arboreal habitats in South and Southeast Asia (Peng et al., 1991). According to recent studies, tree shrews are the closest living relatives of primates (Janecka et al., 2007, Kriegs et al., 2007, Fan et al., 2013, McCollum and Roberts, 2014). Many researchers focus on stress-related brain structures of tree shrews using chronic psychosocial stress model, such as the BST, amygdala, hippocampus, and prefrontal cortex (Flugge, 1996, Fuchs and Flugge, 2003, Kozicz et al., 2008, Zambello et al., 2010). However, no information is available regarding the connections between the BST and the latter regions in the tree shrew brain. Many publications have reported on the brain neuroanatomy of tree shrews (Tupaia belangeri) (Airaksinen et al., 1989, Kozicz et al., 2008, Rice et al., 2011, Ni et al., 2014, Ni et al., 2015, Zhou and Ni, 2016). According to the atlas of the tree shrew brain, the accurate stereotaxic coordinates of the BST are available now, including the bed nucleus of the stria terminalis, anterior part (STA), bed nucleus of the stria terminalis, posterior part (STP), and bed nucleus of the stria terminalis, ventral part (STV) (Zhou and Ni, 2016). In the present study, we aim to examine the afferent projections that target the BST throughout the brain of tree shrews (Tupaia belangeri chinensis) using the retrograde tracer Fluoro-Gold (FG). Here, we provide a complete brain map of retrogradely labeled neurons that project to the BST and demonstrate the densities of labeled neurons in the afferent regions.

Material and Methods Animals Adult male tree shrews (Tupaia belangeri chinensis) from the breeding colony in the Animal House Center of the Kunming Institute of Zoology, Kunming, P.R. China were used. The tree shrews were housed in animal facilities under a 12-hour light/dark cycle (lights on at 8:00 a.m.) at 24 ± 2°C and with ad libitum access to food and water. All animal procedures in the present study were performed in accordance with the University of Science and Technology of China Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Science and Technology of China. All efforts were made to minimize animal suffering as well as the number of animals used. FG tracing and antibody characterization The FG tracing technique has been extensively tested in tree shrews (Remple et al., 2007, Chomsung et al., 2008) and other mammals (Rosa and Tweedale, 2000, Radley et al., 2009, Zingg et al., 2014). The FG antibody characterization has been previously confirmed by using immunohistochemistry with blocking the signal through preadsorption of the FG antibody with FG (Lee et al., 2009). Surgery Three tree shrews were anesthetized with pentobarbital sodium (80 mg/kg, i.p.) and positioned in a stereotaxic apparatus (Stoelting, Stoelting Company, USA). A sagittal incision was made in the scalp. A glass micropipette (outer diameter, approximately 50 µm) was filled with 2% FG solution (Fluorochrome, Denver, CO, USA) and connected to a 1-µl Hamilton syringe that was controlled by an UltraMicroPump (Model: UMP3; World Precision Instruments, Inc. Sarasota, FL USA) and a Micro4 MicroSyringe Pump Controller (Model: UMC4; World Precision Instruments, Inc. Sarasota, FL, USA). For retrograde labeling of BST-projecting neurons, unilateral pressure injections of 2% FG solution (Schmued and Fallon, 1986) were made into the STV and STA in volumes of 80–100 nl using the following coordinates taken from a tree shrew brain atlas (Zhou and Ni, 2016): anteroposterior, +0.34 mm; mediolateral, +1.50 mm; dorsoventral, -6.70 mm from dura. Following an injection, the syringe needle was left in place for 10 min, and all animals were allowed to survive for 7 days. Tissue preparation After the end of the survival period, the tree shrews were deeply anesthetized with pentobarbital sodium (80 mg/kg, i.p.) and perfused with 0.9% saline followed by 4% paraformaldehyde in phosphate-buffer (0.1 M; pH 7.4). After perfusion, the brains were removed and post-fixed by immersion in the same fixative overnight at 4°C. Then, the brains were soaked in 15% sucrose in phosphate-buffered saline (PBS: 0.1 M; pH 7.4) until the tissues sank. This was followed by a soak in a 30% sucrose solution in PBS. The tissues were frozen and sectioned (40 µm) on a Leica microtome

(Leica CM1950, Germany), and the sections were stored at -20°C until use. One series of sections throughout the BST were mounted onto glass slides in a 0.5% gelatin solution and allowed to air-dry overnight. Then, the sections were washed and coverslipped with an 80% glycerin solution. A confocal laser scanning microscope (LSM710, Carl Zeiss, Maple Grove, MN, USA) was used to determine the fluorescence of the FG tracers using the 10x objective. Nissl staining One series of frozen sections was mounted on gelatin-coated slides and dried at 37°C overnight. The slides were rinsed twice with PBS and then stained in 0.02% thionin acetate salt (Sigma-Aldrich) solution for 15-20 minutes. Subsequently, the sections were rinsed quickly in distilled water, dehydrated in ethanol-xylene, coverslipped and visualized on a Zeiss Axioskop2 microscope (Carl Zeiss, USA). Immunohistochemistry Another two series of adjacent sections were taken at the level of the olfactory bulb, forebrain, midbrain and hindbrain. The sections were processed for immunohistochemistry, as previously described (Ni et al., 2014). Briefly, the floating sections were rinsed and treated with 0.3% hydrogen peroxide and 0.5% triton X-100 in PBS to quench the endogenous peroxidase activity. Then, following a blocking step with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBST (PBS containing 0.5% triton X-100), the floating sections were incubated with primary antibodies of (i) rabbit anti-FG (Fluorochrome, LLC; 52-9600, at 1:400 dilution) in PBST containing 5% normal goat serum for 72 h at 4°C. Amplification was performed with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA, USA) and avidin-biotin peroxidase complex (1:200; Vector Laboratories). Chromogen development was performed with 0.05% 3,3’-diaminobenzidine (Sigma-Aldrich). The sections were mounted onto object glasses in a 0.5% gelatin solution and allowed to air-dry overnight before being dehydrated in ethanol-xylene and coverslipped using Permount. Photographs were taken using a Zeiss Axioskop2 microscope (Carl Zeiss, Maple Grove, MN, USA). One series of sections following FG immunoperoxidase labeling were stained with a 0.02% thionin acetate salt solution as described above. Then, photographs were taken of the sections using a Zeiss Axioskop2 microscope (Carl Zeiss, Maple Grove, MN, USA). Artwork and digital photomicrographs The sections were treated with Nissl staining to designate the neuroanatomical structures of the tree shrew brain. The designation of neuroanatomical structures is based on the atlas for tree shrews (Zhou and Ni, 2016). The FG-labeled cells were classified as absent (0), sparse (1), moderate (2), or extensive (3) as described previously (Shu et al., 2015). Each number represents the relative level of FG-labeled neurons in the brain nucleus, from 0 - no FG staining, 1 - less than three labeled neurons per 10,000 um2, 2 - more than three but less than ten labeled neurons per

10,000 um2, 3 - presence of more than ten labeled neurons per 10,000 um2. The data presented here were collected from three tree shrews. The degree of immunostaining in each brain region was assessed by the same observer. Light microscopic images were captured using an Axiocam HRc digital microscope camera (Carl Zeiss). All digital photomicrographs were adjusted with Adobe Photoshop CS5 (Adobe Systems, USA) using the brightness/contrast, crop and image size commands. All line drawings were created using Adobe Illustrator CS5 (Adobe Systems, USA).

Results BST anatomy According to the cytoarchitecture of the BST in tree shrews, the subregions in the BST were mapped from rostral to caudal (Fig. 1, 2, 3). The STA and STP are divided into lateral and medial parts, which are ventral to the lateral ventricle. The STV are also located within the rostral part of the BST, which is ventral to the STA and surrounds the anterior commissure (ac; Fig. 1, 2). FG tracer delivery sites Schematic drawings show the structure and placement of the BST in the tree shrew brain (Fig. 1A). To identify potential BST afferent cell populations throughout the tree shrew brain, FG tracers were injected by pressure into the STV and STA. In our experiments, the retrograde tracer delivery sites were restricted to the STV and STA, but avoided diffusion into adjacent nuclei. In spite of all these exertions, only a few tracers were spread into an adjacent area. The three cases (no. 13DEC06, no. 13DEC17, and no. 14APR17) with the most accurate tracer delivery sites for analysis of retrograde labeling were selected for the analysis. Actually, the three 'accurate' injections involve parts of the BST including lateral BST and the medial BST. In case no. 13DEC06, the FG tracers were accurately delivered in the central part of the STV and STA (Fig. 1B, C). In case no. 13DEC17, the tracer delivery site was located within the caudal part of the STV and STA (Fig. 2A). The shadow zones depict the diffuse spread of FG from the centers of each injection site in the BST (Fig. 2). The injection site of the tree shrew (no. 13DEC17) did not pass the anterior commissure (Fig. 2A, 2B). In case no. 14APR17, the FG tracers were centered in the rostral part of the STV and STA (Fig. 2C). Because of the difficulty of injecting FG into the entirety of the BST without spreading into adjacent structures, the FG injection sites were restricted to most regions of the STV and STA in three representative experiments. The BST afferent cell populations were present in both rostral and caudal regions in the tree shrew brain (Fig. 3S1-S29). Retrograde labeling in the rhinencephalon and telencephalon In the rhinencephalon, some retrogradely labeled cells were observed in the anterior olfactory nucleus, external part (AOE; Fig. 3S1). This is presumably the result of the injection needle passing through the ac, which may have led to the deposit of some FG tracers in the ac. There were no FG-labeled neurons in the other nuclei of the rhinencephalon. In the telencephalon of tree shrews, a high density of labeled cells was seen in many brain regions, including the cerebral cortex and amygdala (Fig. 4; Table 1). The basal ganglia, septal and preoptic regions also contained sparsely labeled cells. Generally, the number of ipsilateral FG-labeled neurons was greater than the number of contralateral FG-labeled neurons in the regions observed in these experiments (Fig. 4). In the cerebral cortex, a large number of FG-labeled neurons were found in the dorsal frontal cortex (DFC; Fig. 3S1), orbital frontal cortex (OFC; Fig. 3S2), infralimbic

cortex (IL; Fig. 3S3, 5A), piriform cortex (Pir; Fig. 3S3, S4), entorhinal cortex (Ent; Fig. 3S15-S18), and ventral subiculum (VS; Fig. 5B, 6A). These neurons were mainly distributed in ipsilaterally projecting regions. A dense cluster of FG-labeled neurons was present in the OFC, IL, Ent, and VS (Fig. 4). In these regions, some labeled neurons were also observed on the contralateral side. The prelimbic cortex (PrL) contained no BST-projecting cells in our experiments. A lower density of labeled neurons was found in the cortex (OFC, IL, Ent, VS) of the tree shrew no. 13DEC17 compared to no. 13DEC06 and no. 14APR17 (Fig. 4). Another prominent concentration of FG labeling was seen in the amygdala (Fig. 3). A large number of darkly labeled neurons were found throughout the rostrocaudal extent of the amygdala, including the anterior amygdaloid area (AA; Fig. 3S7), central amygdaloid nucleus (CeA; Fig. 3S9, S10, 5C), medial amygdaloid nucleus (MeA; Fig. 3S9, S10), basomedial amygdaloid nucleus (BMA; Fig. 3S9, S10), and amygdalohippocampal area (AHi; Fig. 3S13). A sparse to moderate density of weakly labeled neurons were found in the nucleus of the lateral olfactory tract (LOT; Fig. 3S7), sublenticular extended amygdala (EA; Fig. 3S7), anterior cortical amygdaloid nucleus (ACo; Fig. 3S8, S9), and basolateral amygdaloid nucleus (BLA; Fig. 5C). However, the contralateral amygdala contained a small number of weakly labeled neurons (Fig. 4). Similar distribution patterns of retrograde labeling were present in the amygdala of all three tree shrews (Fig. 4). A few FG-labeled neurons were scattered in the basal ganglia, including the interstitial nucleus of the posterior limb of the anterior commissure (IPAC; Fig. 3S6), ventral pallidum (VP; Fig. 3S7), claustrum (Cl; Fig. 4), and accumbens nucleus (Acb; Fig. 3S5). Similarly, a low density of labeled neurons was observed in the lateral septal nucleus (dorsal, intermediate, and ventral), medial septal nucleus (MS), and septofimbrial nucleus (SFi; Fig. 4). The preoptic regions and nucleus of the diagonal band (NDB) contained a sparse to moderate density of FG-labeled neurons (Fig. 4), whereas few, if any, labeled neurons were found on the contralateral side of these regions. Retrograde labeling in the diencephalon In the hypothalamus, the major regions projecting to the BST included the lateral hypothalamic area (LHA; Fig. 3S11), ventromedial hypothalamic nucleus (VMH; Fig. 3S11), dorsomedial hypothalamic nucleus (DMH; Fig. 3S12), perifornical nucleus (PeF; Fig. 3S12), supramammillary nucleus (Fig. 3S13, S14), mammillary nucleus (Fig. 3S13), and peduncular part of the lateral hypothalamus (PLH; Fig. 3S14). These regions contained a sparse to moderate density of labeled neurons (Fig. 4). A higher density of FG-labeled neurons was seen in the ventromedial hypothalamic nucleus (VMH), perifornical nucleus (PeF), and medial part of supramammillary nucleus (SuMM) of tree shrew no. 14APR17 (Fig. 4). A moderate number of labeled neurons were found in the paraventricular thalamic nucleus (Fig. 3S15), ventral posteromedial thalamic nucleus (VPM; Fig. 5D), and posterior complex of the thalamus (Po; Fig. 3S17). A small number of labeled cells were located in intermediodorsal thalamic nucleus (IMD; Fig. 4). Sparsely labeled

neurons were observed on the contralateral side of the paraventricular thalamic nucleus, VPM, and Po (Fig. 4). Retrograde labeling in the mesencephalon In the mesencephalon, many FG-labeled neurons projecting to the BST were present from rostral to caudal, especially in the ventral tegmental area (VTA; Fig. 3S16-S21). A moderate to high density of retrogradely labeled neurons were found in the rostral part of the ventral tegmental area (VTAR; Fig. 4) and the parabrachial pigmented nucleus of the ventral tegmental area (PBP; Fig. 4). A sparse to moderate density of BST-projecting neurons were located in the pedunculopontine tegmental nucleus (PTg; Fig. 3S19), retrorubral field (RRF; Fig. 3S19), periaqueductal gray (PAG; Fig. 4), and dorsal raphe nucleus (DR; Fig. 3S20-S22). Moreover, there were sparse densities of FG-labeled neurons observed in the rostral linear nucleus of the raphe (RLi; Fig. 3S16), caudal linear nucleus of the raphe (CLi; Fig. 3S19, S20), interfascicular nucleus (IF; Fig. 3S17), mesencephalic reticular formation (mRt; Fig. 3S18), and rhabdoid nucleus (Rbd; Fig. 3S21). Several labeled neurons were seen in the contralateral VTAR and contralateral PBP (Fig. 3S16, S18). However, the contralateral mesencephalon in other samples contained relatively few labeled neurons (Fig. 4). FG injections made in the BST of different tree shrews showed identical projection patterns but with different densities of labeled cells (Fig. 4). Retrograde labeling in the metencephalon and myelencephalon In the metencephalon and myelencephalon, a large number of labeled neurons projecting to the BST were concentrated in the lateral parabrachial nucleus (LPB; Fig. 4) and medial parabrachial nucleus (MPB; Fig. 5E). A sparse to moderate density of labeled neurons were dispersed in the laterodorsal tegmental nucleus (LDTg; Fig. 3S22), locus coeruleus (LC; Fig. 3S23), intermediate reticular nucleus (IRt; Fig. 3S24-29), and nucleus of the solitary tract (Sol; Fig. 5F, 6B). These labeled neurons were mainly located in the medial part of the Sol (Fig. 5F). A small, dense cluster of FG-labeled cells was present in the ventrolateral reticular nucleus (VLN; Fig. 3S27). In addition, a few retrogradely labeled cells were found in the pontine reticular nucleus, oral part (PnO; Fig. 3S20), pontine reticular nucleus, caudal part (PnC; Fig. 3S22), supratrigeminal nucleus (Su5; Fig. 3S22), gigantocellular reticular nucleus (Gi; Fig. 3S25), and parvicellular reticular nucleus (PCRt; Fig. 5F). The FG-labeled projection patterns were virtually identical in these experiments (Fig. 4; Table 1).

Discussion These results present a complete distribution map of the BST-projecting neurons in the tree shrew brain from rostral to caudal regions. The data indicate the BST receives afferent inputs from the forebrain (telencephalon and diencephalon), midbrain (mesencephalon), and hindbrain (metencephalon and myelencephalon). Furthermore, the densities of FG-labeled cells were analyzed in various brain regions of each tree shrew, and all three tree shrews had accurate needle placements during the injections. The differences in retrograde labeling intensities observed in our experiments is probably due to the different areas of FG tracer diffusion in the BST. Subdivisions of the BST and potential functions In our present study, the anatomy of architecture of the BST in tree shrews is consistent with previous studies of the hamster (Wood and Swann, 2005). The BST subdivisions proposed for the tree shrew are compared with those of the rat brain (Paxinos and Watson, 2007) as shown in Table 2. Much literature indicates that different subdivisions of the BST are differently connected with different parts of the brain in rats (Dong et al., 2000, Dong and Swanson, 2004a, 2006a, Shin et al., 2008). The neural circuits in anteromedial area of the BST are involved in coordinating neuroendocrine, autonomic, and behavioral or somatic responses associated with maintaining energy balance homeostasis (Dong and Swanson, 2006a). The neural circuits in posterior part of the BST are implicated in the regulation of defensive and reproductive behaviors (Dong and Swanson, 2004b). The lateral BST and other parts of the central extended amygdala are related to fear and anxiety (Khoshbouei et al., 2002, deCampo and Fudge, 2013), while the medial BST and other parts of the medial extended amygdala are related to social behavior (Hines et al., 1992, Wood and Swann, 2005, Bienkowski and Rinaman, 2013). Detailed comparisons of afferent projections into the BST of tree shrews are made with those of other animals (Table 3). Inputs from the anterior olfactory nucleus and cerebral cortex The density of FG-labeled neurons in the AOE, OFC, IL, Ent, VS, and IPAC of the tree shrew (no. 13DEC17) is significantly lower than the others (no. 14APR17 and no. 13DEC06). This is presumably the result of the injection needles passing through the ac of tree shrews (no. 14APR17 and no. 13DEC06). These regions may project to the BST through the ac (Lent and Guimaraes, 1990, 1991, Shammah-Lagnado et al., 1999). The prefrontal cortex and VS provide strong inputs to the BST, which has been implicated in stress regulation (Herman et al., 2005, Radley and Sawchenko, 2011, Herman, 2012). The BST in the tree shew brain receives dense inputs from neurons in the IL, but not those in the adjacent PrL. Some findings in rodents are similar to our present results using retrograde tracing (Vertes, 2004, Bienkowski and Rinaman, 2013), whereas other reports support the innervation of the BST from the PrL using retrograde and anterograde tracing methods (Radley and Sawchenko, 2011, Radley et al., 2013). The VS controls the phasic activity of dopamine neurons in the ventral tegmental area by inducing NMDA-dependent long-term potentiation in the BST, which is involved in cocaine-induced locomotor activity (Glangetas et al., 2015). Inputs from the amygdala, basal ganglia, septal and preoptic regions

The major inputs to the BST originate from the amygdala of tree shrews, which is similar to the results reported in other mammals (Price and Amaral, 1981, Shin et al., 2008, Pardo-Bellver et al., 2012). The BST is heavily innervated by the amygdala and also projects to the brainstem circuitry that mediates fear, stress, and anxiety (Walker et al., 2003). Previous papers have described that the ‘extended amygdala’ is comprised of the BST and its sublenticular extension into the centromedial amygdala (Alheid and Heimer, 1988, Freedman and Shi, 2001, Fudge and Haber, 2001). Some neurons in the EA also project to the BST in tree shrews. Recent study supports the EA is strongly and reciprocally connected to both BST and centromedial amygdala, which reveals the EA’s potential importance in information processing and relay between the two structures (Oler et al., 2016). The study by Oler et al. (2016) highlights the potential importance of the EA for understanding EA functions in relation to adaptive and maladaptive anxiety. Furthermore, the results of an optogenetic study suggested that the BST exerts an inhibitory influence over fear responses to discrete conditioned cues via the BST projection to the CeA (Gungor et al., 2015). In our experiments, the preoptic area has limited projections to the BST. Previous results indicate the neural circuits of the medial preoptic area and BST are involved in maternal responsiveness (Numan and Numan, 1997, McHenry et al., 2015). The BST-projecting neurons are sparse in the basal ganglia and septal regions, which suggests that the BST integrates these inputs for regulation of social and reward behaviors (Compaan et al., 1993, Desai et al., 2013). Sparse neurons in the VP project to the BST, which indicates this neural circuit may play a role in controlling avoidance behavior (Saga et al., 2016). The connection between NDB and BST appears to modulate cognitive behavior (Mufson et al., 1993). Inputs from the hypothalamus and thalamus The BST receives a small number of projections from the hypothalamic regions in tree shrews. Previous studies in the rodent provide a catalog of various inputs to a subregion of the BST that also sends efferent projections to many regions (Gray and Magnuson, 1987, Dong and Swanson, 2003, 2006b, Shin et al., 2008). Although the BST projects to the hypothalamic paraventricular nucleus (PVN), it receives no projections from the PVN (Dong and Swanson, 2006a, Shin et al., 2008). Some of the peptidergic neurons (orexin, melanin-concentrating hormone, cocaine/amphetamine-regulated transcript, and agouti-related protein) in the hypothalamus send inputs to the BST (Shin et al., 2008). These brain circuits are implicated in the regulation of food intake, energy balance, and arousal (Williams et al., 2001, Chou et al., 2003). The BST is also innervated by the neurons in the supramammillary nucleus. The supramammillary nucleus is presumably a component in the circuitry for reward and conditioned taste aversions (Ikemoto et al., 2003, Yasoshima et al., 2005). In the thalamus, the paraventricular thalamic nucleus is the main parts of BST-projecting circuitry. The paraventricular thalamic nucleus sends heavy projections to the BST (Moga et al., 1995). Moreover, the paraventricular thalamic nucleus receives intense hypothalamic projections from peptidergic neurons, including neurons that contain serotonin, orexin, and corticotropin-releasing hormone (Hsu and Price, 2009). These findings suggest that the paraventricular thalamic

nucleus may contribute to transmitting and regulating stress-related signals to the BST (Hsu et al., 2014). The BST is also strongly innervated by the Po and VPM, which were the first described. Stimulation on VPM in rats results in turning behavior without training (Xu et al., 2016). Inputs from the mesencephalon, metencephalon and myelencephalon The BST of tree shrews receives mainly strong innervation from the VTA. These BST-projecting neurons are dopaminergic in the VTA (Moreno et al., 2012, Aransay et al., 2015). The non-dopaminergic neurons in the VTA receive glutamatergic and GABAergic projections from the BST (Jennings et al., 2013, Kudo et al., 2014). Thus, BST neurons projecting to the VTA may participate in integrating reward-related information (Jalabert et al., 2009, Jennings et al., 2013). Neuroanatomical evidence for such reciprocal projections indicate these connections may play a crucial role in balancing reward circuitry and affect. The BST-projecting neurons in the VTA and IF may regulate social behavior (Tang et al., 2014). Disconnecting the ventral BST from the RRF has long-lasting mating deficits in rats (Finn and Yahr, 2005). Furthermore, the BST receives a strong projection from the DR, PAG, and linear nucleus of the raphe, which were also reported in other mammals (Vertes, 1991, Petit et al., 1995, Halberstadt and Balaban, 2008). A recent study suggests PAG/DR dopamine neurons are likely involved in nociception modulation, potentially via activation of a major projection structure downstream from the BST and through dopamine and glutamate release (Li et al., 2016). The vasoactive intestinal polypeptide-like neurons in the DR and CLi project to the BST, which was demonstrated using a double immunohistochemical method (Petit et al., 1995). The BST-projecting neurons in the PTg may be involved in the stress-related locomotor response (Jerzemowska et al., 2013). The BST receives sparse projections from the Rbd of tree shrews. In addition, the parabrachial nucleus, especially the MPB, projects heavily to the BST in tree shrews. The alpha(2A)-adrenergic receptors in the BST could filter excitatory transmissions in the BST by inhibiting a component of the parabrachial nucleus input (Flavin et al., 2014). Previous studies have shown that the parabrachial nucleus contributes to feeding behavior (Andrade et al., 2004, Swick et al., 2015). Connections between the BST and LC are present in tree shrews, which are probably involved in the emotional and stress-related behaviors (Van Bockstaele et al., 2001, Soya et al., 2013). The PCRt neurons that project to the BST may be involved in control of orofacial movements (Tsumori et al., 2010). The BST is heavily innervated by the Sol and reticular nucleus. Anatomical evidence of direct projections from the Sol to the BST was observed in the rat by Ricardo and Koh (1978). The neurons in the Sol that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) project mainly to the BST, which may be important for understanding their potential role in the brain circuitry responsible for sodium appetite (Geerling and Loewy, 2006). The numerous retrogradely labeled neurons in the VLN appears to be A1 noradrenergic (Shin et al., 2008). The VLN is involved in rapid eye movement sleep (Stettner et al., 2013). The sparse BST-projecting neurons are present in the IRt of tree shrews. Previous report suggests that the medullary dorsal reticular nucleus receives small projections from the BST of rats (Almeida et al., 2002).

Activity-dependent changes of the inputs to the BST The BST plays several roles in integrating and processing information received in response to various stress factors (Choi et al., 2007, Radley and Sawchenko, 2011). Many factors could alter the afferent projections to the BST. The bacterial endotoxin (lipopolysaccharide) treatment strongly recruits brain regions with inhibitory input to the BST, whereas sources of excitatory input were either not activated or less robustly activated (Bienkowski and Rinaman, 2011). Recent evidence shows that chronic variable stress decreases the dendritic branching and spine density of BST-projecting cells in the medial prefrontal cortex and produces a selective loss of the mature mushroom-shaped spines of these cells (Radley et al., 2013). Previous results show that adrenalectomy significantly decreases inputs from the BST to the PVN, particularly to the corticotrophin-releasing hormone neurons (Mulders et al., 1997). This implies that a corticosteroid imbalance following adrenalectomy influences the afferent projections to the BST and likely results in a loss of efferent projections from the BST. In summary, the brain regions that send afferent projections to the BST are located throughout the brain and are topographically organized in tree shrews. The present results provide the first detailed whole-brain mapping of BST-projecting neurons in the tree shrew brain. The BST is densely innervated by the prefrontal cortex, Ent, VS, amygdala, ventral tegmental area, and parabrachial nucleus. Moreover, moderate projections to the BST are sent from the MPO, supramammillary nucleus, paraventricular thalamic nucleus, PAG, DR, LC, Sol, and VLN. In addition, different densities of BST-projecting neurons in various regions were examined in the three tree shrew brains that contained the most accurately placed tracer delivery sites. Afferent projections to the BST were identified in the VP, NDB, VPM, Po, IF, RRF, Rbd, IRt, and PCRt. Although these BST-projecting regions are not found in other animals, the results should been confirmed by injecting anterograde tracer into each labeled brain structure of tree shrews respectively. These results highlight the potential roles of these brain circuits in the regulation of numerous physiological and behavioral processes including stress, reward, food intake, and arousal.

Acknowledgments This study was supported by the Strategic Priority Research Program of the Chinese Academy of Science (XDB02030001), the National Natural Science Foundation of China (31500859, 91432305, and 81271492) and the Fundamental Research Funds for the Central Universities (WK2070000058). Conflicts of interest All authors claim that there are no conflicts of interest. Authors and Contributors RJ Ni was involved in designing of the experiment, tissue processing, histochemical analysis, collection and interpretation of data, and writing the manuscript. JN Zhou was responsible for experiment designing and manuscript preparation. PH Luo was involved in tissue processing and data interpretation. YM Shu and JT Chen were involved in data collection, histochemical analysis and manuscript preparation.

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FIGURE LEGENDS Figure 1. Unilateral pressure injection of Fluoro-Gold (FG) retrograde tracer into the bed nucleus of the stria terminalis of the tree shrew brain (no. 13DEC06). (A) Schematic drawings of needle placements in the bed nucleus of the stria terminalis, ventral part (STV) (the drawings were adapted from The Tree Shrew (Tupaia belangeri chinensis) Brain in Stereotaxic Coordinates (Zhou and Ni, 2016)). (B) FG immunohistochemical labeling revealed that FG injection produced a highly localized, dense tracer deposit in the STV. (C) Neurons labeled with FG fluorescence (blue) were distributed in the STV (dense) and STA. Scale bars = 200 µm in B, C. Figure 2. Schematic drawings showing needle placements in the bed nucleus of the stria terminalis, ventral part (STV) of the tree shrew brain (the drawings adapted from The Tree Shrew (Tupaia belangeri chinensis) Brain in Stereotaxic Coordinates (Zhou and Ni, 2016)). The shadow zones represent the region of Fluoro-Gold (FG) tracer diffusion at the injection sites of two tree shrews, no. 13DEC17 (A) and no. 14APR17 (C). Nissl staining (blue) following FG immunoperoxidase labeling (black) showed individual brain structures and injection site of the tree shrew, no. 13DEC17 (B). Scale bar = 200 µm in B. Figure 3. Camera lucida drawings of histological sections from rostral to caudal regions containing FG-labeled neurons (black dots) following FG injections into the bed nucleus of the stria terminalis of tree shrews. These tree shrew brain drawings were adapted from the atlas of Zhou and Ni (2016). Representative coronal sections were selected from two tree shrew brains (no. 14APR17 and no. 13DEC17) and are referenced to the bregma and interaural line (distance indicated at two lower corners, the corresponding atlas level from The Tree Shrew (Tupaia belangeri chinensis) Brain in Stereotaxic Coordinates (Zhou and Ni, 2016)). The density of dots represents the relative density of cells in the areas. Each dot represents approximate five FG-labeled neurons. For abbreviations, see Abbreviations. Figure 4. Density of FG-labeled cells in each brain structure of three tree shrews (no. 14APR17, no. 13DEC17, and no. 13DEC06) are displayed in a graph. The color of each box represents the relative density of labeled cells in each brain region, e.g., 0—no FG labeling, 1—sparse, 2—moderate, 3—extensive. Each number represents the relative level of FG-labeled neurons in the brain nucleus, from 0 - no FG staining, 1 - less than three labeled neurons per 10,000 um2, 2 - more than three but less than ten labeled neurons per 10,000 um2, 3 - presence of more than ten labeled neurons per 10,000 um2. The minimum (Min) and maximum (Max) values indicate the range of density in selected brain regions of individual animals. Figure 5. BST-projecting neurons were darkly and densely labeled in the infralimbic cortex (IL, A), amygdala (B), ventral subiculum (VS, C), ventral posteromedial thalamic nucleus (VPM, D), medial parabrachial nucleus (MPB, E), and solitary nucleus (Sol, F). The dashed line marks the boundaries between brain regions (B, E, F). Scale bars = 100 µm in C-F; 200 µm in A, B. Figure 6. BST-projecting neurons (black dots) were labeled in the ventral subiculum (VS, A), entorhinal cortex (Ent, A), and solitary nucleus (Sol, B). Nissl staining (blue)

following FG immunoperoxidase labeling (black dots) showed individual brain structures. The dash line marks the boundaries between brain regions. Scale bars = 100 µm in C-F; 200 µm in A, B.

Graphical Abstract Image: Here we provided density of Fluoro-Gold (FG)-labeled cells in each brain structure of three tree shrews. The color of each box represents the relative density of labeled cells in each brain region, e.g., 0—no FG labeling, 1—sparse, 2—moderate, 3—extensive.

Highlights: 1. The first detailed whole-brain mapping of BST-projecting neurons in tree shrews. 2. The BST-projecting regions were firstly identified in the tree shrew brain. 3. The density of BST-projecting neurons in whole-brain regions was analyzed.

Table 1. Density of FG-labeled cells in each brain structure of three tree shrews (no. 14APR17, no. 13DEC17, and no. 13DEC06). no. 14APR17

Brain region

ipsilateral

Min

Ma x

no. 13DEC17

contralater al Min

Ma x

ipsilateral

Min

Ma x

no. 13DEC06

contralater al Min

Ma x

ipsilateral

Min

Ma x

contralater al Min

Ma x

anterior olfactory nucleus, external part

1

2

0

1

1

1

0

1

1

2

0

1

1

1

0

1

0

1

0

0

1

2

0

1

1

3

1

1

1

1

0

0

2

3

1

2

2

3

1

2

1

2

1

1

2

3

1

1

1

1

0

0

1

1

0

0

1

2

0

1

0

1

0

1

0

1

0

0

0

1

0

1

2

3

1

1

1

2

0

1

2

3

1

1

2

3

1

1

2

2

1

1

2

3

1

1

1

1

0

0

0

1

0

0

1

1

0

1

ventral pallidum (VP)

1

1

0

1

1

1

0

0

1

1

0

1

claustrum (Cl)

0

1

0

0

0

0

0

0

0

1

0

1

1

1

0

1

0

1

0

0

1

1

0

1

2

3

0

1

1

2

1

1

1

2

1

1

1

1

0

1

1

2

1

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

2

0

1

1

2

1

1

1

1

0

1

1

2

0

1

2

3

1

1

2

3

1

1

(AOE) dorsal frontal cortex (DFC) orbital frontal cortex (OFC) infralimbic cortex (IL) piriform cortex (Pir) perirhinal cortex (PRh) entorhinal cortex (Ent) ventral subiculum (VS) interstitial nucleus of the posterior limb of the anterior commissure (IPAC)

accumbens nucleus (Acb) anterior amygdaloid area (AA) nucleus of the lateral olfactory tract (LOT) sublenticular extended amygdala (EA) anterior cortical amygdaloid nucleus (ACo) medial amygdaloid nucleus (MeA)

basomedial amygdaloid nucleus

2

3

0

1

2

3

0

1

2

3

1

1

1

1

0

1

0

1

0

1

0

1

0

1

1

2

0

1

2

3

1

1

2

3

1

1

2

3

0

1

2

2

1

1

1

2

0

1

0

1

0

1

0

0

0

0

0

1

0

0

1

1

0

1

1

1

0

1

1

1

0

1

1

2

0

1

1

1

0

1

1

1

0

1

1

1

0

1

0

1

0

0

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

0

1

0

0

1

1

0

1

1

2

0

0

1

2

0

1

1

1

0

1

1

2

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

1

1

1

1

0

1

1

1

0

1

1

1

0

1

1

2

0

1

1

1

0

1

0

1

0

1

0

1

0

0

0

1

0

0

0

1

0

0

1

2

0

1

1

1

0

1

1

1

0

1

1

2

1

1

1

1

0

1

1

1

0

1

1

2

0

1

1

2

1

1

1

2

0

1

(BMA) basolateral amygdaloid nucleus (BLA) central amygdaloid nucleus (CeA) amygdalohippocampa l area (AHi) lateral septal nucleus, dorsal part (LSd) lateral septal nucleus, ventral part (LSv) lateral septal nucleus, intermediate part (LSi) medial septal nucleus (MS) septofimbrial nucleus (SFi) nucleus of the diagonal band (NDB) stria terminalis (st) medial preoptic area (MPO) lateral preoptic area (LPO) lateral hypothalamic area (LHA) ventromedial hypothalamic nucleus (VMH) dorsomedial hypothalamic nucleus (DMH) perifornical nucleus (PeF) supramammillary nucleus, medial part (SuMM) supramammillary nucleus, lateral part (SuML)

medial mammillary nucleus, lateral part

0

1

0

0

0

1

0

0

0

1

0

0

0

1

0

1

1

1

0

1

0

1

0

0

0

1

0

0

0

1

0

1

1

1

0

1

1

2

1

1

1

2

1

1

1

2

1

1

1

1

1

1

1

2

1

1

1

2

1

1

1

2

1

1

1

2

1

1

1

2

1

1

2

2

1

1

1

1

0

1

1

2

1

1

2

3

1

1

1

2

1

1

2

3

1

1

1

2

0

1

1

1

0

1

0

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

2

1

0

1

1

1

0

1

0

1

0

1

1

1

0

1

2

2

1

1

1

2

1

1

1

2

0

1

2

3

1

1

1

2

1

1

1

2

0

0

1

2

0

1

1

1

0

1

1

1

0

1

1

2

0

1

1

1

0

1

1

1

0

1

1

1

0

1

0

1

0

1

1

1

0

1

(ML) lateral mammillary nucleus (LM) posterior hypothalamic nucleus (PH) peduncular part of lateral hypothalamus (PLH) paraventricular thalamic nucleus, anterior part (PVA) paraventricular thalamic nucleus, posterior part (PVP) posterior complex of the thalamus (Po) ventral posteromedial thalamic nucleus (VPM) intermediodorsal thalamic nucleus (IMD) rostral linear nucleus of the raphe (RLi) caudal linear nucleus of the raphe (CLi) interfascicular nucleus (IF) ventral tegmental area, rostral part (VTAR) parabrachial pigmented nucleus of the VTA (PBP) pedunculopontine tegmental nucleus (PTg) retrorubral field (RRF) mesencephalic reticular formation

(mRt) periaqueductal gray (PAG) rhabdoid nucleus (Rbd) dorsal raphe nucleus (DR)

1

2

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

2

1

1

1

1

0

1

2

1

0

1

1

1

0

1

0

1

0

1

0

1

0

1

0

1

0

0

0

1

0

1

0

1

0

0

1

2

1

1

1

2

1

1

2

3

1

1

2

3

1

2

2

3

1

2

2

3

1

1

1

2

1

1

1

2

0

1

1

1

0

1

1

2

1

1

1

1

0

1

0

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

0

1

0

0

0

1

0

0

0

1

0

0

0

1

0

0

0

1

0

0

0

1

0

0

1

2

1

1

2

2

1

1

2

2

1

1

2

2

0

1

1

2

0

1

1

2

0

1

1

2

1

1

1

1

0

1

1

1

0

1

pontine reticular nucleus, oral part (PnO) pontine reticular nucleus, caudal part (PnC) lateral parabrachial nucleus (LPB) medial parabrachial nucleus (MPB) locus coeruleus (LC) laterodorsal tegmental nucleus (LDTg) supratrigeminal nucleus (Su5) gigantocellular reticular nucleus (Gi) parvicellular reticular nucleus (PCRt) nucleus of the solitary tract (Sol) ventrolateral reticular nucleus (VLN) intermediate reticular nucleus (IRt)

These data were showed in Figure 4. Each number represents the relative level of FG-labeled neurons in the brain nucleus, from 0 - no FG staining, 1 - less than three labeled neurons per 10,000 um2, 2 - more than three but less than ten labeled neurons per 10,000 um2, 3 - presence of more than ten labeled neurons per 10,000 um2. The minimum (Min) and maximum (Max) values indicate the range of density in selected brain regions of individual animals.

Table 2. Comparison of BST subdivisions proposed for the tree shrew (present study) with principal atlases of the rat brain (Paxinos and Watson, 2007). Tree shrew

STA: bed nucleus of the stria terminalis, anterior part

STV: bed nucleus of the stria terminalis, ventral part

STAM: bed nucleus of the stria terminalis, anteromedial part

STAL: bed nucleus of the stria terminalis, anterolateral part

STP: bed nucleus of the stria terminalis, posterior part

Rat (Paxinos and Watson, 2007) STD: bed nucleus of the stria terminalis, dorsal part STMA: bed nucleus of the stria terminalis, medial division, anterior part STLD: bed nucleus of the stria terminalis, lateral division, dorsal part STLJ: bed nucleus of the stria terminalis, lateral division, juxtacapsular part STLI: bed nucleus of the stria terminalis, lateral division, intermediate part STLP: bed nucleus of the stria terminalis, lateral division, posterior part STLV: bed nucleus of the stria terminalis, lateral division, ventral part STMV: bed nucleus of the stria terminalis, medial division, ventral part Fu: bed nucleus of stria terminalis, fusiform part STMAM: bed nucleus of the stria terminalis, medial division, anteromedial part STMAL: bed nucleus of the stria terminalis, medial division, anterolateral part STLP: bed nucleus of the stria terminalis, lateral division, posterior part STLD: bed nucleus of the stria terminalis, lateral division, dorsal part STLJ: bed nucleus of the stria terminalis, lateral division, juxtacapsular part STMP: bed nucleus of the stria terminalis, medial division, posterior part STMPM: bed nucleus of the stria terminalis, medial division, posteromedial part STMPI: bed nucleus of the stria terminalis, medial division, posterointermediate part STMPL: bed nucleus of the stria terminalis, medial division, posterolateral

STPM: bed nucleus of the stria terminalis, posteromedial part

STPL: bed nucleus of the stria terminalis, posterolateral part

part STLP: bed nucleus of the stria terminalis, lateral division, posterior part STMPM: bed nucleus of the stria terminalis, medial division, posteromedial part STMPI: bed nucleus of the stria terminalis, medial division, posterointermediate part STMPL: bed nucleus of the stria terminalis, medial division, posterolateral part STLP: bed nucleus of the stria terminalis, lateral division, posterior part

Table 3. Comparison of afferent projections into the BST of tree shrews (present study) with those of other animals. Brain region anterior olfactory nucleus, external part (AOE) subependymal zone/rhinocele (SEZ/RC)

Tree shrew

Rat (Shin et al., 2008)

Other references
(rabbit; Broadwell, 1975)





frontal cortex (FC)




infralimbic cortex (IL)




piriform cortex (Pir)





(rabbit; Kapp et al., 1985)

(pigeon; Atoji et al., 2006)


agranular insular cortex (AI) endopiriform nucleus, dorsal part (EPd)




entorhinal cortex (Ent)




ventral subiculum (VS) interstitial nucleus of the posterior limb of the anterior commissure (IPAC) ventral pallidum (VP) claustrum (Cl) substantia innominate (SI) accumbens nucleus (Acb) anterior amygdaloid area (AA) nucleus of the lateral olfactory tract (LOT) sublenticular extended amygdala (EA) anterior cortical amygdaloid nucleus (ACo) medial amygdaloid nucleus (MeA) basomedial amygdaloid nucleus (BMA) basolateral amygdaloid nucleus (BLA) central amygdaloid nucleus (CeA) amygdalohippocampal area (AHi) lateral septal nucleus, dorsal part





(rat; McDonald and Mascagni, 1997)

(rat; Shammah-Lagnado et al., 2001)












(rat; Weller and Smith, 1982)
(monkey; Oler et al., 2016)





































(LSd) lateral septal nucleus, ventral part (LSv) lateral septal nucleus, intermediate part (LSi) medial septal nucleus (MS) septofimbrial nucleus (SFi) nucleus of the diagonal band (NDB) stria terminalis (st) medial preoptic area (MPO) lateral preoptic area (LPO) lateral hypothalamic area (LHA) ventromedial hypothalamic nucleus (VMH) dorsomedial hypothalamic nucleus (DMH) perifornical nucleus (PeF)



















































(rat; Vertes, 1992)

supramammillary nucleus (SuM)




mammillary nucleus (MN) parasubthalamic nucleus (PSTN) posterior hypothalamic nucleus (PH) peduncular part of lateral hypothalamus (PLH) paraventricular thalamic nucleus, anterior part (PVA) paraventricular thalamic nucleus, posterior part (PVP) posterior complex of the thalamus (Po) ventral posteromedial thalamic nucleus (VPM) intermediodorsal thalamic nucleus (IMD) central medial thalamic nucleus (CM) rostral linear nucleus of the raphe (RLi) caudal linear nucleus of the raphe (CLi) interfascicular nucleus (IF)
























































ventral tegmental area, rostral part (VTAR) parabrachial pigmented nucleus of the VTA (PBP) pedunculopontine tegmental nucleus (PTg) retrorubral field (RRF) mesencephalic reticular formation (mRt) periaqueductal gray (PAG) rhabdoid nucleus (Rbd) dorsal raphe nucleus (DR) pontine reticular nucleus (PN) lateral parabrachial nucleus (LPB) medial parabrachial nucleus (MPB) locus coeruleus (LC) laterodorsal tegmental nucleus (LDTg) supratrigeminal nucleus (Su5) gigantocellular reticular nucleus (Gi) parvicellular reticular nucleus (PCRt) nucleus of the solitary tract (Sol) ventrolateral reticular nucleus (VLN) intermediate reticular nucleus (IRt)




















































(rat; Ohtake, 1992)


















Index of abbreviations 2 3 10N 12n 12N 3N 3PC 3V 4n 4N 4V 6n 6N 7N 7n 8cn 8vn AA Ab AB Ac ac Acb AcbC AcbSh aci ACo acp AD AHi AHy alv AM Amb AmbC AOD AOE AOL AOM AOV

layer 2 of cortex layer 3 of cortex dorsal motor nucleus of vagus root of hypoglossal nerve hypoglossal nucleus oculomotor nucleus oculomotor nucleus, parvicellular part 3rd ventricle trochlear nerve trochlear nucleus 4th ventricle root of abducens nerve abducens nucleus facial nucleus facial nerve cochlear root of the vestibulocochlear nerve vestibular root of the vestibulocochlear nerve anterior amygdaloid area auditory belt area accessory basal nucleus auditory core area anterior commissure accumbens nucleus accumbens nucleus, core accumbens nucleus, shell anterior commissure, intrabulbar part anterior cortical amygdaloid nucleus anterior commissure, posterior part anterodorsal thalamic nucleus amygdalohippocampal area anterior hypothalamic area alveus of the hippocampus anteromedial thalamic nucleus ambiguus nucleus ambiguus nucleus, compact part anterior olfactory nucleus, dorsal part anterior olfactory nucleus, external part anterior olfactory nucleus, lateral part anterior olfactory nucleus, medial part anterior olfactory nucleus, ventral part

AP APT APTD APTV Aq Arc ArcP ASt AV B BLA BMA CA1 CA2 CA3 cc CC Cd CeA Cg CG Cl CLi CM CnF cp csc Cu cu D3V das DCN DFC DG DLG DMH DpG DpWh DR DS dsc DTg

area postrema anterior pretectal nucleus anterior pretectal nucleus, dorsal part anterior pretectal nucleus, ventral part aqueduct arcuate hypothalamic nucleus arcuate hypothalamic nucleus, posterior part amygdalostriatal transition area anteroventral thalamic nucleus brachium of the superior colliculus basolateral amygdaloid nucleus basomedial amygdaloid nucleus field CA1 of the hippocampus field CA2 of the hippocampus field CA2 of the hippocampus corpus callosum central canal caudate nucleus central amygdaloid nucleus cingulate cortex central gray claustrum caudal linear nucleus of the raphe central medial thalamic nucleus cuneiform nucleus cerebral peduncle commissure of the superior colliculus cuneate nucleus cuneate fasciculus dorsal 3rd ventricle dorsal acoustic stria dorsal cochlear nucleus dorsal frontal cortex dentate gyrus of the hippocampus dorsal lateral geniculate nucleus dorsomedial hypothalamic nucleus deep gray layer of the superior colliculus deep white layer of the superior colliculus dorsal raphe nucleus dorsal subiculum dorsal spinocerebellar tract dorsal tegmental nucleus

dtgx E EA EAC Ect ECu EGP Ent EPl EPlA EW f fi fmi fr g7 GCL Gem Gi GL GlA Gr GrA GrDG Hip IAM ic IC ICj icp IF IG IGL IGP IL IM IMD InC InG Ins Int InWh

dorsal tegmental decussation ependyma and subependymal layer sublenticular extended amygdala sublenticular extended amygdala, central part ectorhinal cortex external cuneate nucleus external globus pallidus entorhinal cortex external plexiform layer of the olfactory bulb external plexiform layer of the accessory olfactory bulb Edinger-Westphal nucleus fornix fimbria of the hippocampus forceps minor of the corpus callosum fasciculus retroflexus genu of the facial nerve granule cell layer of the olfactory bulb gemini hypothalamic nucleus gigantocellular reticular nucleus glomerular layer of the olfactory bulb glomerular layer of the accessory olfactory bulb gracile nucleus granule cell layer of the accessory olfactory bulb granular layer of the dentate gyrus hippocampus interanteromedial thalamic nucleus internal capsule inferior colliculus islands of Calleja inferior cerebellar peduncle interfascicular nucleus indusium griseum intergeniculate leaf internal globus pallidus infralimbic cortex intercalated amygdaloid nucleus, main part intermediodorsal thalamic nucleus interstitial nucleus of Cajal intermediate gray layer of the superior colliculus insular cortex interposed cerebellar nucleus intermediate white layer of the superior colliculus

IO IPA IPAC IPC IPl IPL IPR IRd IRt IRv IT Lat LC LD LDTg lfp LHA LHb LHbL LHbM LL ll LM LMoL lo LOT LPB LPO LRt LSd LSi LSS LSv LV M1 M2 MCL mcp MD MdD MdV ME

inferior olive interpeduncular nucleus, apical subnucleus interstitial nucleus of the posterior limb of the anterior commissure interpeduncular nucleus, caudal subnucleus internal plexiform layer of the olfactory bulb interpeduncular nucleus, lateral subnucleus interpeduncular nucleus, rostral subnucleus infraradiata dorsalis intermediate reticular nucleus infraradiata ventral area inferior temporal cortex lateral cerebellar nucleus locus coeruleus laterodorsal thalamic nucleus laterodorsal tegmental nucleus longitudinal fasciculus of the pons lateral hypothalamic area lateral habenular nucleus lateral habenular nucleus, lateral part lateral habenular nucleus, medial part nucleus of the lateral lemniscus lateral lemniscus lateral mammillary nucleus lacunosum moleculare layer of the hippocampus lateral olfactory tract nucleus of the lateral olfactory tract lateral parabrachial nucleus lateral preoptic area lateral reticular nucleus lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral stripe of the striatum lateral septal nucleus, ventral part lateral ventricle primary motor cortex secondary motor cortex mitral cell layer of the olfactory bulb middle cerebellar peduncle mediodorsal thalamic nucleus medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part median eminence

me5 Me5 MeA Med MFC MG MGD MGV MHb MiA MiTg ml ML mlf mlx MM MnR MoDG MPA MPB MPO MPT MRe mRt MS mt MT Mx NDB oc oc/dsc och OFC ONL Op opt OPT Or OV P5 PaA PAG

mesencephalic trigeminal tract mesencephalic trigeminal nucleus medial amygdaloid nucleus medial cerebellar nucleus medial frontal cortex medial geniculate nucleus medial geniculate nucleus, dorsal part medial geniculate nucleus, ventral part medial habenular nucleus mitral cell layer of the accessory olfactory bulb microcellular tegmental nucleus medial lemniscus medial mammillary nucleus, lateral part medial longitudinal fasciculus medial lemniscus decussation medial mammillary nucleus, medial part median raphe nucleus molecular layer of the dentate gyrus medial preoptic area medial parabrachial nucleus medial preoptic nucleus medial pretectal nucleus mammillary recess of the 3rd ventricle mesencephalic reticular formation medial septal nucleus mammillothalamic tract medial terminal nucleus of the accessory optic tract matrix region of the medulla nucleus of the diagonal band olivocerebellar tract olivocerebellar tract and dorsal spinocerebellar tract optic chiasm orbital frontal cortex olfactory nerve layer optic nerve layer of the superior colliculus optic tract olivary pretectal nucleus oriens layer of the hippocampus olfactory ventricle (olfactory part of lateral ventricle) peritrigeminal zone paraventricular hypothalamic nucleus, anterior part periaqueductal gray

PBG PBP PC Pc pc PCRt Pd Pe PeF PF PG PH PHA Pir1 PLH Pn PN PnC PnO Po PoDG PoMn Post pPAG PPc PPd PPr Pr Pr5 PrC PrEW PRh PrL PrS pRt PT PTg Pu Pv PvA PVA PVN

parabigeminal nucleus parabrachial pigmented nucleus of the VTA paracentral thalamic nucleus central nucleus of the pulvinar posterior commissure parvicellular reticular nucleus dorsal nucleus of the pulvinar periventricular hypothalamic nucleus perifornical nucleus parafascicular thalamic nucleus pineal gland posterior hypothalamic nucleus posterior hypothalamic area piriform cortex, layer 1 peduncular part of lateral hypothalamus pontine nuclei paranigral nucleus of the VTA pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior complex of the thalamus polymorph layer of the dentate gyrus posteromedian thalamic nucleus postsubiculum p1 periaqueductal gray posterior parietal caudal area posterior parietal dorsal area posterior parietal rostral area prepositus nucleus principal sensory trigeminal nucleus precommissural nucleus pre-Edinger-Westphal nucleus perirhinal cortex prelimbic cortex presubiculum p1 reticular formation paratenial thalamic nucleus pedunculopontine tegmental nucleus putamen ventral nucleus of the pulvinar parietal ventral area paraventricular thalamic nucleus, anterior part paraventricular hypothalamic nucleus

PVP py Py pyx Rad Rbd Re Rh RLi RMC Ro RPa RPC RRF rs RSg Rt RtTg S1 S2 s5 SCC SCN scp scpd SFi SFO SGe SHi SHy sm SNC SNL SNR SO Sol SON Sp5 sp5 SPTg SpVe st

paraventricular thalamic nucleus, posterior part pyramidal tract pyramidal cell layer of the hippocampus pyramidal decussation radiatum layer of the hippocampus rhabdoid nucleus reuniens thalamic nucleus rhomboid thalamic nucleus rostral linear nucleus of the raphe red nucleus, magnocellular part nucleus of Roller raphe pallidus nucleus red nucleus, parvicellular part retrorubral field rubrospinal tract retrosplenial granular cortex reticular thalamic nucleus reticulotegmental nucleus of the pons primary somatosensory cortex secondary somatosensory cortex sensory root of the trigeminal nerve somatosensory caudal cortex suprachiasmatic nucleus superior cerebellar peduncle superior cerebellar peduncle, descending limb septofimbrial nucleus subfornical organ supragenual nucleus septohippocampal nucleus septohypothalamic nucleus stria medullaris of the thalamus substantia nigra, compact part substantia nigra, lateral part substantia nigra, reticular part superior olive solitary nucleus supraoptic nucleus spinal trigeminal nucleus spinal trigeminal tract subpeduncular tegmental nucleus spinal vestibular nucleus stria terminalis

STh StHy STA STAL STAM STP STPL STPM STV str Su5 Sub SubC SuG SuM SuML SuMM sumx TC tfp TI TS tth Tu Tu1 tz Tz V1 V2 VCA VCN Ves VL VLG VLN VM VMH VP VPM VPO VPPC VPT

subthalamic nucleus striohypothalamic nucleus bed nucleus of the stria terminalis, anterior part bed nucleus of the stria terminalis, anterolateral part bed nucleus of the stria terminalis, anteromedial part bed nucleus of the stria terminalis, posterior part bed nucleus of the stria terminalis, posterolateral part bed nucleus of the stria terminalis, posteromedial part bed nucleus of the stria terminalis, ventral part superior thalamic radiation supratrigeminal nucleus submedius thalamic nucleus subcoeruleus nucleus superficial gray layer of the superior colliculus supramammillary nucleus supramammillary nucleus, lateral part supramammillary nucleus, medial part supramammillary decussation temporal cortex transverse fibers of the pons temporal inferior area triangular septal nucleus trigeminothalamic tract olfactory tubercle layer 1 of olfactory tubercle trapezoid body nucleus of the trapezoid body primary visual cortex secondary visual cortex ventral cochlear nucleus, anterior part ventral cochlear nucleus vestibular nucleus ventrolateral thalamic nucleus ventral lateral geniculate nucleus ventrolateral reticular nucleus ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventral pallidum ventral posteromedial thalamic nucleus ventral periolivary nucleus ventral posterior nucleus of the thalamus, parvicellular part ventral posterior thalamic nucleus

VS vsc VTAR VTg vtgx VTM xscp ZI Zo

ventral subiculum ventral spinocerebellar tract ventral tegmental area, rostral part ventral tegmental nucleus ventral tegmental decussation ventral tuberomammillary nucleus decussation of the superior cerebellar peduncle zona incerta zonal layer of the superior colliculus