Liang and Paxinos Molecular Brain (2020) 13:67 https://doi.org/10.1186/s13041-020-00602-8 RESEARCH Open Access Afferents of the mouse linear nucleus Huazheng Liang1,2* and George Paxinos2 Abstract The linear nucleus (Li) was identified in 1978 from its projections to the cerebellum. However, there is no systematic study of its connections with other areas of the central nervous system possibly due to the challenge of injecting retrograde tracers into this nucleus. The present study examines its afferents from some nuclei involved in motor and cardiovascular control with anterograde tracer injections. BDA injections into the central amygdaloid nucleus result in labeled fibers to the ipsilateral Li. Bilateral projections with an ipsilateral dominance were observed after injections in a) jointly the paralemniscal nucleus, the noradrenergic group 7/ Köllike -Fuse nucleus/subcoeruleus nucleus, b) the gigantocellular reticular nucleus, c) and the solitary nucleus/the parvicellular/intermediate reticular nucleus. Retrogradely labeled neurons were observed in Li after BDA injections into all these nuclei except the central amygdaloid and the paralemniscal nuclei. Our results suggest that Li is involved in a variety of physiological functions apart from motor and balance control it may exert via its cerebellar projections. Keywords: linear nucleus, hindbrain, amygdala, paralemniscal nucleus, subcoeruleus nucleus, reticular formation, solitary nucleus Introduction of Li to the cerebellum which is important for move- The linear nucleus (Li) is found in the medullary reticu- ment control [7], it is expected that Li has a role in lar formation surrounding the middle segment of the movement. Apart from cerebellar efferents, neither the compact part of the ambiguus nucleus (AmbC) in dorsal, efferents nor afferents of Li have been rigorously studied. medial and/or lateral aspects. It resembles an upside However, incidental observations from earlier studies down L or a capital pi (Π) in Nissl stained or retrograde suggest several candidate structures becaue these struc- tracer labeled sections [37]. This part of the brain con- tures provide inputs to the medullary reticular formation tains clusters of nuclei with diverse functions centered where Li is located. These include the central nucleus of on speech, swallowing, cardiovascular and respiratory the amygdala [14, 48], periaqueductal gray [45], the soli- control and is expected to have different functional in- tary nucleus (Sol) [2], Köllike-Fuse nucleus (KF) [17] puts from the rest of the brain. and the lateral vestibular nucleus (LVe) [4]. Further The linear nucleus (Li) was first identified in 1978 by studies using retrograde tracers also suggest that the Li horseradish peroxidase retrograde labelling from rat projects to the raphe nuclei [13, 18], the lateral paragi- cerebellar tactile regions [47]. Since then it has been gantocellular reticular nucleus (LPGi) [28], the peduncu- confirmed that Li projects to the cerebellar cortex [9, lopontine tegemental nucleus (PPTg) [42], and the 36], especially the paramedian lobule, crus2 [36], and septal area [46]. The majority of the abovementioned deep cerebellar nuclei [38]. Given the strong projections nuclei have recognized roles in movement control, con- sistent with the likely role of the projections from Li to * Correspondence: [email protected] the cerebellum. Thus, some of these nuclei may provide 1Department of Neurology, Translational Research Institute of Brain and relay signals to the cerebellum through Li. Some of these ’ Brain-like Intelligence, Shanghai Fourth People s Hospital Affiliated to Tongji regions such as the amygdala, the solitary nucleus, raphe University School of Medicine, 1878 North Sichuan Road, Hongkou District, Shanghai 200081, China nuclei also have established roles in cardiovascular 2Neuroscience Research Australia, Sydney, NSW 2031, Australia © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liang and Paxinos Molecular Brain (2020) 13:67 Page 2 of 11 control and pain modulation, suggesting that Li may used for each nucleus. As a control group, two mice re- share these functions, if these regions are confirmed to ceived BDA injections into the cisterna magna and project to the Li. Notably, each of these regions also pro- others received BDA injections to adjacent brain nuclei. ject to the spinal cord to regulate these functions. In all cases, the needle of the Hamilton syringe was left The Li can be identified by the expression of genes in- in place for 15 min after the injection and then the skin cluding slc17a7 (http://mouse.brain-map.org/gene/show/ was sutured, topical antibiotic tetracycline was sprayed 48802) and zic 1 (http://mouse.brain-map.org/experiment/ over the site of incision, buprenorphine was injected show/72103843) relative to other parts of the medullary re- subcutaneously. ticular formation. We have also shown in the adult rodent brain that seipin, a lipid metabolism related protein, is Tissue preparation expressed in Li but is also expressed in surrounding regions The mice were anesthetized with a lethal dose of pento- including the gigantocellur reticular nucleus (Gi) [26]. barbital solution (0.1 ml, 200 mg/ml) 3 weeks after HCN4, potassium/sodium hyperpolarization-activated stereotaxic injections along with 5 mice that did not cyclic nucleotide-gated channel 4, was first identified undergo tracer injections. They were then flushed with in the heart and brain [29]. It is involved in pace- 30 ml of 0.9 % normal saline containing heparin (150 maker activities in many brain areas including the IU/mouse; Sigma) using a peristaltic pump (entry cerebellum [6, 21]. The present study investigated through the left ventricle of the heart), followed by 60 whether HCN4 is expressed in Li. ml of 4 % paraformaldehyde (Sigma) (in 0.1 M phos- The main objective of the study is to examine afferents phate buffer). The brain and spinal cord were removed to Li from the central amygdaloid nucleus, KF, the para- and postfixed in 4 % paraformaldehyde for 2 h at 4 °C, lemniscal nucleus (PL) which is rostral and adjacent to and then transferred to 30% sucrose (in 0.1 M PB solu- KF, Gi, Sol and adjacent parvicellular/intermediate re- tion). After 48 h, brains were cut into 40 μm thick sec- ticular nucleus (PCRt/IRt). All of these nuclei project to tions using a Leica CM 1950 cryostat. Every second the spinal cord and are potentially involved in motor section from the brain was used for anterograde tracing and cardiovascular control [22–25]. studies. Materials and methods Immunohistochemistry Animals Peroxidase immunohistochemistry for potassium/so- C57BL/6 mice of 12-14 weeks of age, weighing 25-30 g dium hyperpolarization-activated cyclic nucleotide-gated were used and obtained from the Animal Resource channel 4 (HCN4) was undertaken on half of the brain Centre, Western Australia. sections of five mice that did not undergo surgery. Sec- tions were washed and treated with 1 % H2O2 in 50 % Anterograde tracing ethanol before being transferred into 5 % goat serum in Mice were anesthetized by an intraperitoneal injection of 0.1 M PB to block non-specific antigen binding sites. ketamine (80 mg/kg) and xylazine (5 mg/kg) diluted in The sections were incubated in the primary anti-HCN4 normal saline and placed in a mouse stereotaxic instru- (rabbit, 1:500, Alomone, APC-052) solution overnight ment (Kopf Instruments, Tujunga, CA, USA). The skull and subsequently in the secondary antibody (biotinylated surface was exposed and a hole was made above the nu- goat anti-rabbit IgG; Sigma, 1:200) for 2 h. The sections clei of interest at the following coordinates [35] (antero- were then washed and transferred to an extravidin per- posterior, medio-lateral, dorso-ventral coordinates, re- oxi- dase solution (Sigma, 1:1000) for 2 h. Finally, the spectively: central amygdaloid nucleus- bregma: -0.82 to sections were incubated in a 3,3,-diaminobenzidine -1.58 mm, midline: +2.0 to +2.6 mm, surface: -4.25 to (DAB) reaction complex (Vector lab, Burlingame, CA, -4.75 mm; paralemniscal nucleus-Bregma: -4.16 to -4.72 USA) until an optimal colour developed. At the end of mm, midline:+1.0 to +1.25 mm, surface: -2.175 to -2.5 the procedure, the sections were mounted and dehy- mm; KF- Bregma:-4.83-5.33mm, midline:+1.0-1.75mm, drated before being coverslipped. surface:-3.0-4.25mm; solitary nucleus and parvicelluar/ Sections from BDA-injected mice were washed and intermediate reticular nucleus:-6.59-7.07mm, midline: treated with 1% H2O2 in 50 % ethanol before being +0.75-1.5mm, surface: -2.75-3.5mm; gigantocellular re- transferred to 5 % goat serum in 0.1 M PB to block the ticular nuclei: Bregma:-5.63 to -7.19 mm, midline:0–1.25 non-specific sites. After 2 h, sections were incubated in mm, surface:-3.40 to -4.85 mm). The target areas were an extravidin peroxidase solution (Sigma, 1:1,000) for injected with 10-20 nl of biotinylated dextran amine 2h.