Edited by: Yun-Qing Li, Fourth Military Medical University, China
Reviewed by: Xu-Feng Huang, University of Wollongong, Australia; Adhil Bhagwandin, University of Cape Town, South Africa
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The cerebrospinal fluid (CSF)-contacting nucleus is a unique nucleus in the brain. It is located within the ventral gray of the lower portion of the aqueduct (Aq) and upper portion of the fourth ventricle (4V) floor (Song et al.,
The diencephalon is located between the cerebral cortex and the mesencephalon and is covered by the cortex. It can be divided into five parts: dorsal thalamus, epithalamus, hypothalamus, metathalamus, and subthalamus. The dorsal thalamus serves as a relay center for the transmission of sensory and motor messages from the medulla oblongata and spinal cord to the cerebrum. The hypothalamus is a high center of visceral function modulation, and it is extremely important for the maintenance of homeostasis. The metathalamus processes the visual and auditory information to the cortex. The epithalamus (mainly habenula) is involved in reward processing and affective control (Hikosaka,
One of the aims of Neuroscience is to unveil the neural networks between different types of neurons to understand the functions of the brain (Watabe-Uchida et al.,
Specific pathogen-free (SPF) grade Sprague–Dawley rats (weight 250 ± 50 g) were acquired from the Experimental Animal Centre of Xuzhou Medical University. Rats successfully injected with the tracer into the CSF-contacting nucleus were used for observation and analysis (
Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and heads were fixed on a stereotaxic instrument (Stoelting 51700, USA). A 1% CB solution (0.2 μl, Sigma, USA) was injected into the core of the CSF-contacting nucleus (Bregma: 8,242 ± 183 μm, Lateral: 92 ± 6 μm, Depth: 6,451 ± 109 μm; Song et al.,
Seven to 10 days after the injection of the tracer, rats were perfused and sacrificed. Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.) and perfused with 300 ml of phosphate-buffered saline (0.01 M PBS, pH 7.4), followed by 4% paraformaldehyde in 0.2 M phosphate buffer (300 ml, pH 7.4). The whole brain and spinal cord were isolated and sectioned coronally on a cryostat (Leica CM1900, Germany) at 40 μm. All sections were kept in sequence and numbered. In this study, only the diencephalon regions were captured and analyzed.
All sections were examined with CB immunofluorescence (rabbit anti-CB primary antibody diluted in 1:600, Abcam; donkey anti-rabbit Alexa Fluor 488 secondary antibody diluted in 1:200, Life Technologies, Carlsbad, CA, USA). Sections were mounted in sequence on slides, counterstained with DAPI and coverslipped. Diencephalon sections were imaged with a fluorescence microscope (Leica DM6, Germany) and a confocal laser microscope (Zeiss, Germany). The cell density of CB-positive neurons (cell number/0.2 mm2 area) in each brain region was calculated using Image-Pro Plus 7.0 software. The density of CB-positive neurons was classified as sparse, moderate and dense according to the densities: <5, 6–10 and >10, respectively.
The CB-positive neurons were aligned, segmented and registered according to the rat common reference atlas (Paxinos and Watson,
Injections of the CB tracer produced dense positive staining (green). The tracer was confined within the boundary of the CSF-contacting nucleus, where the microsyringe needle tract can be seen to be located at the core of the CSF-contacting nucleus (
Image of the cholera toxin B subunit (CB)-tracer injection into the cerebrospinal fluid (CSF)-contacting nucleus.
After the retrograde tracer was injected into the CSF-contacting nucleus, it was transported retrogradely along the axons, and neuron somata projecting from the diencephalon was detected.
In the diencephalon, the retrogradely labeled neurons appeared fusiform or polygon-shaped with different neuron sizes and clear processes. Some neurons were bipolar neurons with two obvious processes, while others were multipolar neurons with abundant dendrites (
The cellular morphology of retrogradely labeled neurons in the diencephalon
The entire diencephalon projections to the CSF-contacting nucleus could be identified by positive-labeled neurons and were mainly located in the hypothalamus, epithalamus, and subthalamus. Few or no positive-labeled neurons were identified in the dorsal thalamus and metathalamus.
In the epithalamus, CB-positive neurons were identified in the medial habenular nucleus (MHb), in the medial part of the lateral habenular nucleus (LHbM) and the lateral part of the lateral habenular nucleus (LHbL). Among them, the MHb and LHbM have strong connections, while the LHbL has sparse projections to the CSF-contacting nucleus (
Distribution of CB-positive neurons in the epithalamus
In the subthalamus, CB-positive neurons were observed in the zona incerta (ZI) and subthalamic nucleus (STh). The ZI sends moderate projections, and STh sends sparse connections to the CSF-contacting nucleus (
Distribution of CB-positive neurons in the subthalamus
Most of the connections between the diencephalon and the CSF-contacting nucleus were observed in the hypothalamus. A total of 38 sub-regions in the hypothalamus formed projections of the CSF-contacting nucleus. These include: the paraventricular hypothalamic nucleus (Pa), periventricular hypothalamic nucleus (Pe), anterior hypothalamic area (AH), lateroanterior hypothalamic nucleus (LA), suprachiasmatic nucleus (SCh), supraoptic nucleus (SO), supraoptic nucleus, retrochiasmatic part (SOR), episupraoptic nucleus (ESO), subparaventricular zone of the hypothalamus (SPa), retrochiasmatic area (RCh), retrochiasmatic area lateral part (RChL), lateral hypothalamic area (LH), accessory neurosecretory nuclei (ANS), dorsal hypothalamic area (DA), stigmoid hypothalamic nucleus (Stg), arcuate hypothalamic nucleus (Arc), dorsomedial hypothalamic nucleus (DM), ventromedial hypothalamic nucleus (VMH), A11 dopamine cells (A11), A13 dopamine cells (A13), medial tuberal nucleus (MTu), terete hypothalamic nucleus (Te), paraterete nucleus (PTe), perifornical nucleus (PeF), subincertal nucleus (SubI), posterior hypothalamic nucleus (PH), dorsal part of the posterior hypothalamic area (PHD), ventral part of the pre-mammillary nucleus (PMV), dorsal part of the pre-mammillary nucleus (PMD), dorsal tuberomammillary nucleus (DTM), ventral tuberomammillary nucleus (VTM), lateral mammillary nucleus (LM), parasubthalamic nucleus (PSTh), gemini hypothalamic nucleus (Gem), sub-mammillothalamic nucleus (SMT), prerubral field (PR), rostral interstitial nucleus of medial longitudinal fasciculus (RI), and fields of Forel (F;
Distribution of CB-positive neurons in the hypothalamus Part I
Distribution of CB-positive neurons in hypothalamus Part II
Distribution of CB-positive neurons in hypothalamus Part III
Among the hypothalamic regions, the Pa, AH, LA, SCh, SO, RCh, RChL, LH, ANS, DA, STg, Arc, DM, VMH, A11, A13, MTu, Te, PeF, SubI, PH, PHD, PMV, PMD, DTM, and LM send strong projections to the CSF-contacting nucleus; the SOR, SPa, PTe, VTM, PSTh, Gem, and SMT send moderate projections; the Pe, ESO, PR, RI, and F send sparse connections to the CSF-contacting nucleus (
In summary, CB-positive neurons were distributed in three functional areas including 43 sub-regions in the diencephalon and ranged from sparse, moderate and dense. CB-positive neurons were mainly located in the epithalamus, subthalamus, and hypothalamus. The dorsal thalamus and metathalamus did not contain CB-positive neurons.
The distribution of CB-positive neurons throughout the diencephalon was 3D reconstructed. The density of the connections became clear in a 3D view. Red areas were dense connections (MHb, LHbM, Pa, AH, LA, SCh, SO, RCh, RChL, LH, ANS, DA, Stg, Arc, DM, VMH, A11, A13, MTu, Te, PeF, SubI, PH, PHD, PMV, PMD, DTM, and LM); green areas were moderate connections (ZI, SOR, SPa, PTe, VTM, PSTh, Gem, and SMT); and blue areas were sparse connections (LHbL, STh, Pe, ESO, PR, RI, and F;
Three-Dimensional (3D) view of the diencephalon connection patterns to the CSF-contacting nucleus
In the diencephalon, the CB positive neurons were found in three functional areas including 43 sub-regions. The number of projections from these regions to the CSF- contacting nucleus is shown in
Diencephalic CB-positive neuronal input to the CSF-contacting nucleus (mean ± SD,
The CSF-contacting nucleus is a unique nucleus in the brain. This nucleus has non-synaptic connections between the CSF-contacting neurons and blood vessels and the CSF, and plays an important role in the regulation of body fluids; it has also synaptic connections between the CSF-contacting and non-CSF-contacting neurons, and it carries out nervous crosstalk in the brain. The unique anatomical features of the CSF-contacting nucleus imply that it may be a key structure bridging the nervous-and humor- regulating systems. The connections between the CSF-contacting neurons and blood vessels and CSF have been described previously (Zhang et al.,
Our results indicate that the CSF-contacting nucleus receives extensive projections from three functional areas including 43 sub-regions of the diencephalon (
The schematic diagram of projections from functional areas in the diencephalon to the CSF-contacting nucleus. Among them, epithalamus contains three sub-regions, the hypothalamus contains 38 sub-regions, and subthalamus contains two sub-regions.
The CSF-contacting nucleus receives dense projections from the hypothalamus, which might participate in homeostasis modulation. The role of homeostasis, modulated by the nervous, fluid, and immune systems, is to maintain the coordinated function of organs and systems in life activities, and a healthy physiological equilibrium in a changing world (Burdakov,
It is well established that hypothalamic regions play an important role in the regulation of feeding behavior, which contributes to processes of energy homeostasis (Flier and Maratos-Flier,
Hypothalamic nuclei, such as the SO, LH and PSTh, can modulate the fluid homeostasis and project extensively to the CSF-contacting nucleus. The SO can sense the plasma osmolality and stabilize it
The hypothalamic SCh and SPa have connections to the CSF-contacting nucleus and can both modulate the biological rhythm. The SCh is known as a master brain clock controlling the circadian rhythms. Body activities synchronize the metabolism, cognition and various behaviors to the environmental day-night cycle (Mohawk et al.,
The hypothalamus is a higher center of visceral activity modulation. Activation of the hypothalamus can produce significant autonomic responses. Among the hypothalamic regions and nuclei that mediate the visceral activity regulation are the Pa, DM, VMH, VTM, PeF, PH and PSTh, which project extensively to the CSF-contacting nucleus. The Pa is regarded as an integrative region that modulates the sympathetic outflow and cardiovascular activity (Coote,
The CSF-contacting nucleus receives input from hypothalamic Pa, SO and DA and may participate in stress. Stress responses activate the hypothalamic-pituitary-adrenal (HPA) axis, where Pa can release hormones, such as the corticotropin-releasing hormone (CRH) and AVP, that affect biological activities (Joseph and Whirledge,
Both the LHb and MHb in the epithalamus have strong projections to the CSF-contacting nucleus. Lesion and genetic studies in mice and zebrafish respectively showed neuron burst firing under depression, which can be significantly reversed with antidepressants (Kim et al.,
The CSF-contacting nucleus receives input from the Pa, SO, A11, ZI, and STh and may participate in pain modulation. The Pa is involved in visceral hypersensitivity as revealed by colorectal distension (CRD; Zhang et al.,
The epithalamus (MHb and LHb) and LH in the hypothalamus project to the CSF-contacting nucleus and may participate in drug addiction. The MHb plays a significant role in drug addiction, especially nicotine and opioid addiction because it contains a high density of nicotinic acetylcholine receptors and μ opioid receptors (Fowler and Kenny,
The CSF-contacting nucleus receives input from the LH, PeF, and ZI and may participate in sleeping and arousal. Different neuron types in the tuberomammillary nucleus and LH are implicated in the sleep/wakefulness regulation (Saito et al.,
In this study, we used a tract-tracing method to reveal the CSF-contacting nucleus input patterns from the diencephalon. The unique morphological feature of the CSF-contacting nucleus is that the somata are located in the brain parenchyma and can receive input from the above diencephalon areas; the processes can form synaptic and non-synaptic connections with non-CSF-contacting neurons, CSF, or plasma. Circuits forming between the diencephalon→CSF-contacting nucleus→non-CSF-contacting neurons may participate in the regulation of life activities
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
All animal experiments were approved by and performed in accordance with the guidelines of the Committee for Ethical Use of Laboratory Animals of Xuzhou Medical University.
S-YS and L-CZ designed the study and prepared the manuscript. S-YS, YL, X-MZ, Y-HL, C-YB, C-JS, JH, and J-LC conducted the studies. All authors read and approved the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank all members of the L-CZ group at Xuzhou Medical University.