Next Article in Journal
CRKL, AIFM3, AIF, BCL2, and UBASH3A during Human Kidney Development
Next Article in Special Issue
Prenatal and Postnatal Methyl-Modulator Intervention Corrects the Stress-Induced Glucocorticoid Response in Low-Birthweight Rats
Previous Article in Journal
Enzyme Therapy: Current Challenges and Future Perspectives
Previous Article in Special Issue
Differential Effects of Fkbp4 and Fkbp5 on Regulation of the Proopiomelanocortin Gene in Murine AtT-20 Corticotroph Cells
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 22
Issue 17
10.3390/ijms22179180
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at −25 °C for Several Years

by
Akito Otubo
1,
Sho Maejima
1,
Takumi Oti
1,2,
Keita Satoh
1,3,
Yasumasa Ueda
4,5,
John F. Morris
6,
Tatsuya Sakamoto
1 and
Hirotaka Sakamoto
1,6,*
1
Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan
2
Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, Japan
3
Department of Anatomy, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
4
Department of Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
5
Department of Physiology, Kansai Medical University, Shinmachi, Hirakata, Osaka 573-1191, Japan
6
Department of Physiology, Anatomy & Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(17), 9180; https://doi.org/10.3390/ijms22179180
Submission received: 5 August 2021 / Accepted: 21 August 2021 / Published: 25 August 2021
(This article belongs to the Special Issue Molecular Mechanisms Underlying Stress Response and Resilience)
Browse Figures
Versions Notes

Abstract

:
Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at −25 °C for several years (4–6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

1. Introduction

Studies using macaque monkeys as model non-human primates are essential because of their high applicability to human clinical research which often requires the testing of experimental therapies in primates. However, primate research is now stringently controlled by law around the world [1]. Rodents, in which research is less restricted, are often not sufficiently analogous to humans in their physiology to allow an extrapolation from rodent to human. Medical science is urged to make minimal use of animals such as primates, and basic studies on non-human primates appear to be permitted only if the work could not be carried out in any other species. Thus, although tissues from primates are often essential to achieve relevant results, there are considerable problems in their use.
Arginine vasopressin (AVP), an anti-diuretic hormone, is released, not only into the blood stream, but also into the central nervous system where, in mammals, it has been shown to be important for stress coping, aggression, courtship behavior, learning, bonding, and various socio-sexual behaviors [2]. AVP is produced mainly by magnocellular neurosecretory neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus [3]. The AVP gene encodes a precursor containing AVP, AVP-associated neurophysin II (NPII), and a glycopeptide copeptin [4,5,6]. The expression and release of AVP by magnocellular neurosecretory neurons in the SON and PVN are regulated by physiological conditions, including plasma osmotic pressure and blood pressure [7]. The magnocellular axons project primarily via the internal layer of the median eminence to the posterior pituitary where they release AVP into the systemic circulation. In addition, some parvocellular neurons in the PVN produce AVP and project into extrahypothalamic areas where the AVP and/or other co-packaged molecules regulate brain function as neuromodulators [8].
Corticotrophin-releasing factor (CRF) is a strong stimulator of adrenocorticotrophic hormone (ACTH) secretion from the anterior pituitary when released onto portal capillaries in the median eminence in response to the stress [9]. A population of parvocellular neurons in the anteromedial part of the PVN produces CRF and may, depending on the extent of stress, also produce AVP. Both peptides released into the hypothalamo-hypophysial portal circulation play an important synergistic role in stress resilience [10,11,12,13]. Because intense AVP- and CRF-immunoreactivity have both been observed in the external layer of the macaque median eminence, the peptides are probably co-released into the portal circulation to amplify ACTH release from the primate anterior pituitary [14,15].
In rodents, the presence of glutamate-immunoreactivity in magnocellular neuroendocrine cells of the SON suggests that AVP neurons also produce glutamate as a neurotransmitter [16,17]. Within the neurosecretory endings of the posterior pituitary, glutamate immunoreactivity is specifically localized to electron-lucent microvesicles with no overlap onto the dense-cored neurosecretory vesicle (dcv) population in rats [16]. Immunocytochemical co-localization of CRF and the vesicular glutamate transporter 2 (VGLUT2) in the locus coeruleus of rats suggests that the co-release of CRF and glutamate may function to regulate postsynaptic targets [18]. It is currently unclear whether glutamate has a similar or other functions in the primate hypothalamo-pituitary paraventricular AVP/CRF system.
Tissues, especially those of the brain, fixed in formaldehyde without glutaraldehyde have long been thought to be unsuitable for electron microscopic analysis by reason of the weaker cross-linking action of formaldehyde, e.g., [19,20]. Here we report the immunoelectron microscopic characterization of AVP-producing neurons in the primate hypothalamo-pituitary axis tissue fixed by perfusion with formaldehyde and stored at −25 °C for several years. Special attention was paid to the size of dcv in AVP-producing magno- and parvocellular neurons and to the colocalization of CRF with AVP-related gene products in the dcv. We show that immunoelectron microscopy of formaldehyde-fixed tissue can confirm the well-known size difference in dcv between magno- and parvocellular AVP neurons in Japanese macaque monkeys. Furthermore, we show that, in formaldehyde-fixed stored neural tissue of macaque monkeys, both AVP/CRF and VGLUT2/glutamate can be co-localized in both the magnocellular endings of the posterior pituitary and the parvocellular endings in the external layer of the median eminence.

2. Results

2.1. Antibody Characterization and the Expression of VGLUT2 at the Protein Level in the Posterior Pituitary

Full details of all the antibodies used in this study are shown in Table 1. We first validated by Western blot analysis the specificity for the VGLUT2 and NPII antibodies in Japanese macaque monkeys. Western blot analysis demonstrated the specificity for the guinea pig polyclonal antibody against VGLUT2 and the expression of VGLUT2 at the protein level in the posterior pituitary; a single strong immunoreactive band was detected at the expected molecular weight of ~56 kDa [21] (Figure 1). The mouse monoclonal antibody against AVP-neurophysin II (NPII) also detected a single major band at the expected molecular weight of ~12 kDa on the blots of posterior pituitary [14,22] (Figure 1).

2.2. Anatomy of AVP-Producing Neurons in the Macaque Hypothalamus

Immunostaining for NPII was performed to study the localization of AVP neurons in the hypothalamus of Japanese macaque monkeys. Numerous magnocellular (large-sized) AVP neurons were observed in the PVN (Figure 2A) and SON (Figure 2B). Parvocellular (small-sized) AVP neurons were also observed in the PVN (Figure 2C). The median eminence can be divided into an internal and an external layer. The internal layer is the zone through which the axons of magnocellular neurons densely project into the posterior pituitary (Figure 2D) [3,8,15]. The external layer of the median eminence is where the axons of many parvocellular neurosecretory neurons, including parvocellular AVP/CRF neurons of the PVN, end on hypothalamo-hypophysial portal vessels and release anterior pituitary-hormone-regulating hormones (Figure 2D) [3,8,15].
In this study, immunoelectron microscopic characterization of AVP-producing neurons was performed in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at −25 °C for several years. We found that both ultrastructure and immunoreactivity are sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Immunoelectron microscopy of the posterior pituitary showed many AVP dcv containing both NPII (PS41)- and copeptin (CP8)-immunoreactivity (Figure 3A,B). In other neighboring endings, no NPII- or copeptin-immunoreactivity was detectable (Figure 3C). In the median eminence, many dcv showing PS41/CP8 double-immunoreactivity were observed in both the internal and external layers (Figure 4). However, the AVP dcv in the internal layer (magnocellular) of the median eminence (Figure 4B) were larger than those in the external layer (parvocellular) of the median eminence (Figure 4D).

2.3. Size Characterization of Dcv

Approximately 200 dcv in the neurosecretory axons in the posterior pituitary (magnocellular), the internal layer of the median eminence (magnocellular), and the external layer of the median eminence (parvocellular) were selected and their major axis diameters were measured. The average of major axis was 196 ± 2 nm in the posterior pituitary, 197 ± 2 nm in the internal layer of the median eminence, but significantly smaller (93 ± 1 nm) in the external layer of the median eminence (Figure 5; Table 2).
Next, we measured the diameter of dcv in the perikarya of randomly selected SON and PVN AVP neurosecretory neurons (at ×5000 magnification; Table 3). In the SON (7 cell bodies), the average diameter of dcv contained in the cell body was 204 ± 12 nm (11 vesicles), confirming that, as expected, all AVP neurons in the SON are magnocellular (Figure 6; Table 3). The 18 AVP neurons in the PVN selected for analysis formed two distinct groups: those with larger dcv (180 ± 19 nm; 11 vesicles in Figure 7A,B) identified as magnocellular; and those with smaller dcv (102 ± 10 nm; 14 vesicles in Figure 7C,D) identified as parvocellular neurons.

2.4. A Subpopulation of Parvocellular AVP Neurons in the PVN Co-Expresses CRF

In the PVN, of the 18 AVP neuronal cell bodies analyzed, 6 were identified as magnocellular on the basis of vesicle size; none of these showed any CRF-immunoreactivity. Of the 12 PVN perikarya identified as parvocellular on the basis of vesicle size, a subpopulation (especially in the parvocellular part of the PVN) co-expressed CRF (Figure 8) and such neurons were more intermingled with magnocellular neurons in the macaque monkey than in rodent PVN as reported previously [14,25,33]. The terminal regions of parvocellular AVP/CRF neurons in the external layer of the median eminence were next analyzed (Figure 9). In these cells, the major axis diameter of the sparse dcv was 106 ± 3 nm (16 vesicles) (Figure 10) for the vesicles in the CRF-immunoreactive terminals, comparable to the 113 ± 9 nm (12 vesicles) in the cell body (Figure 11). In two putative parvocellular neurons (#24 and #25), the mean major axis of the dcv appeared slightly larger which might relate to the maturity of the vesicles (Table 3) [34].

2.5. Both Magno- and Parvocellular AVP Neurons Are Glutamatergic

We performed immunoelectron microscopy for l-glutamate in the posterior pituitary in order to determine its occurrence in the terminal regions of magnocellular AVP neurons. The immunoreactivity for glutamate was associated primarily with the clusters of small electron-lucent vesicles in the axonal endings of AVP neurons of posterior pituitary (Figure 12A,B). In contrast, NPII-immunoreactivity was associated with dcv (Figure 12A,B). Next, we examined the terminal regions of parvocellular axons in the median eminence. Triple staining for CRF/NPII/glutamate (Figure 12C,D) showed that, as in the posterior pituitary, glutamate-immunoreactivity was associated with the clusters of electron-lucent microvesicles in the parvocellular endings which contained AVP/CRF-positive dcv (Figure 12C,D) and in other similar endings of the terminals of other parvocellular neurosecretory neurons. Due to the mild formaldehyde fixation, the membrane of single microvesicles could not be clearly distinguished. However, a comparison with glutaraldehyde-fixed material leaves no doubt that these are clusters of microvesicles. Similar results were obtained by immunoelectron microscopy for VGLUT2 in both the posterior pituitary and the external layer of the median eminence (Figure 13). Very few gold particles were associated with other intra-varicosity organelles, such as the mitochondria, and background labelling over glial and endothelial elements was also extremely low. Immunoelectron microscopy studies were repeated independently at least three times using different monkeys and always produced similar results.

3. Discussion

It is often assumed that glutaraldehyde, a cross-linking fixative, is essential for the preservation of fine ultrastructure [19,20] and that formaldehyde-only fixed tissues are therefore inappropriate for electron microscopic analyses. However, the use of glutaraldehyde often results in a reduction of immunoreactivity and, for this reason, formaldehyde-fixed tissues without glutaraldehyde are used in most cases for immunohistochemical analysis at the light microscopic level [19,20]. The current study demonstrates that, in the macaque brain, interpretable ultrastructure and adequate ultrastructural immunoreactivity are both preserved in brain tissue even after long-term storage at −25 °C for conventional light microscopy, provided that the fixed tissues are post-fixed with 0.1% glutaraldehyde after the long-term storage. Thus, formalin-fixed macaque brains stored for a long period, at least up to six-years in this study, can be used for immunoelectron microscopic analysis, suggesting that not only macaque brain, but also postmortem formalin-fixed human brain tissue could be amenable to immunoelectron microscopic analysis. It is also possible that it could be preserved over decades in antifreeze solution. Furthermore, the ultrastructure of tissues from rare animals such as endangered species might be studied in this way even if they had been prepared for light microscopic immunohistochemistry and stored at −25 °C in antifreeze solution.
The methodology used in this study has some obvious advantages in the ultrastructural analysis of the nervous system, in which tissue specimens are necessarily small, particularly where the brain is large, as in macaque monkeys and humans. After confirmation of the specificity of antibodies at the light microscopic level, post-embedding immunoelectron microscopic analysis can readily be performed on adjacent sections. Furthermore, because identification of brain nuclei and/or cells is easier at the light microscopic level, they can then be precisely targeted for post-embedding immunoelectron microscopy.
The size of dcv contained in the terminals of AVP neurons was quantified and compared among the posterior pituitary and internal/external layers of the median eminence. This showed, also for the macaque, the expected significant difference in size of the dcv (Figure 14). It is known that the posterior pituitary and the internal layer of the median eminence contain the axons of magnocellular AVP neurons of the PVN and SON, whereas the external layer of the median eminence is the terminal region of parvocellular AVP neurons of the PVN [3,8,15] and other parvocellular neurons influence the anterior pituitary. In the dcv of the internal layer of the median eminence through which the magnocellular axons pass en route to the posterior pituitary, the AVP precursor should be processed sufficiently to reveal both prominent NPII- and copeptin-immunoreactivity in almost all of the axons [14,15,33]. In the macaque hypothalamus, quantifying only the size of the dcv in the cell body or its axon allows us to determine the magnocellular or parvocellular identity of the cell. Finally, our study confirms at the electron microscopic level that certain axonal endings in the external layer of the median eminence of macaques are derived from parvocellular AVP neurons of the PVN.
It has been reported that rodent AVP neurons use glutamate as a neurotransmitter [16,17,35,36]. In this study, we examined the detectability, and therefore the expression of glutamate and VGLUT2 in the macaque posterior pituitary. We first demonstrated by Western blotting that VGLUT2 was expressed in the macaque posterior pituitary, suggesting that magnocellular AVP (and also oxytocin) neurons use glutamate as a neurotransmitter in Japanese macaque monkeys. Immunoelectron microscopic analysis revealed that the AVP/CRF-immunoreactivity was restricted to the dcv, but the VGLUT2-immunoreactivity was associated with the smaller, electron-lucent microvesicles (Figure 14). Similarly, immunoreactivity for glutamate itself was essentially restricted to regions containing clusters of microvesicles in the axonal endings of AVP neurons in both the posterior pituitary and external layer of the median eminence (Figure 14). Because the antibody for glutamate, an amino acid, is raised against the cross-linking site of glutaraldehyde as the antigen, it is thought to be essential that glutaraldehyde is included in the fixative prior to glutamate immunocytochemistry [16,17,31]. In our study, although glutaraldehyde was not included in the primary perfusion fixative, post-fixation of the formaldehyde-fixed tissue with glutaraldehyde enabled us to detect glutamate by immunoelectron microscopy even after prolonged storage. Therefore, the subcellular localization of amino acids appears, surprisingly, to be preserved for a long period even after conventional formaldehyde fixation. These results suggest that the immunoelectron microscopic detection of amino acids including glutamate and GABA would also be possible in conventional formaldehyde-fixed and stored human brain tissue.
Our results in macaque tissue show that glutamate as an excitatory neurotransmitter is in microvesicles both in magno- and parvocellular AVP neurons. Although the localization of glutamate receptors has been reported in the neural lobe, median eminence and anterior pituitary, a clear role for glutamate secretion at these sites remains unknown [37,38]. Salt loading has been shown to produce robust increases in the VGLUT2 mRNA of AVP neuron perikarya and in VGLUT2-immunoreactivity in the rat posterior pituitary [39], suggesting that an osmotic challenge could produce an increase in glutamate release in the primate magnocellular AVP system (Figure 14). Similarly, a role in the anterior pituitary of glutamate released from parvocellular AVP/CRF neurons into the hypothalamo-hypophysial portal veins remains unclear. Functional implications for glutamate release at the terminals of both magno- and parvocellular AVP neurons remain to be explored.

4. Materials and Methods

4.1. Animals

Three male (2–9-year-old, weight 2.3–12.6 kg) and four female (9–11-year-old, weight 7.2–10.8 kg) Japanese macaque monkeys (Macaca fuscata) were used in this study. Macaques were maintained in a temperature-controlled (22–24 °C) room under a daily photoperiod of 12:12 h light/dark cycle (lights off at 8:00 p.m.). These animals were checked and shown to be free of specific pathogens. Food and water were available ad libitum. All animals were kept in individual cages. The housing and experimental protocols followed the guidelines of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and were in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Okayama University (Okayama, Japan), by Tokyo Medical and Dental University (Tokyo, Japan), and by National Institute for Physiological Sciences (Okazaki, Japan). All efforts were made to minimize animal suffering and reduce the number of animals used in this study.

4.2. Tissue Processing

Hypothalamic sections and posterior pituitaries were obtained from four macaque monkeys (2 females and 2 males) as described previously [14]. Briefly, macaques were deeply anaesthetized with an overdose of sodium pentobarbital (~100 mg/kg body weight), and transcardially perfused with physiological saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4). After perfusion, brains and pituitaries were immediately removed and immersed in the same fixative for ~16 h at 4 °C. Coronal 30-µm-thick sections of the hypothalamus were prepared with a Linear-Slicer (PRO10, Dosaka EM, Kyoto, Japan). Preparations were rinsed with PB and then immersed in antifreeze solution (30% glycerol, 30% ethylene glycol, 0.36% NaCl, and 0.05% NaN3 in 40 mM PB) for storage at −25 °C until use. All the initial fixation was done 4–6 years before the present set of experiments. For Western blot analysis, three female macaques were sacrificed by blood loss under deep pentobarbital anesthesia (see above). Posterior pituitaries were quickly removed, frozen immediately in powdered dry ice, and stored at −80 °C until use.

4.3. Immunofluorescence

The long-term stored, formaldehyde-fixed sections were rinsed with phosphate-buffered saline (PBS) containing 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) (PBST) five times for 10 min each. After blocking nonspecific binding with 1% normal goat serum and 1% BSA in PBST for 30 min at room temperature, the sections were incubated with mouse monoclonal anti-AVP-NPII antibody (PS41; 1:1000 dilution) and with rabbit antiserum directed against copeptin (1: 10,000 dilution) or with rabbit antiserum directed against CRF (1: 20,000 dilution) for 4–5 days at 4 °C. The mouse monoclonal NPII antibody has previously shown to be specific for AVP neurons in rodents [26,27] and macaque monkeys [14]. The copeptin7–14 (ATQLDGPA) fragment, used as an antigen to produce our copeptin antiserum, is well conserved in mammals. It is identical in rats [40], mice [41], macaque monkeys [14], and humans [40]. The copeptin antiserum has also been characterized previously by our immunohistochemistry [22], Western [23] and dot blot [22] analyses. The rabbit polyclonal antiserum against rat/human CRF (PBL rC70), which was kindly donated by Wylie Vale, has been characterized previously [14,22,24]. It was directed against the rat form of CRF, which is identical to the human form, and has also been characterized in non-human primates [14,25]. Alexa Fluor 546-linked goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-linked goat anti-rabbit IgG (Molecular Probes) were used for detection at 1:1000 dilution. Immunofluorescence studies were repeated independently at least three times using different macaques and always produced similar results. Immunostained sections were viewed by confocal laser scanning microscopy (FluoView 1000, Olympus, Tokyo, Japan). The information for antibodies used in this study is shown in Table 1.

4.4. Post-Embedding Immunoelectron Microscopy

The long-term stored, formaldehyde-fixed sections were rinsed with PBS five times for 10 min each, and re-fixed in 4% PFA + 0.1% glutaraldehyde in 0.1 M PB for 4–6 h at 4 °C. Sections were then rinsed with 0.1 M PB three times for 5 min each, and were dehydrated through increasing concentrations of methanol, flat-embedded in LR Gold resin (Electron Microscopy Sciences, Hatfield, PA, USA) by gently pressing against the bottom of the flat-bottom-capsule with a pre-prepared resin block, and polymerized under UV lamps at −25 °C for 24 h. Ultrathin sections (70 nm in thickness) were collected on nickel grids coated with (or without for triple labeling) a collodion film, rinsed with PBS several times, then incubated with 2% normal goat serum and 2% BSA in 50 mM Tris(hydroxymethyl)-aminomethane-buffered saline (TBS; pH 8.2) for 20 min to block non-specific binding. The sections were then incubated with the mouse monoclonal antibody against NPII (1:200 dilution), with the rabbit polyclonal antisera against CRF (1:5000 dilution), copeptin (1:100 dilution), l-glutamate (1:10 dilution, Abcam, Cambridge, UK), and/or with the guinea pig polyclonal antibody against VGLUT2 (1:20 dilution, Synaptic Systems, Göttingen, Germany) for 1 h at room temperature. After incubation with the primary antibodies, the sections were washed with PBS, then incubated with a goat antibody against rabbit IgG conjugated to 5 or 10 nm gold particles (1:50 dilution, BBI Solutions, Cardiff, UK) and a goat antibody against mouse IgG conjugated to 15 nm gold particles (1:50 dilution, BBI Solutions) and/or a goat antibody against guinea pig IgG conjugated to 5 nm gold particles (1:50 dilution, Nanoprobes, Yaphank, NY, USA) for 1 h at room temperature. The Abcam antibody to glutamate was raised in rabbit against l-glutamate conjugated to glutaraldehyde as the immunogen. The manufacturer’s information shows no measurable cross-reactivity detected against glutamate in peptides or proteins but modest cross-reactivity against d-glutamate. The specificity of this antibody was confirmed by immunostaining in the rat retina [31]. To intensify the detectability of the immunoreaction for CRF in the cell body, a streptavidin-biotin intensification kit (Nichirei, Tokyo, Japan) was used. Tissues were first incubated with the biotinylated goat anti-rabbit IgG antibody for 10 min at room temperature, followed by incubation in avidin-biotin-horseradish peroxidase (HRP) complex solution for 5 min at room temperature. The sections were then washed with PBS, incubated with the goat antibody against HRP conjugated to 6 nm gold particles (1:50 dilution, Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 1 h at room temperature.
Triple immunoelectron microscopy with antibodies against CRF, NPII, and glutamate was performed by using the front and back of ultrathin sections mounted on nickel grids without a supporting film. First, immunocytochemistry with a pair of primary antibodies (CRF and NPII) was performed on one side of the section and detected using 15 nm (mouse) and 10 nm (rabbit) colloidal gold particles (BBI Solutions), respectively. Next, immunocytochemistry with the other primary rabbit antibody (against glutamate) was performed on the other side of the section and detected by use of 5 nm colloidal gold particles (BBI Solutions). Immunoelectron microscopy studies were repeated independently at least three times using different macaques and always produced similar results.
Finally, the sections were contrasted with uranyl acetate and lead citrate and viewed using an H-7650 (Hitachi, Tokyo, Japan) or JEM-1010 (JEOL, Tokyo, Japan) electron microscope operated at 80 kV. The information for antibodies used in this study is shown in Table 1.

4.5. Western Blotting

The lysates derived from posterior pituitaries were boiled in 10 µL sample buffer containing 62.5 mM trishydroxymethyl-aminomethane-HCl (Tris-HCl; pH. 6.8), 2% SDS, 25% glycerol, 10% 2-mercaptoethanol, and a small amount of bromophenol blue. These samples were run on a 4–20% SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA) from the gel by a semidry blotting apparatus (Bio-Rad Laboratories). The blotted membranes were blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, Tokyo, Japan) for 30 min at room temperature and incubated overnight at 4 °C with anti-AVP-NPII mouse monoclonal antibody (PS41; 1:1000 dilution) or anti-VGLUT2 guinea pig polyclonal antibody (1:10,000 dilution) in Can Get Signal Solution 1 (TOYOBO). The blotted membranes were rinsed three times with 0.05% Tween 20 in Tris-HCl-buffered saline (TBST) and incubated with horseradish peroxidase-conjugated goat polyclonal antibody against mouse IgG (Bio-Rad Laboratories) or guinea pig IgG (Bioss Inc., Woburn, MA, USA) 1:10,000 dilution in Can Get Signal Solution 2 (TOYOBO) for 1 h at room temperature. After washing for three times with TBST, blots were visualized by Immun-Star WesternC Chemiluminescence Kit (Bio-Rad Laboratories). The information for antibodies used in this study is shown in Table 1. Western blotting analyses were repeated independently at least three times using different macaques and always produced similar results.

4.6. Statistical Analysis

All data values were expressed as mean ± standard error of the mean (SEM) and were analyzed using Statcel4 software. Statistical significance was determined as p < 0.05 using one-way analysis of variance (ANOVA). When significant difference was found by ANOVA, the post hoc Tukey–Kramer test was performed. All the various analyses in this study were conducted “blind”. All electron micrographs were coded and evaluated without the knowledge of the tissue, and the code was not broken until the analysis was complete.

5. Conclusions

We report the immunoelectron microscopic characterization of AVP-producing neurons in primate hypothalamo-pituitary systems by the use of formaldehyde-fixed tissues stored at −25 °C for up to six years in this study. The size difference in dcv between magno- and parvocellular AVP neurons in Japanese macaque monkeys was determined (Figure 14). Furthermore, we have determined that, despite the prolonged fixation and storage, the size of the magno- and parvocellular dcv was virtually identical to that observed in freshly fixed and embedded tissue [8,23,42,43,44,45]. We also demonstrate by immunoelectron microscopy that both magno- and parvocellular AVP neurons are glutamatergic in primates (Figure 14). These results show, for the macaque brain, that interpretable neuronal ultrastructure and sufficient immunoreactivity for several relevant proteins are preserved even after a long-term storage of tissues fixed in formaldehyde for light microscopy. They indicate that this methodology could be applied to human post-mortem brain and, therefore, be very useful in translational research.

Author Contributions

Conceptualization, H.S.; methodology, A.O., S.M., T.O., K.S., Y.U. and H.S.; validation, A.O. and H.S.; data curation, A.O. and H.S.; formal analysis, A.O. and H.S.; investigation, A.O., S.M., T.O., K.S. and H.S.; resources, T.O., K.S., Y.U. and H.S.; software, A.O.; visualization, A.O. and H.S.; writing—original draft preparation, H.S.; writing—review and editing, J.F.M.; interpreted the data and provided advice and equipment, Y.U., J.F.M. and T.S.; supervision and project administration, H.S.; funding acquisition, T.O., T.S. and H.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) KAKENHI [to H.S.; 15K15202, 15H05724, 15KK025708, 16H06280 (Advanced Bioimaging Support; ABiS); to T.S.; 21H02520; to T.O.; 20K15837], by Grant-in-Aid for Scientific Research on Innovative Areas “Singularity Biology (No.8007)” of MEXT, Japan (to T.S.; 21H00428), and by the Research Grant from the Suzuken Memorial Foundation, Japan (to H.S.; 19-085). T.O. and K.S. were supported by Research Fellowships of JSPS for Young Scientists.

Institutional Review Board Statement

This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committees of Okayama University-National Institute for Physiological Sciences joint research (Protocol Code MOFF-np-2014-008 and date of approval: 20 April 2015) and Tokyo Medical and Dental University (protocol code A2018-241C and date of approval: 18 October 2018).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in the manuscript. All relevant data of this manuscript are available from the corresponding author upon reasonable request.

Acknowledgments

Tissues of Nihonzaru (Japanese macaque monkeys) were provided by National Institutes of Natural Sciences (NINS) and Kyoto University Primate Research Institute (KUPRI) with support in part by the National Bio-Resource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank Narumi Katsuyama (Tokyo Medical and Dental University, Japan), Kei Tamaura and Ray Nomura for their technical support. We are grateful to Wylie Vale for the supply of an antiserum against CRF.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

ANOVAanalysis of variance
AVParginine vasopressin
CRFcorticotrophin-releasing factor
dcvdense-cored neurosecretory vesicle
HRPhorseradish peroxidase
NPneurophysin
PBphosphate buffer
PBSphosphate-buffered saline
PFAparaformaldehyde
PVDFpolyvinylidene difluoride
PVNparaventricular nucleus
SEMstandard error of the mean
SONsupraoptic nucleus
Tris-HCltrishydroxymethyl-aminomethane-HCl
TBSTTris-HCl-buffered saline
VGLUT2vesicular glutamate transporter 2

References

  1. Abbott, A. Biomedicine: The changing face of primate research. Nature 2014, 506, 24–26. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Walum, H.; Young, L.J. The neural mechanisms and circuitry of the pair bond. Nat. Rev. Neurosci. 2018, 19, 643–654. [Google Scholar] [CrossRef] [PubMed]
  3. Buijs, R.M.; De Vries, G.J.; Van Leeuwen, F.W.; Swaab, D.F. Vasopressin and oxytocin: Distribution and putative functions in the brain. Prog. Brain Res. 1983, 60, 115–122. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Breslow, E. Structure and folding properties of neurophysin and its peptide complexes: Biological implications. Regul. Pept. 1993, 45, 15–19. [Google Scholar] [CrossRef]
  5. Davies, J.; Waller, S.; Zeng, Q.; Wells, S.; Murphy, D. Further delineation of the sequences required for the expression and physiological regulation of the vasopressin gene in transgenic rat hypothalamic magnocellular neurones. J. Neuroendocrinol. 2003, 15, 42–50. [Google Scholar] [CrossRef]
  6. Land, H.; Schutz, G.; Schmale, H.; Richter, D. Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor. Nature 1982, 295, 299–303. [Google Scholar] [CrossRef]
  7. Koshimizu, T.A.; Nakamura, K.; Egashira, N.; Hiroyama, M.; Nonoguchi, H.; Tanoue, A. Vasopressin V1a and V1b receptors: From molecules to physiological systems. Physiol. Rev. 2012, 92, 1813–1864. [Google Scholar] [CrossRef] [Green Version]
  8. Morris, J. Neurosecretory Vesicles: Structure, Distribution, Release and Breakdown. In Neurosecretion: Secretory Mechanisms; Lemons, J.R., Dayanithi, G., Eds.; Springer Nature Switzerland: Cham, Switzerland, 2020; Volume 8, pp. 81–102. [Google Scholar]
  9. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 1981, 213, 1394–1397. [Google Scholar] [CrossRef] [PubMed]
  10. Fellmann, D.; Bugnon, C.; Bresson, J.L.; Gouget, A.; Cardot, J.; Clavequin, M.C.; Hadjiyiassemis, M. The CRF neuron: Immunocytochemical study. Peptides 1984, 5 (Suppl. 1), 19–33. [Google Scholar] [CrossRef]
  11. Itoi, K.; Talukder, A.H.; Fuse, T.; Kaneko, T.; Ozawa, R.; Sato, T.; Sugaya, T.; Uchida, K.; Yamazaki, M.; Abe, M.; et al. Visualization of corticotropin-releasing factor neurons by fluorescent proteins in the mouse brain and characterization of labeled neurons in the paraventricular nucleus of the hypothalamus. Endocrinology 2014, 155, 4054–4060. [Google Scholar] [CrossRef]
  12. Mouri, T.; Itoi, K.; Takahashi, K.; Suda, T.; Murakami, O.; Yoshinaga, K.; Andoh, N.; Ohtani, H.; Masuda, T.; Sasano, N. Colocalization of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of the human hypothalamus. Neuroendocrinology 1993, 57, 34–39. [Google Scholar] [CrossRef]
  13. Whitnall, M.H.; Mezey, E.; Gainer, H. Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles. Nature 1985, 317, 248–250. [Google Scholar] [CrossRef]
  14. Otubo, A.; Kawakami, N.; Maejima, S.; Ueda, Y.; Morris, J.F.; Sakamoto, T.; Sakamoto, H. Vasopressin gene products are colocalised with corticotrophin-releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata). J. Neuroendocrinol. 2020, 32, e12875. [Google Scholar] [CrossRef] [PubMed]
  15. Zimmerman, E.A.; Carmel, P.W.; Husain, M.K.; Ferin, M.; Tannenbaum, M.; Frantz, A.G.; Robinson, A.G. Vasopressin and neurophysin: High concentrations in monkey hypophyseal portal blood. Science 1973, 182, 925–927. [Google Scholar] [CrossRef] [PubMed]
  16. Meeker, R.B.; Swanson, D.J.; Greenwood, R.S.; Hayward, J.N. Ultrastructural distribution of glutamate immunoreactivity within neurosecretory endings and pituicytes of the rat neurohypophysis. Brain Res. 1991, 564, 181–193. [Google Scholar] [CrossRef]
  17. Meeker, R.B.; Swanson, D.J.; Hayward, J.N. Light and electron microscopic localization of glutamate immunoreactivity in the supraoptic nucleus of the rat hypothalamus. Neuroscience 1989, 33, 157–167. [Google Scholar] [CrossRef]
  18. Valentino, R.J.; Rudoy, C.; Saunders, A.; Liu, X.B.; Van Bockstaele, E.J. Corticotropin-releasing factor is preferentially colocalized with excitatory rather than inhibitory amino acids in axon terminals in the peri-locus coeruleus region. Neuroscience 2001, 106, 375–384. [Google Scholar] [CrossRef]
  19. Maunsbach, A.B. The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. II. Effects of varying osmolality, ionic strength, buffer system and fixative concentration of glutaraldehyde solutions. J. Ultrastruct. Res. 1966, 15, 283–309. [Google Scholar] [CrossRef]
  20. Maunsbach, A.B. The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J. Ultrastruct. Res. 1966, 15, 242–282. [Google Scholar] [CrossRef]
  21. Balaram, P.; Hackett, T.A.; Kaas, J.H. Differential expression of vesicular glutamate transporters 1 and 2 may identify distinct modes of glutamatergic transmission in the macaque visual system. J. Chem. Neuroanat. 2013, 50–51, 21–38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Kawakami, N.; Otubo, A.; Maejima, S.; Talukder, A.H.; Satoh, K.; Oti, T.; Takanami, K.; Ueda, Y.; Itoi, K.; Morris, J.F.; et al. Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin. J. Comp. Neurol. 2021, 529, 1372–1390. [Google Scholar] [CrossRef] [PubMed]
  23. Satoh, K.; Oti, T.; Katoh, A.; Ueta, Y.; Morris, J.F.; Sakamoto, T.; Sakamoto, H. In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: Response to osmotic stimulation. FEBS J. 2015, 282, 2488–2499. [Google Scholar] [CrossRef]
  24. Sawchenko, P.E.; Swanson, L.W.; Vale, W.W. Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat. Proc. Natl. Acad. Sci. USA 1984, 81, 1883–1887. [Google Scholar] [CrossRef] [Green Version]
  25. Foote, S.L.; Cha, C.I. Distribution of corticotropin-releasing-factor-like immunoreactivity in brainstem of two monkey species (Saimiri sciureus and Macaca fascicularis): An immunohistochemical study. J. Comp. Neurol. 1988, 276, 239–264. [Google Scholar] [CrossRef] [PubMed]
  26. Wang, H.; Ward, A.R.; Morris, J.F. Oestradiol acutely stimulates exocytosis of oxytocin and vasopressin from dendrites and somata of hypothalamic magnocellular neurons. Neuroscience 1995, 68, 1179–1188. [Google Scholar] [CrossRef]
  27. Castel, M.; Morris, J.F.; Whitnall, M.H.; Sivan, N. Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques. Cell Tissue Res. 1986, 243, 193–204. [Google Scholar] [CrossRef]
  28. Castel, M.; Morris, J.F. The neurophysin-containing innervation of the forebrain of the mouse. Neuroscience 1988, 24, 937–966. [Google Scholar] [CrossRef]
  29. Frahm, S.; Antolin-Fontes, B.; Gorlich, A.; Zander, J.F.; Ahnert-Hilger, G.; Ibanez-Tallon, I. An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence. eLife 2015, 4, e11396. [Google Scholar] [CrossRef]
  30. Mills, F.; Globa, A.K.; Liu, S.; Cowan, C.M.; Mobasser, M.; Phillips, A.G.; Borgland, S.L.; Bamji, S.X. Cadherins mediate cocaine-induced synaptic plasticity and behavioral conditioning. Nat. Neurosci. 2017, 20, 540–549. [Google Scholar] [CrossRef] [Green Version]
  31. Sun, D.; Vingrys, A.J.; Kalloniatis, M. Metabolic and functional profiling of the normal rat retina. J. Comp. Neurol. 2007, 505, 92–113. [Google Scholar] [CrossRef] [PubMed]
  32. Hou, S.; Duale, H.; Cameron, A.A.; Abshire, S.M.; Lyttle, T.S.; Rabchevsky, A.G. Plasticity of lumbosacral propriospinal neurons is associated with the development of autonomic dysreflexia after thoracic spinal cord transection. J. Comp. Neurol. 2008, 509, 382–399. [Google Scholar] [CrossRef] [Green Version]
  33. Kawata, M.; Sano, Y. Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macaca fuscata). Anat. Embryol. 1982, 165, 151–167. [Google Scholar] [CrossRef]
  34. Cannata, M.A.; Morris, J.F. Changes in the appearance of hypothalamo-neurohypophysial neurosecretory granules associated with their maturation. J. Endocrinol. 1973, 57, 531–538. [Google Scholar] [CrossRef] [PubMed]
  35. Ziegler, D.R.; Cullinan, W.E.; Herman, J.P. Distribution of vesicular glutamate transporter mRNA in rat hypothalamus. J. Comp. Neurol. 2002, 448, 217–229. [Google Scholar] [CrossRef]
  36. Zhang, L.; Hernandez, V.S.; Zetter, M.A.; Eiden, L.E. VGLUT-VGAT expression delineates functionally specialised populations of vasopressin-containing neurones including a glutamatergic perforant path-projecting cell group to the hippocampus in rat and mouse brain. J. Neuroendocrinol. 2020, 32, e12831. [Google Scholar] [CrossRef]
  37. Hrabovszky, E.; Liposits, Z. Glutamatergic phenotype of hypothalamic neurosecretory systems: A novel aspect of central neuroendocrine regulation. Ideggyogy Sz. 2007, 60, 182–186. [Google Scholar] [PubMed]
  38. Brann, D.W. Glutamate: A major excitatory transmitter in neuroendocrine regulation. Neuroendocrinology 1995, 61, 213–225. [Google Scholar] [CrossRef] [PubMed]
  39. Hrabovszky, E.; Csapo, A.K.; Kallo, I.; Wilheim, T.; Turi, G.F.; Liposits, Z. Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus. Neurochem. Int. 2006, 48, 753–761. [Google Scholar] [CrossRef]
  40. Rehbein, M.; Hillers, M.; Mohr, E.; Ivell, R.; Morley, S.; Schmale, H.; Richter, D. The neurohypophyseal hormones vasopressin and oxytocin. Precursor structure, synthesis and regulation. Biol. Chem. Hoppe-Seyler 1986, 367, 695–704. [Google Scholar] [CrossRef]
  41. Hara, Y.; Battey, J.; Gainer, H. Structure of mouse vasopressin and oxytocin genes. Brain Res. Mol. Brain Res. 1990, 8, 319–324. [Google Scholar] [CrossRef]
  42. Naugle, M.M.; Lozano, S.A.; Guarraci, F.A.; Lindsey, L.F.; Kim, J.E.; Morrison, J.H.; Janssen, W.G.; Yin, W.; Gore, A.C. Age and Long-Term Hormone Treatment Effects on the Ultrastructural Morphology of the Median Eminence of Female Rhesus Macaques. Neuroendocrinology 2016, 103, 650–664. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Holmes, R.L. Comparative Observations on Inclusions in Nerve Fibres of the Mammalian Neurohypophysis. Z Zellforsch Mikrosk Anat. 1964, 64, 474–492. [Google Scholar] [CrossRef] [PubMed]
  44. Armstrong, W.E.; Tian, M. Separate ultrastructural distributions of neurophysin and C-terminal glycopeptide within dense core vesicles in rat neural lobe. Brain Res. 1991, 562, 144–148. [Google Scholar] [CrossRef]
  45. Morris, J.F. Hormone storage in individual neurosecretory granules of the pituitary gland: A quantitative ultrastructural approach to hormone storage in the neural lobe. J. Endocrinol. 1976, 68, 209–224. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Ijms 22 09180 g001 550
Figure 1. Western immunoblotting of vesicular glutamate transporter 2 (VGLUT2) and vasopressin-associated neurophysin (NPII). The number on the left indicates the molecular weight (kDa). Extracts of protein from the posterior pituitary of the Japanese macaque monkey were transferred onto polyvinylidene difluoride membranes and probed with the guinea pig polyclonal antiserum against VGLUT2 or with the mouse monoclonal antibody against NPII. The antisera recognized a single major band at the expected molecular weight of VGLUT2 (~56 kDa) or NPII (~12 kDa) on a Western blot of the macaque posterior pituitary.
Figure 1. Western immunoblotting of vesicular glutamate transporter 2 (VGLUT2) and vasopressin-associated neurophysin (NPII). The number on the left indicates the molecular weight (kDa). Extracts of protein from the posterior pituitary of the Japanese macaque monkey were transferred onto polyvinylidene difluoride membranes and probed with the guinea pig polyclonal antiserum against VGLUT2 or with the mouse monoclonal antibody against NPII. The antisera recognized a single major band at the expected molecular weight of VGLUT2 (~56 kDa) or NPII (~12 kDa) on a Western blot of the macaque posterior pituitary.
Ijms 22 09180 g001
Ijms 22 09180 g002 550
Figure 2. Immunofluorescence for vasopressin-associated neurophysin (NPII) in the macaque hypothalamus. NPII-immunoreactivity was observed in the paraventricular nucleus (PVN) of the hypothalamus; PaM, magnocellular part of the PVN (A); supraoptic nucleus (B); PaPV, parvocellular part of the PVN, ventral division (C); and median eminence (ME) (D). Opt, optic nerve; InL, internal layer of the ME; ExL, external layer of the ME. Scale bar, 100 µm.
Figure 2. Immunofluorescence for vasopressin-associated neurophysin (NPII) in the macaque hypothalamus. NPII-immunoreactivity was observed in the paraventricular nucleus (PVN) of the hypothalamus; PaM, magnocellular part of the PVN (A); supraoptic nucleus (B); PaPV, parvocellular part of the PVN, ventral division (C); and median eminence (ME) (D). Opt, optic nerve; InL, internal layer of the ME; ExL, external layer of the ME. Scale bar, 100 µm.
Ijms 22 09180 g002
Ijms 22 09180 g003 550
Figure 3. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque posterior pituitary. Numerous neurosecretory vesicles located in some of the varicosities were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined areas in (A) are enlarged in (B,C). In contrast, in other presumably oxytocin-containing varicosities, no NPII/copeptin-immunoreactivity was detected (A,C). Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Figure 3. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque posterior pituitary. Numerous neurosecretory vesicles located in some of the varicosities were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined areas in (A) are enlarged in (B,C). In contrast, in other presumably oxytocin-containing varicosities, no NPII/copeptin-immunoreactivity was detected (A,C). Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Ijms 22 09180 g003
Ijms 22 09180 g004 550
Figure 4. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the internal layer (InL; (A,B)) and external layer (ExL; (C,D)) of the macaque median eminence. Numerous dense-cored neurosecretory vesicles located in some of the varicosities were immunopositive for both copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) and (C) is enlarged in (B) and (D), respectively. Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Figure 4. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the internal layer (InL; (A,B)) and external layer (ExL; (C,D)) of the macaque median eminence. Numerous dense-cored neurosecretory vesicles located in some of the varicosities were immunopositive for both copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) and (C) is enlarged in (B) and (D), respectively. Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Ijms 22 09180 g004
Ijms 22 09180 g005 550
Figure 5. Bee-swarm and box plots displaying the major axis of dense-cored neurosecretory vesicles distributed in the posterior pituitary (black), internal layer of the median eminence (ME) (red), and external layer of the ME (green) in macaque monkeys. The major axis of dense-cored vesicles in the posterior pituitary and the internal layer of the ME (~200 nm) was significantly larger than that of the vesicles in the external layer of the ME (~100 nm). * p < 0.05, vs. posterior pituitary and internal layer of the ME.
Figure 5. Bee-swarm and box plots displaying the major axis of dense-cored neurosecretory vesicles distributed in the posterior pituitary (black), internal layer of the median eminence (ME) (red), and external layer of the ME (green) in macaque monkeys. The major axis of dense-cored vesicles in the posterior pituitary and the internal layer of the ME (~200 nm) was significantly larger than that of the vesicles in the external layer of the ME (~100 nm). * p < 0.05, vs. posterior pituitary and internal layer of the ME.
Ijms 22 09180 g005
Ijms 22 09180 g006 550
Figure 6. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque supraoptic nucleus (SON). Many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Figure 6. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque supraoptic nucleus (SON). Many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Ijms 22 09180 g006
Ijms 22 09180 g007 550
Figure 7. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque paraventricular nucleus (PVN) of the hypothalamus. (A) In the cell body of a putative magnocellular vasopressin neuron, many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). (C) Similar results are obtained in the cell body of a putative parvocellular vasopressin neuron. The size of dense-cored vesicles in (A,B) was larger than that of vesicles in (C,D). The outlined areas are enlarged. Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Figure 7. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and copeptin in the macaque paraventricular nucleus (PVN) of the hypothalamus. (A) In the cell body of a putative magnocellular vasopressin neuron, many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for copeptin (10-nm gold particles) and NPII (15-nm gold particles). (C) Similar results are obtained in the cell body of a putative parvocellular vasopressin neuron. The size of dense-cored vesicles in (A,B) was larger than that of vesicles in (C,D). The outlined areas are enlarged. Scale bars, 200 nm, and 100 nm in enlarged images. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Ijms 22 09180 g007
Ijms 22 09180 g008 550
Figure 8. Double-label immunofluorescence for corticotrophin-releasing factor (CRF) and vasopressin-associated neurophysin (NPII) in the paraventricular nucleus (PVN) of the macaque hypothalamus; PaM, magnocellular part of the PVN (A); PaPV, parvocellular part of the PVN, ventral division (B). Immunoreactivity against CRF (green) and NPII (magenta) is merged in each right-hand panel (overlap; white). The outlined areas in merged images are enlarged. Arrows in (A; merged) indicate perikarya single-immunopositive for NPII in the PaM. Arrows in (B; merged) indicate doubly immunopositive for CRF and NPII in the PaPV. Scale bars, 100 µm, and 10 µm in enlarged images.
Figure 8. Double-label immunofluorescence for corticotrophin-releasing factor (CRF) and vasopressin-associated neurophysin (NPII) in the paraventricular nucleus (PVN) of the macaque hypothalamus; PaM, magnocellular part of the PVN (A); PaPV, parvocellular part of the PVN, ventral division (B). Immunoreactivity against CRF (green) and NPII (magenta) is merged in each right-hand panel (overlap; white). The outlined areas in merged images are enlarged. Arrows in (A; merged) indicate perikarya single-immunopositive for NPII in the PaM. Arrows in (B; merged) indicate doubly immunopositive for CRF and NPII in the PaPV. Scale bars, 100 µm, and 10 µm in enlarged images.
Ijms 22 09180 g008
Ijms 22 09180 g009 550
Figure 9. Double-label immunofluorescence for copeptin (A) or corticotrophin-releasing factor (CRF) (B) and vasopressin-associated neurophysin (NPII) in the macaque median eminence (ME). Immunoreactivity against copeptin (A; green) or CRF (B; green) and NPII (magenta) is merged in each right-hand panel (overlap; white). The outlined areas in merged images are enlarged. Arrowheads in (A) indicate tissue doubly immunopositive for copeptin and NPII. Scale bars, 50 µm, and 10 µm in enlarged images. InL, internal layer of the ME; ExL, external layer of the ME.
Figure 9. Double-label immunofluorescence for copeptin (A) or corticotrophin-releasing factor (CRF) (B) and vasopressin-associated neurophysin (NPII) in the macaque median eminence (ME). Immunoreactivity against copeptin (A; green) or CRF (B; green) and NPII (magenta) is merged in each right-hand panel (overlap; white). The outlined areas in merged images are enlarged. Arrowheads in (A) indicate tissue doubly immunopositive for copeptin and NPII. Scale bars, 50 µm, and 10 µm in enlarged images. InL, internal layer of the ME; ExL, external layer of the ME.
Ijms 22 09180 g009
Ijms 22 09180 g010 550
Figure 10. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and corticotrophin-releasing factor (CRF) in the external layer (ExL) of the macaque median eminence. In some axonal varicosities, numerous dense-cored neurosecretory vesicles were doubly immunopositive for CRF (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Figure 10. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and corticotrophin-releasing factor (CRF) in the external layer (ExL) of the macaque median eminence. In some axonal varicosities, numerous dense-cored neurosecretory vesicles were doubly immunopositive for CRF (10-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion; MV, clustered microvesicles.
Ijms 22 09180 g010
Ijms 22 09180 g011 550
Figure 11. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and corticotropin-releasing factor (CRF) in the paraventricular nucleus (PVN) of the hypothalamus in a macaque monkey. (A) In the cell body of a possible parvocellular vasopressin neuron, many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for CRF (6-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Figure 11. Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and corticotropin-releasing factor (CRF) in the paraventricular nucleus (PVN) of the hypothalamus in a macaque monkey. (A) In the cell body of a possible parvocellular vasopressin neuron, many dense-cored neurosecretory vesicles located in the cell body were doubly immunopositive for CRF (6-nm gold particles) and NPII (15-nm gold particles). The outlined area in (A) is enlarged in (B). Scale bars, 200 nm, and 100 nm in the enlarged image. Arrows indicate dense-cored neurosecretory vesicles. m, mitochondrion.
Ijms 22 09180 g011
Ijms 22 09180 g012 550
Figure 12. Immunoelectron microscopy for glutamate (Glu) in the posterior pituitary (PP) (A) and external layer of the median eminence (ExL) (C) in a macaque monkey. (B) Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and Glu shows that Glu-immunoreactivity is associated with microvesicles in the macaque posterior pituitary. (D) Triple-label immunoelectron microscopy for NPII/corticotrophin-releasing factor (CRF)/Glu indicates that Glu-immunoreactivity is associated with electron-lucent small vesicles in the axonal endings, in the NPII/CRF-double positive endings. The outlined areas are enlarged. Scale bars, 100 nm. Green arrowheads indicate NPII-immunoreactivity; blue arrows indicate CRF-immunoreactivity; magenta arrowheads indicate Glu-immunoreactivity. m, mitochondrion; MV, clustered microvesicles.
Figure 12. Immunoelectron microscopy for glutamate (Glu) in the posterior pituitary (PP) (A) and external layer of the median eminence (ExL) (C) in a macaque monkey. (B) Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and Glu shows that Glu-immunoreactivity is associated with microvesicles in the macaque posterior pituitary. (D) Triple-label immunoelectron microscopy for NPII/corticotrophin-releasing factor (CRF)/Glu indicates that Glu-immunoreactivity is associated with electron-lucent small vesicles in the axonal endings, in the NPII/CRF-double positive endings. The outlined areas are enlarged. Scale bars, 100 nm. Green arrowheads indicate NPII-immunoreactivity; blue arrows indicate CRF-immunoreactivity; magenta arrowheads indicate Glu-immunoreactivity. m, mitochondrion; MV, clustered microvesicles.
Ijms 22 09180 g012
Ijms 22 09180 g013 550
Figure 13. Immunoelectron microscopy for vesicular glutamate transporter 2 (VGLUT2) in the posterior pituitary (PP) (A) and external layer of the median eminence (ExL) (C) in a macaque monkey. (B) Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and VGLUT2 shows that VGLUT2-immunoreactivity is associated with clustered microvesicles (MV) in the posterior pituitary. (D) Double-label immunoelectron microscopy for NPII/VGLUT2 indicates that VGLUT2-immunoreactivity is associated with the membrane of electron-lucent small vesicles in the vasopressin endings. The outlined areas are enlarged. Scale bars, 100 nm. Green arrowheads indicate NPII-immunoreactivity; magenta arrowheads indicate VGLUT2-immunoreactivity. m, mitochondrion; MV, clustered microvesicles.
Figure 13. Immunoelectron microscopy for vesicular glutamate transporter 2 (VGLUT2) in the posterior pituitary (PP) (A) and external layer of the median eminence (ExL) (C) in a macaque monkey. (B) Double-label immunoelectron microscopy for vasopressin-associated neurophysin (NPII) and VGLUT2 shows that VGLUT2-immunoreactivity is associated with clustered microvesicles (MV) in the posterior pituitary. (D) Double-label immunoelectron microscopy for NPII/VGLUT2 indicates that VGLUT2-immunoreactivity is associated with the membrane of electron-lucent small vesicles in the vasopressin endings. The outlined areas are enlarged. Scale bars, 100 nm. Green arrowheads indicate NPII-immunoreactivity; magenta arrowheads indicate VGLUT2-immunoreactivity. m, mitochondrion; MV, clustered microvesicles.
Ijms 22 09180 g013
Ijms 22 09180 g014 550
Figure 14. Working model showing that the size difference in dense-cored neurosecretory vesicles (dcv) between magnocellular (~200 nm) and parvocellular (~100 nm) vasopressin (AVP)- and/or corticotrophin-releasing factor (CRF)-producing neurons in the hypothalamus of the Japanese macaque monkey. We also demonstrate that both magno- and parvocellular AVP/CRF neurons are glutamatergic in primates. AP, anterior pituitary; ME, median eminence; MV, clustered microvesicles; PP, posterior pituitary; PVN, paraventricular nucleus; SON, supraoptic nucleus.
Figure 14. Working model showing that the size difference in dense-cored neurosecretory vesicles (dcv) between magnocellular (~200 nm) and parvocellular (~100 nm) vasopressin (AVP)- and/or corticotrophin-releasing factor (CRF)-producing neurons in the hypothalamus of the Japanese macaque monkey. We also demonstrate that both magno- and parvocellular AVP/CRF neurons are glutamatergic in primates. AP, anterior pituitary; ME, median eminence; MV, clustered microvesicles; PP, posterior pituitary; PVN, paraventricular nucleus; SON, supraoptic nucleus.
Ijms 22 09180 g014
Table
Table 1. Primary antibodies used in this study.
Table 1. Primary antibodies used in this study.
AntigenDescriptionSource, Host Species, Cat #, or Code #Working DilutionReference #RRID
CopeptinSynthetic peptide mapping at the amino acids 7–14 of human/mouse copeptinGenerated by our laboratory, rabbit polyclonal, CP81:10,000 (IF)
1:100 (EM)		[14,22,23]AB_2722604
CRFHuman/rat CRF coupled to human alpha-globulins via bis-diazotized benzidineDonated by Dr. W. Vale, rabbit polyclonal, PBL rC701:20,000 (IF)
1:5000 (EM)		[14,24,25]AB_2314234
NPIISoluble proteins extracted from the posterior pituitary of the ratATCC, mouse monoclonal, PS41, CRL-17991:1000 (IF)
1:200 (EM)
1:1000 (WB)		[14,26,27,28]AB_2313960
VGLUT2Recombinant protein corresponding to amino acids 510–582 of the rat VGLUT2Synaptic Systems, guinea pig polyclonal, 135 4041:20 (EM)
1:10,000 (WB)		[29,30]AB_887884
l-Glutamatel-Glutamate conjugated to glutaraldehydeAbcam, rabbit polyclonal, ab94401:10 (EM)[31,32]AB_307256
Abbreviations: ATCC, American Type Culture Collection; CRF, corticotropin-releasing factor; EM, immunoelectron microscopy; IF, immunofluorescence; NPII, arginine vasopressin-associated neurophysin II; RRID, research resource identifier; VGLUT2, vesicular glutamate transporter 2; WB, Western blotting; #, number.
Table
Table 2. Identification of cell types (magno- or parvocellular neurons) based on the major axis of dense-cored neurosecretory vesicles.
Table 2. Identification of cell types (magno- or parvocellular neurons) based on the major axis of dense-cored neurosecretory vesicles.
RegionMajor Axis (nm)Magno or ParvoNumber of Vesicles ExaminedIdentification
Posterior pituitary196 ± 2Magno205 AVP
Internal ME197 ± 2Magno237AVP without CRF
External ME93 ± 1 *Parvo212CRF
AVP, arginine vasopressin; internal ME, internal layer of the median eminence; CRF, corticotrophin-releasing factor; external ME, external layer of the median eminence. * p < 0.05, vs. posterior pituitary and internal layer of the median eminence. All data values are presented as mean ± standard error of the mean (SEM).
Table
Table 3. Characterization of cell types (magno- or parvocellular neurons) based on the major axis of dense-cored neurosecretory vesicles.
Table 3. Characterization of cell types (magno- or parvocellular neurons) based on the major axis of dense-cored neurosecretory vesicles.
Cell BodyMajor Axis (nm)Magno or ParvoNumber of Vesicles ExaminedSON or PVNIdentification
#1187 ± 5 *Magno29SONAVP
#2175 ± 6 *Magno35SONAVP
#3189 ± 10 *Magno24SONAVP
#4181 ± 5 *Magno26SONAVP
#5171 ± 7 *Magno25SONAVP
#6195 ± 6 *Magno30SONAVP
#7205 ± 6 *Magno46SONAVP
#8188 ± 5 *Magno49PVNAVP
#9170 ± 5 *Magno20PVNAVP
#10169 ± 8 *Magno22PVNAVP
#11172 ± 4 *Magno24PVNAVP
#12184 ± 5 *Magno25PVNAVP
#13182 ± 5 *Magno21PVNAVP
#14103 ± 5 †Parvo38PVNAVP
#1598 ± 4 †Parvo42PVNCRF
#16109 ± 4 †Parvo51PVNCRF
#1793 ± 5 †Parvo24PVNCRF
#18107 ± 5 †Parvo40PVNCRF
#19103 ± 4 †Parvo35PVNCRF
#20110 ± 7 †Parvo42PVNCRF
#21108 ± 5 †Parvo77PVNCRF
#2293 ± 4 †Parvo81PVNCRF
#2395 ± 5 †Parvo30PVNCRF
#24120 ± 6 *,†(Parvo)40PVNCRF
#25113 ± 3 *,†(Parvo)89PVNCRF
AVP, arginine vasopressin; CRF, corticotrophin-releasing factor; SON, supraoptic nucleus; PVN, paraventricular nucleus; #, number. * p < 0.05, vs. external layer of the median eminence; † p < 0.05, vs. posterior pituitary and internal layer of the median eminence. All data values are presented as mean ± standard error of the mean (SEM).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Otubo, A.; Maejima, S.; Oti, T.; Satoh, K.; Ueda, Y.; Morris, J.F.; Sakamoto, T.; Sakamoto, H. Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at −25 °C for Several Years. Int. J. Mol. Sci. 2021, 22, 9180. https://doi.org/10.3390/ijms22179180

AMA Style

Otubo A, Maejima S, Oti T, Satoh K, Ueda Y, Morris JF, Sakamoto T, Sakamoto H. Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at −25 °C for Several Years. International Journal of Molecular Sciences. 2021; 22(17):9180. https://doi.org/10.3390/ijms22179180

Chicago/Turabian Style

Otubo, Akito, Sho Maejima, Takumi Oti, Keita Satoh, Yasumasa Ueda, John F. Morris, Tatsuya Sakamoto, and Hirotaka Sakamoto. 2021. "Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at −25 °C for Several Years" International Journal of Molecular Sciences 22, no. 17: 9180. https://doi.org/10.3390/ijms22179180

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop