Variation of pro‐vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin‐related glycopeptide copeptin

Arginine vasopressin (AVP) is synthesized in parvocellular‐ and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre‐pro‐AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N‐terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII‐expressing neurons exhibited strong N‐terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII‐immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin‐immunoreactivity co‐localized with NPII‐immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa‐d‐arginine amide resulted in a marked reduction of copeptin‐immunoreactivity in the NPII‐immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro‐AVP could explain the disproportionally low levels of N‐terminal copeptin expression in rodent AVP (NPII)‐expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-D-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.  Whitnall, Ozato, & Gainer, 1985;Castel & Morris, 1988;Otero-Garcia et al., 2014). Axons of AVP-producing neurons, originating from the magnocellular neurons in the PVN and the SON, traverse the internal layer of the median eminence (ME) and project to the posterior pituitary, where AVP is released into the systemic circulation from the axon terminals (Burbach, Luckman, Murphy, & Gainer, 2001). Production of AVP in the PVN and the SON, as well as its release from the posterior pituitary, is regulated by osmotic pressure and/or body fluid volume (Koshimizu et al., 2012;Murphy, Waller, Fairhall, Carter, & Robinson, 1998). AVP is also released from the dendrites of magnocellular neurons into hypothalamic neuropil, and is involved in the autocrine regulation of magnocellular neurons and paracrine regulation of other neurons in a variety of functions (Johnson & Young, 2017;Ludwig & Leng, 2006). Although SCN neurons are not part of neuroendocrine systems, AVP is also produced in the dorsomedial (shell) part of the SCN (Golombek & Rosenstein, 2010;Ramkisoensing & Meijer, 2015). Physiological roles of AVP in the SCN are still obscure, but it has been hypothesized to be involved in diverse mechanisms in the brain, which are related to the control of circadian rhythm (Gizowski, Zaelzer, & Bourque, 2016;Trudel & Bourque, 2010).
In the PVN, corticotropin-releasing factor (CRF)-producing neurons, located mainly in the anteromedial part of the nucleus, comprise a third population of neuroendocrine neurons that is capable of producing AVP (Fellmann et al., 1984;Itoi et al., 2014;Mouri et al., 1993;Whitnall, 1993). These neurons are parvocellular neurons which project to the external layer of the ME, where CRF and AVP are released into the pituitary portal vessels from nerve endings terminating on the capillary bed (Fellmann et al., 1984). Both CRF and AVP stimulate adrenocorticotropin (ACTH) secretion from corticotrophs in the anterior pituitary, and they act synergistically in ACTH secretion when applied together to anterior pituitary cells (Fischman & Moldow, 1984;Gillies, Linton, & Lowry, 1982;Hashimoto, Murakami, Hattori, & Ota, 1984;Liu et al., 1983;Vale et al., 1983). The concentration of AVP in the pituitary portal vessels increases when animals are stressed (Plotsky, 1987) or have undergone adrenalectomy (Plotsky & Sawchenko, 1987). In rats, CRF and AVP are co-packaged into the same neurosecretory vesicles in the CRF-and AVP-double positive ( + ) subpopulation of axons in the ME, where they can be co-released from the nerve endings of the parvocellular neurons (Whitnall, Mezey, & Gainer, 1985). Therefore, both CRF and AVP are regarded as physiological secretagogues for ACTH in the pituitary (Mouri et al., 1993;Whitnall, 1993).
A fourth population of AVP-producing neurons in the PVN is located in the posterolateral part of the nucleus. These cells are categorized as parvocellular neurons, and they project to extrahypothalamic areas and to the spinal cord, and are thought to be involved in the regulation of autonomic output (Sawchenko, 1987).
After removal of the signal-peptide in the endoplasmic reticulum, the precursor protein is packaged into neurosecretory vesicles in the Golgi apparatus (Burbach et al., 2001;Davies et al., 2003). It is then subjected to post-translational cleavage by enzymes (prohormone convertases) and co-packaged in vesicles during its axonal transport to terminals (Hook et al., 2008). In the magnocellular neurons which project to the posterior pituitary, PC1/3, PC2, furin, and PC7 were all strongly expressed. In the parvocellular CRF neurons, however, there was little or no expression of PC1 although PC2, furin, and PC7 were expressed there (Dong et al., 1997). At this moment, we have no knowledge of proteases in parvocellular AVP neurons that project to other regions of the brain. It is not yet clear what enzyme(s) cleaves copeptin from the NP moiety of the pre-pro-AVP, either. Copeptin constitutes the C-terminal part of the AVP precursor, and in magnocellular neurons, its cleavage from the precursor is completed at the level of posterior pituitary (Morgenthaler, Struck, Jochberger, & Dunser, 2008). Copeptin is also glycosylated, which is important for the stability of the peptide in the circulation (Bhandari et al., 2009;Blanchard et al., 2013;Bolignano et al., 2014;Wuttke et al., 2013).
Activation of the AVP neurons projecting to the neurohypophysis stimulates secretion of both AVP and copeptin into systemic circulation on an equimolar basis (Morgenthaler et al., 2008). Activation of CRF/AVP neurons projecting to the ME are primarily stimulated by stress, and particular chronic stress, leading to increases in AVP expression in these neurons (de Goeij, Jezova, & Tilders, 1992;Sawchenko, Arias, & Mortrud, 1993). However, it has not been elucidated whether AVP and copeptin are released simultaneously into the portal capillaries.
Copeptin is produced as a co-product of AVP and NPII, as described above, and the AVP gene is expressed in all populations of AVP-producing neurons in the hypothalamus, i.e., the magnocellular neurons in the PVN and the SON, the parvocellular CRF neurons in the PVN, the dorsomedial group of SCN neurons, and the preautonomic neurons in the posterolateral part of the PVN. In the present immunofluorescence study, we used either an N-terminal copeptin antiserum or a C-terminal antiserum, together with an NPII antiserum, to examine the presence of copeptin in the hypothalamus of mice, rats, and macaque monkeys by immunofluorescence, and elucidated the co-localization of copeptin with AVP/NPII in somata of hypothalamic neuroendocrine neurons, as well as in their nerve endings in the ME. Furthermore, an immunoelectron microscopic analysis was carried out in the mouse to examine the subcellular localization of copeptin. Special attention was paid to the co-localization of copeptin with AVP/NPII within secretory vesicles. Remarkably, copeptin immunoreactivity was not uniform in different populations of AVP-producing neurons. In addition, there was a marked species difference in copeptin-immunoreactivity in the parvocellular neurosecretory neurons in the PVN.
These animals were shown to be free of specific pathogens. Food and water were available ad libitum. The housing and experimental protocols followed the guideline 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 Tohoku University (Miyagi, Japan), by Kyoto Prefectural University of Medicine (Kyoto, Japan), and by Kansai Medical University (Osaka, Japan). All efforts were made to minimize animal suffering and reduce the number of animals used in this study.

| Tissue preparation
Mice, rats, and monkeys were anesthetized with an overdose of sodium pentobarbital (50-90 mg/kg body weight), and transcardially perfused with physiological saline followed by 4% paraformaldehyde (PFA) (for immunohistochemistry and immunofluorescence; 17 male and four female mice; four CRF-VenusΔNeo adult male mice; four male rats; two male and two female Japanese macaque monkeys) or by 4% PFA + 0.1% glutaraldehyde (for electron microscopic studies; five male mice), both in 0.1 M phosphate buffer (PB, pH 7.4). Brains and neurohypophyses were immediately removed and immersed in 4% PFA in 0.1 M PB for 16-24 hr (for immunohistochemistry and immunofluorescence) at 4 C, or in 4% PFA + 0.1% glutaraldehyde in 0.1 M PB for 3 hr at room temperature (for electron microscopy). Tissues for immunofluorescence were then cryoprotected by immersion in 25% sucrose in 0.1 M PB at 4 C until they sank, then quickly frozen using powdered dry ice and cut into 30-μm-thick coronal sections on a cryostat (CM3050 S; Leica, Nussloch, Germany). For electron microscopy, the fixed mouse brains were sectioned in the frontal plane at 200 μm thickness with a Linear-Slicer (PRO10; Dosaka EM, Kyoto, Japan) and were immersed in 0.1 M PB.

| Immunohistochemistry and immunofluorescence
For immunohistochemistry, endogenous peroxidase activity was quenched by incubation with 1% H 2 O 2 in absolute methanol for 20 min and the sections were then rinsed with PBS three times for 5 min each. These processes were omitted for immunofluorescence.
Specific staining for AVP and CRF were abolished by preadsorption of the antisera for 1 hr at room temperature with 50 μg/ml synthetic AVP or mouse CRF before use. We further confirmed by Western blot analysis the specificity for the NPII antibody. The monoclonal antibody (PS41) labeled a single band at approximately the expected molecular weight of 11 kDa on the blots of mouse, rat, and macaque monkey posterior pituitary.
Western blot analysis also showed the specificity for the NPI antibody (PS60) in the mouse posterior pituitary. A goat polyclonal antiserum for copeptin (m-20) raised against an oligopeptide near the C-terminus of copeptin of mouse origin at a dilution of 1:15,000 was also used as described previously (Itoi et al., 2014).
Control procedures for the DAB method were performed using pre-absorption of the working dilution (CP-8; 1:20,000 or m-20; 1:30,000) of the primary antiserum with a saturating concentration of the antigen (50 μg/ml; N-terminus fragment of mouse copeptin 7-14 [ATQLDGPA] or C-terminus fragment of mouse copeptin 20-39 [RLVQLAGTRESVDSAKPRVY]) for 1 hr at room temperature before use. Immunostained sections were observed under the Olympus FSX100 microscope (Tokyo, Japan).
To detect the Venus signals in tissues from CRF-VenusΔNeo mice, we performed immunofluorescence for detection of green fluorescent protein (GFP) to intensify the Venus signal (for localizing CRF neurons) as described previously (Kono et al., 2017). Primary rat monoclonal anti-GFP antibody (1:10,000 dilution) (Nacalai Tesque, Kyoto, Japan; RRID: AB_10013361) was used. Double immunofluorescence for GFP and NPII or copeptin was performed as described above. Alexa  Table 1.
The data are presented as the mean ± SEM and were analyzed using Statcel4 software. Statistical analyses of the proportion of copeptin-negative ( − ) /NPII + neurons were performed using one-way analysis of variance (ANOVA) (in male mice or rats) or two-way ANOVA (sex difference and right/left difference in mice). When significance was found using ANOVA, the post hoc Bonferroni/Dunn test was performed.

| Post-embedding immunoelectron microscopy
Preparations were dehydrated through increasing concentrations of methanol, embedded in LR Gold resin (Electron Microscopy Sciences, PA), and polymerized under ultraviolet lamps at −20 C for 24 hr.
Ultrathin sections (70 nm in thickness) were collected on nickel grids coated with a Collodion film (or without for triple labeling), 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 30 min to block nonspecific binding. The sections were then incubated with the rabbit polyclonal antiserum against N-terminal copeptin (CP8, 1:100 dilution), or the goat polyclonal antiserum against C-terminal copeptin (m-20, 1:1,000 dilution), and the PS41 mouse monoclonal antibody against NPII (1:200 dilution) (Castel et al., 1986) for 2 hr at room temperature. The rabbit polyclonal antiserum against rat CRF was also used at 1:5,000 for 2 hr at room temperature. For double immunoelectron microscopy, after incubation with the primary antibodies, the sections were washed with PBS, then incubated with a goat antibody against rabbit IgG conjugated to 10 nm gold particles (1:50 dilution) (BBI Solutions, Cardiff, UK) and/or a goat antibody against mouse IgG conjugated to 15 nm gold particles (1:50 dilution) (BBI Solutions) for 1 hr at room temperature.
Triple immunoelectron microscopy with antibodies for AVPassociated NPII, oxytocin-associated NPI, and copeptin was performed using the front and back of ultrathin sections mounted on nickel grids. First, immunocytochemistry with 2 primary antibodies (anti-NPII; PS41 and copeptin) was performed on one side of the section and detected using 15 and 10 nm colloidal gold particles.
Next, the other antibody (anti-NPI; PS60) was detected using 5 nm colloidal gold particles on the other side of the section. 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 antibodies used in this study are shown in Table 1.

| Intracerebroventricular injection of a prohormone convertase inhibitor, hexa-D-arginine amide
Adult male mice were anesthetized with an intraperitoneal injection of medetomidine hydrochloride (0.3 mg/kg body weight), midazolam (4 mg/kg body weight), and butorphanol tartrate (5 mg/kg body weight) and then placed in the stereotaxic apparatus for the intracerebroventricular injection of the protease inhibitor (furin inhibitor II) The needle of a syringe (701 RN) (Hamilton, Reno, NV) filled with D6R was inserted into the right lateral ventricle (location of the needle tip: −0.4 mm caudal to the bregma, −1.0 mm lateral to the midline, 2.0 mm below the dura). After inserting the needle, D6R (9 μg in 2 μl PBS) was infused into the lateral ventricle at a flow rate of 1 μl/min (n = 6). Control mice were infused with the same volume of PBS (n = 4). Mice were perfused with fixative 18 hr after D6R or PBS injection, and then brains were removed. Double immunofluorescence for NPII and copeptin was then performed as described above.

| Western blotting
Adult male mice were sacrificed by decapitation to obtain the fresh brains. Brains were cut into 400 μm thick coronal sections using a vibrat- were blotted onto the PVDF membrane. The blotted membrane was immunostained as described above.
3 | RESULTS Figure 1 shows the amino acid sequences of copeptin in seven mammals including the mouse, rat, and monkey. The epitope against which the CP8 antiserum was raised is indicated by asterisks above the sequences. This region is well conserved across mammals (stars below the sequences) and is identical in the mouse, the rat, the monkey, and the human. The specificity of our custom N-terminal CP8 copeptin antiserum was confirmed by an absorption experiment in which the primary rabbit antiserum against mouse copeptin 7-14 was preabsorbed with an excess amount of mouse copeptin 7-14 (ATQLDGPA), whereas the staining was not affected by treatment with the same concentration of synthetic C-terminus fragment of mouse copeptin 20-39 (RLVQLAGTRESVDSAKPRVY) (Figure 2). Unless otherwise stated the antiserum is the N-terminal CP8 antiserum. AVP neurons were immunoidentified with the anti-NPII antibody that has been well characterized and used as a marker for AVP neurons (Ben- Barak et al., 1985;Castel & Morris, 1988). Because the localization of AVP-and NPII-expressing neurons is identical in the rodent brain ( Figures 3 and 4), NPII-immunofluorescence was used as a marker for AVP neurons in this study.

| Species difference in copeptin expression in parvocellular neurosecretory neurons in the PVN
In the mouse, most magnocellular NPII + cells also exhibit copeptin-immunoreactivity in both the PVN and the SON. In the PVN, however, a major proportion (87%) of NPII + neurons in the medial subgroup (PaMP) of parvocellular neurons did not contain immunodetectable copeptin ( Figure 5; arrowheads) ( Table 2) (Table 3).
In contrast to the parvocellular PVN neurons, almost all NPII + neurons in the SCN (a nucleus that contains small-sized AVP neurons) did exhibit copeptin-immunoreactivity ( Figure 5). As expected, oxytocin neurons identified by NPI immunoreactivity did not exhibit copeptinimmunoreactivity in either the PVN or SON ( Figure 6). Similar results were obtained in the rat (Figure 7), but the proportion of copeptin − / NPII + neurons in the parvocellular PVN was less marked (53%) compared with that in the mouse (Table 2). In the macaque monkey, remarkably, virtually all NPII + neurons in both the magnocellular and No obvious sex difference was observed in monkeys in terms of copeptin-and NPII-immunoreactivity.
In the internal layer of the ME that contains axons of magnocellular neurons originating from the PVN or SON and projecting to the posterior pituitary, virtually all NPII + fibers exhibited copeptin-immunoreactivity in the mouse, rat, and monkey ( Figure 9a).
However, in the external layer of the mouse ME, where the parvocellular CRF axons terminate, copeptin immunoreactivity could be detected in only a small number of NPII + fibers (Figure 9a; arrowheads). In the external layer of the rat ME, co-expression of NPII-and copeptin-immunoreactivity was observed more frequently in the external layer of the ME than in the mouse, but less frequently than in the monkey (Figure 9b; arrowheads). In the macaque monkey, most NPII + fibers were copeptin-immunoreactive in both the internal and external layer of the ME (Figure 9c; arrowheads).

| Expression of NPII or copeptin in parvocellular CRF neurons in mice
Expression of NPII and CP8-detectable copeptin in the parvocellular CRF neurons was examined in the CRF-VenusΔNeo mouse. In the parvocellular PVN, very few (one or two cells per section) Venus + neurons showed NPII-immunoreactivity (Figure 10a; arrowheads) or copeptin-immunoreactivity (Figure 10b; arrowheads). We next studied co-localization of CRF with NPII in the ME of a wild-type mouse.
CRF-immunoreactive fibers were detectable only in the external layer of the ME, and approximately half of the CRF-immunoreactive fibers contained NPII-immunoreactivity (Figure 10c; arrowheads). NPII + fibers were intensively stained in the internal layer of the mouse ME of the wild-type mouse, but were devoid of CRF-immunoreactivity ( Figure 10c). However, NPI + fibers were hardly observed in the external layer of the ME in the mouse (Figure 11).
Preliminary experiments using a copeptin m-20 antiserum raised against an epitope near the C-terminus of mouse copeptin 20-39 revealed copeptin-immunoreactivity in virtually all NPII-expressing magnocellular and parvocellular neurons in the mouse PVN ( Figure 12).
The specificity of the Santa Cruz copeptin antiserum (m-20) was confirmed by an absorption experiment in which the primary goat antiserum against the C-terminus of mouse copeptin was preabsorbed with an excess amount of synthetic C-terminus fragment of mouse copeptin 20-39 (RLVQLAGTRESVDSAKPRVY), whereas the staining was not affected by treatment with the same concentration of mouse N-terminus fragment of mouse copeptin 7-14 (ATQLDGPA) (Figure 2).

| Co-localization of copeptin with NPII within secretory vesicles in mice
Triple immunoelectron microscopy was carried out to examine colocalization of NPII, NPI, and CP8-detectable copeptin within neurosecretory vesicles in the mouse. Both NPII and copeptin were detected within axons of neurosecretory neurons in the posterior pituitary, where they were co-associated within neurosecretory vesicles (Figure 13a,b). NPI was also detected in axon terminals, but no copeptin-immunoreactivity was observed in NPI-labeled terminals adjacent to NPII-labeled terminals (Figure 13c). Numerous neurosecretory vesicles located in cell bodies and dendrites of magnocellular neurons in the SON were immunolabeled for both copeptin and NPII (Figure 14a-c). However, copeptin-immunoreactivity was not observed in immature neurosecretory vesicle cores within the Golgi apparatus ( Figure 15). In magnocellular neurons in the PVN, many neurosecretory vesicles were labeled for both copeptin and NPII and the vesicles were 160 nm in diameter (Figure 16a). The size was similar to that of neurosecretory vesicles in the SON (Figures 14-16). In parvocellular PVN neurons, there were also numerous dense-cored neurosecretory vesicles (100 nm in diameter) that contained NPIIimmunoreactivity, but these vesicles were devoid of copeptinimmunoreactivity (Figure 16b; arrows).
Next, we examined co-localization of NPII and copeptin in the mouse ME. The two layers of the ME, that is, the internal and external layers, investigated in the present immunoelectron microscopy, are shown schematically in Figure 17a. In the internal layer of the ME, immunoreactivity for both NPII and copeptin was intense in neurosecretory axons, and co-associated within the neurosecretory vesicles ( Figure 17b). The size of these vesicles was 160 nm in diameter, characteristic of those of magnocellular neurons in rodents (Armstrong & Tian, 1991;Pow & Morris, 1989;Satoh et al., 2015).
However, in the external layer of the mouse ME, although many NPII + dense-cored vesicles (100 nm in diameter) were present in axon terminals, as reported previously (Shaw, Castel, & Morris, 1987), in most terminals the CP8 antiserum detected little or no copeptin immunoreactivity associated with the vesicles (Figure 17c; white arrows). Many neighboring axons contained dense-cored vesicles that were immunoreactive for neither NPII nor copeptin (Figure 17c; black arrows). In a small proportion of nerve terminals in the external layer of the mouse ME, however, dense-cored vesicles containing both CRF-and NPIIimmunoreactivity ( Figure 17d; white arrows) were present, as expected.

| Effects of intracerebroventricular injection of a prohormone convertase inhibitor, D6R on the immunoreactivity for copeptin and NPII in mice
We hypothesized that processing of AVP precursor may differ between neuronal populations. Therefore, D6R was injected intracerebroventricularly to inhibit the activity of PC1/3, which is a key processing enzyme required for the cleavage of the AVP precursor in the mouse (Figure 18). In the D6R-injected group, many NPII + magnocellular neurons (magenta) exhibited only a faint  (Figure 19).
F I G U R E 1 2 Double-label immunofluorescence for copeptin (by using a goat polyclonal antibody against to near the C-terminus of copeptin of mouse origin) and vasopressin-neurophysin (NPII) in the mouse paraventricular nucleus (PVN) (middle part). Immunoreactivities against copeptin (green) and NPII (magenta) were merged in each right panel, respectively. In the internal layer of the ME (area of the magnocellular axons), AVP (NPII) and copeptin were both detected within the terminals of neurosecretory axons, and co-associated with the neurosecretory vesicle structures in the same terminal. Electron photomicrographs illustrating the presence of AVP (NPII)immunoreactive (signaled by 15-nm gold particles) and copeptin (c) or CRF-immunoreactive (signaled by 10-nm gold particles) (d) on densecored vesicles of nerve endings co-localized in the external layer of the ME. Some vesicles were clearly AVP (NPII)-positive (white arrows), but copeptin could not be detected; vesicles in other terminals were negative for both (black arrows) (c). In contrast, many vesicles which were immunopositive for AVP (NPII) were also positive for CRF (white arrows) (d). Scale bars, 200 nm. 3V, third ventricle; m, mitochondrion F I G U R E 1 8 Double-label immunofluorescence for N-terminal copeptin and vasopressin (AVP)-neurophysin (NPII) in the magnocellular part of the paraventricular nucleus (magno-PVN), the supraoptic nucleus (SON), the suprachiasmatic nucleus (SCN), the parvocellular part of the PVN (parvo-PVN), and the median eminence (ME) in the mouse. Representative results are displayed for control tissues (left; control) and identical areas after the intracerebroventricular administration of a prohormone convertase inhibitor (hexa-D-arginine amide; D6R) (right; D6R-treated). Immunoreactivity against copeptin (green) and AVP (NPII) (magenta) was merged in each panel (overlap white).  (Hook et al., 2008). Cleavage of the monobasic R site may be catalyzed by a different processing enzyme(s), so pro-AVP processing may take place as a two-step reaction, although a specific enzyme for the monobasic R site cleavage has not yet been identified. Therefore, one possible explanation for our finding is that the second cleavage between NPII and copeptin does not take place adequately in parvocellular AVP-expressing neurons in rats and mice, possibly through a paucity of a monobasic R protease, and despite the presence of the K/R protease. Indeed, no effect of D6R injection on AVP staining was found in this study, suggesting that a D6R-sensitive enzyme is not involved in the first step of processing (AVP-cleavage and circularization) in mouse AVP neurons. It has been reported that D6R inhibits PC1/3, but does not affect the activity of PC2 (Cameron, Appel, Houghten, & Lindberg, 2000). As yet, for mice, nothing is known of the concentration of the convertases or whether enzymatic activities, such as PC1 and PC2 in parvocellular PVN neurons differ from those of magnocellular AVP neurons. However, for rats, Dong et al. (1997) and Schafer et al. (1993) have both reported that, in the parvocellular PVN neurons, PC2 expression was greater but PC1 expression was much lower than that in the magnocellular PVN and SON neurons. These results suggest that the differential distribution of prohormone convertases between magnocellular and parvocellular PVN subdivisions could play a role in the processing of AVP prohormone in the different cell types. It is possible that, in the rat and mouse, copeptin had been cleaved enzymatically into smaller fragments that were undetectable by our N-terminal custom copeptin antiserum (CP8). Mass spectrometry analysis might, in theory, be able to demonstrate the fragmentation of copeptin in the parvocellular cell bodies. However, the low region-specific resolution of quantitative mass spectrometry at present precludes demonstration of the fragmentation of copeptin in only a subpopulation of parvocellular PVN neurons. Furthermore, our preliminary observation that copeptin could be detected in PVN parvocellular neurons by the m-20 antiserum, which was raised against an epitope close to the C-terminus of mouse copeptin (see Figure 12), shows that the C-terminus at least is intact and available to the antiserum. Our Western blotting experiment using the two copeptin antisera  showed that neither antiserum detected a positive band at a molecular weight (15 kDa) corresponding to copeptin incorporated as part of the AVP precursor in extracts of either mouse hypothalamus or pituitary. Our dot blot analysis also supports our hypothesis that both copeptin antisera specifically recognize each antigen peptide ( Figure 21). In addition, we previously reported by Western blot analysis that our custom copeptin 7-14 antiserum (CP8) specifically recognizes the copeptin 1-14 (fragment)-GFP fusion protein (Satoh et al., 2015). Furthermore, our post-embedding immunoelectron microscopy showed that immunoreactivity to our custom copeptin 7-14 antiserum (CP8), the Santa Cruz copeptin polyclonal antiserum (m-20), and the anti-NPII monoclonal antibody (PS41) could all be detected in neurosecretory vesicles within magnocellular AVP neurons, but apparently not in immature vesicle cores within the Golgi apparatus. Taken together, these results indicate that both copeptin antisera used in this study recognize only the processed peptide, and do not recognize copeptin when it is still part of a prohormone. In sharp contrast to the lack of copeptin-immunoreactivity in most NPII + parvocellular neurons in the rodent PVN, most parvocellular PVN NPII + neurons in the monkey exhibited prominent copeptin-immunoreactivity, demonstrating a striking species difference in copeptin expression between rodents and the monkey in NPII-immunoreactive CRF parvocellular neurons in the PVN. Taken together, these results suggest that there is a difference between magnocellular and parvocellular neurons in the proteolytic processing of the AVP precursor in rodents, but it remains unclear whether this is simply a difference in the speed of processing, and what might be its functional import.
Glycosylation of copeptin increases the stability of the peptide and its plasma half-life (Bhandari et al., 2009;Blanchard et al., 2013;Bolignano et al., 2014;Wuttke et al., 2013). Therefore, a third possible explanation for the relative lack of CP8-detectable copeptin in rodent parvocellular neurons would be that the glycosylation has not been completed in these neurons, leading to rapid breakdown of copeptin.
Our preliminary finding that the copeptin is, however, detectable with the m-20 C-terminal antiserum makes this possibility unlikely.
It has long been known that a subset of CRF neurons in the PVN co-expresses AVP in rats , mice (Whitnall, 1993), and humans (Mouri et al., 1993;Whitnall, 1993). CRF and AVP are cosecreted from nerve endings in the ME into capillaries, and reach the anterior pituitary via portal vessels to stimulate synergistically the release of ACTH from corticotrophs (Jones & Gillham, 1988 (Whitnall, 1993) or adrenalectomy (Itoi et al., 2014) increases dramatically the number of doubly immunopositive cells. In the external layer of the ME, about half of the parvocellular CRF nerve endings contain detectable AVP in the rat (Whitnall, 1993;Whitnall et al., 1985), whereas in the mouse virtually all CRF neurosecretory axons in the external layer of the ME contain AVP (Whitnall, 1993). In humans, AVP can be detected in all CRF neurons, depending on the premortem conditions (Mouri et al., 1993).
In the present study, very few CRF + neurons in the PVN of the CRF-VenusΔNeo mouse exhibited N-terminal copeptin-immunoreactivity, at least under normal unstressed circadian glucocorticoid levels, a finding consistent with an idea that AVP expression in CRF neurons is sensitive to stress and circulating glucocorticoid concentrations (Itoi et al., 2014). Another subpopulation of CRF + and/or AVP + parvocellular PVN neurons is known to project to autonomic centers in the brainstem and spinal cord, and it is likely that these neurons are unresponsive to negative feedback by glucocorticoids (Sawchenko, 1987). The copeptin status of this subpopulation of neurons remains to be determined.
In the mouse, very little copeptin-immunoreactivity could be identified in nerve terminals of parvocellular CRF neurons at the external layer of the ME with the N-terminal CP8 antiserum, and the reason for this remains unclear. It is reported that, in the ox, copeptin (39aa: 1-39) is metabolized into several small peptides of 10aa (1-10), 18aa (1-18), 17aa (23-39), and 14aa (26-39) residues (Land et al., 1982;Smyth & Massey, 1979). If copeptin in the mouse parvocellular neurons is metabolized rapidly, then this could explain why it was undetectable in most, but not all, nerve fibers in the external ME with the CP8 antiserum. In particular, peptides produced by enzymatic cleavage between 10 [L] and 11 [D] may not be identified because our custom CP8 copeptin antiserum was raised against an epitope that encompasses amino acid residues 7 [A] to 14 [A]. However, copeptin-immunoreactivity was readily detectable with the CP8 antiserum in some NPII-expressing nerve endings at the external layer of the ME in the rat and the monkey, reiterating the species difference in copeptin-immunoreactivity. Furthermore, our preliminary study shows that the m-20 C-terminal antiserum can detect copeptin immunoreactivity in both the cell bodies and the terminals of parvocellular NPII + neurons of mice (Figure 12), which indicates that the C-terminal epitope has not been metabolized to the extent that it is unrecognizable. Interpretation of any apparent differences in immunocytochemical detection of parvocellular neuropeptides between the cell bodies and terminals is difficult because of the concentration of the neurosecretory vesicles in the axon terminals (Morris, 2020). However, relatively slow processing of the pro-AVP precursor could explain why copeptin-immunoreactivity in the ME was more intense than that in the parvocellular PVN perikarya. Although AVP neurons in the SCN are also small-sized neurons, virtually all NPII + SCN neurons were copeptin + /NPII + in both mice and rats. Thus, SCN neurons resemble magnocellular neurons in terms of the N-terminal (CP8) copeptin-immunoreactivity.
Intracerebroventricular administration of a prohormone convertase inhibitor, D6R, resulted in a marked reduction of copeptinimmunoreactivity in the NPII-immunoreactive magnocellular PVN neurons and in SCN neurons in the mice. This result raises the possibility that insufficient processing of the prohormone could explain the lack of N-terminal copeptin immunoreactivity in a subpopulation of NPII (AVP)-expressing parvocellular neurons. Indeed, the second cleavage (between NPII and copeptin residues) is incomplete in birds and amphibians, and a two-domain protein comprising NPII and copeptin has been identified as a "big NP" in the posterior pituitary and in the circulation (Acher, 1980;Michel, Chauvet, Chauvet, & Acher, 1987). The absence of S S bonds in the "big NP" is likely to have resulted in an alteration in three-dimensional structure and could provide another explanation for the lower NPII (PS41) immunostaining of magnocellular neurosecretory neurons in the D6R- pituitary. In addition, D6R is known to be predominantly a furin inhibitor (Cameron et al., 2000), and both Schafer et al. (1993) and Dong et al. (1997) reported the ubiquitous presence of furin in the PVN and SON.
Copeptin has long been regarded as biologically inactive (Morgenthaler et al., 2008). However, recent advances in research on the glycopeptide mean that it is now widely accepted that copeptin possesses multi-faceted biological properties. For example, copeptin is required for correct folding of the AVP precursor, and its absence may lead to an inefficient monomer folding that contributes to the pathogenesis of human hypothalamic diabetes insipidus (Barat, Simpson, & Breslow, 2004). Roles of copeptin in modulating cellular functions have also been explored, and it has been reported that copeptin causes a low amplitude, slow increase of [Ca 2+ ] i in a population of cultured rat hypothalamic neurons (Gao et al., 2008). Copeptin (both copeptin 1-39 [full length] and the C-terminal fragment 22-39) also potentiate the amplitude of excitatory postsynaptic potentials evoked in neurons of the rat lateral septum (van den Hooff, Seger, Burbach, & Urban, 1990), indicating that the C-terminal component of copeptin may modulate neuronal activity in the brain.
In conclusion, N-terminal copeptin-immunoreactivity was not detectable (or was present in disproportionately low concentrations) in a subpopulation of AVP-expressing parvocellular neurons in the PVN of rodents, but was present in virtually all AVP-expressing magnocellular neurons in the PVN and SON. This difference between parvocellular and magnocellular copeptin expression was not observed in the monkey, and was not seen when the copeptin was detected with a C-terminal copeptin antiserum. This study has revealed not only that there are species differences in the processing of the pro-AVP precursor and production of free copeptin, but also that the processing may not be the same in different groups of pro-AVPexpressing cells within a given species. At the moment, there is an increasing use of plasma copeptin measurements as a surrogate marker for AVP measurements in human clinical problems such as diabetes insipidus, sepsis and cardiovascular disease (Morgenthaler et al., 2008). Future research on macaques could provide valuable and relevant information on copeptin processing and plasma copeptin levels under conditions such as dehydration and other stresses. However, relatively little is currently known about the control of pro-AVP precursor processing in humans, the enzymes involved, and the extent to which the various antibodies used to detect copeptin recognize the possible different molecular weight forms. Our results, which reveal for the first time differences among rodents and macaques, strongly suggest that this is an area which deserves further experimental exploration.