Journal of Neuroendocrinology, 2014, 26, 497–509 © 2014 British Society for Neuroendocrinology

ORIGINAL ARTICLE

Activity-Dependent Notch Signalling in the Hypothalamic-Neurohypophysial System of Adult Mouse Brains T. Mannari and S. Miyata Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan.

Journal of Neuroendocrinology

Correspondence to: Dr Seiji Miyata, Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan (e-mail: [email protected]).

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double-labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)- and oxytocin (OXT)-containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT- but not AVP-containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4-immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity-dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH. Key words: neuropetides, oxytocin, vasopressin, notch, plasticity

The hypothalmic-neurohypophysial system (HNS) secretes arginine vasopressin (AVP) and oxytocin (OXT), which regulate body fluid homeostasis, uterine contractions, reproductive behaviour, milk ejection and social behaviours. AVP and OXT are synthesised in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) and are released from axon terminals in the neurohypophysis (NH). In the HNS, it is well known that AVP- and OXT-containing magnocellular neurones show structural reconstruction upon chronic physiological stimulation, such as salt loading, lactation and parturition (1). In the SON and PVN, cellular processes of astrocytes surround the somata and dendrites of magnocellular neurones under normal conditions, whereas their processes withdraw and thereby astrocytic coverage is reduced upon chronic physiological stimulation. The reduction of astrocytic coverage results in an increase in multiple synapses and the direct apposition of the neuronal membrane to promote neuronal excitation and synchronisation (2–4). Several

doi: 10.1111/jne.12172

molecules are proposed to be involved in the neuro-neuronal and neuro-glial interactions that lead to these structural reconstructions, such as polysialic acid neural cell adhesion molecule (PSANCAM) (5,6), F3 (7), opioid-binding cell adhesion molecule (8), tenascin-C (9,10) and chondroitin sulphate proteoglycans (3). Clearance of glutamate from extracellular space is important for sculpting transmitter release (11) and therefore the reduction of astrocytic coverage around magnocellular neurones results in glutamate spilling over the synapse and diffusion into the extracellular space to increase the level of glutamate (12). Inhibition of astrocytic transporter reduces the amplitude of evoked glutamatergic synaptic currents and also the frequency under conditions where astrocytic coverage of magnocellular neurones is maximal (13). Moreover, ambient glutamate facilitates GABA release by acting on presynaptic kainic acid receptors on OXT-containing magnocellular neurones in virgin rats, although the elevated level of glutamate

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reverses to inhibit GABA release via signalling of phospholipase C in lactating animals (12). Another possible explanation for the alteration of glial coverage is the finding that taurine released from astrocytes acts on glycine receptors on magnocellular neurones (14). Pituicytes, neurohypophysial astrocytes, engulf axonal terminals under normal conditions in the NH, whereas cellular processes of pituicytes withdraw to permit direct contact of the axonal terminals of magnocellular neurones with the vascular basement membrane in response to chronic physiological stimulation (15,16). Several plasticity-related molecules are proposed to participate in neurovascular and neuroglial interactions, such as PSA-NCAM (5,6), F3 (5,7), tenascin-C (10) and chondroitin sulphate proteoglycans (17). Notch signalling is well known to function as a master regulator for proliferation and differentiation of neural stem/progenitor cells (18,19). Notch signalling is controlled by the interaction between Notch1-4 on signal receiving cells and their ligands, such as Deltalike ligand (DLL) 1, 3 and 4, as well as Jagged1 and 2, on signal sending cells (20–22). Activation of Notch signalling leads to the ubiquitination of Notch ligand in ligand-expressing cells (23). The binding of Notch and its ligand causes the proteolytic cleavage of Notch via presenilin protease of the c-secretase complex and then Notch intracellular domain (NICD), a cleavage Notch form, translocates to the nucleus to activate the transcription of target genes, including NCAM (24), F3 (25) and tenascin-C (26). In developing brains, the most well-characterised Notch function is the inhibition of neurogenesis, the maintenance of neural progenitor characters, and the fate choice of neurones and glial cells (22). Most studies of Notch signalling in brains have been performed with respect to the proliferation and differentiation of stem/progenitor cells in developing brains and restricted adult brain regions, such as the subgranular zone (SGZ) and subventricular zone (SVZ) (18), although a few studies have reported that Notch signalling regulates synaptic plasticity and behaviour. For example, cleavage of Notch1 is significantly elevated in the SGZ during memory consolidation (27). Notch1 and its ligand Jagged1 are expressed at somatodendrites of pyramidal neurones and axonal terminals in the hippocampal CA1, respectively, and an increase ion synaptic activity activates Notch signalling (28,29). In the HNS, only one study has reported that a Notch ligand, Delta-like 1 homologue (Dlk1), is expressed at somatodendrites in the SON and PVN of the adult mouse brain (30). Although Notch signalling is presumed to be a key regulator in the structural reconstruction of the HNS when considering its functions for intercellular communication and the control of PSANCAM, F3 and tenascin-C expression, very little is known about whether Notch signalling participates in structural plasticity in the HNS of adult mammals. The present study aimed to determine whether Notch signalling participates in neuro-neuronal and/or neuroglial interactions in the HNS of adult mice upon physiological stimulation. First, we carried out immunohistochemical studies of Notch3 and DLL4 to examine their cellular localisation. Second, we examined activity-dependent changes in the expression levels of DLL4 and cleavage forms of Notch3. The present study demonstrated that DLL4 was highly expressed at axonal boutons against AVP- and OXT-containing magnocellular neurones in the SON and PVN, as well as at OXT-containing axonal terminals in the NH. The © 2014 British Society for Neuroendocrinology

expression of Notch3 was observed at somatodendrites of AVPand OXT-containing magnocellular neurones in hypothalamic nuclei and at pituicytes in the NH. Chronic and acute osmotic stimulation reduced the expression level of DLL4 in the hypothalamic nuclei. Moreover, chronic and acute osmotic stimulation increased the level of cleavage forms of Notch3, NICD3 and Notch3 transmembrane subunits (NTM3) in the HNS. Thus, the Notch signalling pathway is possibly involved in changes of intercellular communication during structural reconstruction in the HNS of the adult mouse.

Materials and methods Animals Adult mice (C57BL/6J; 70–84 days old) were used in the experiments. The animals were housed two per cage in a colony room under a 12 : 12 h light/dark cycle and given ad lib. access to commercial chow and tap water. All procedures were carried out in accordance with the National Institute of Health Guidelines and the Guideline for Proper Conduct of Animal Experiments Science Council of Japan to minimise the number of animals used and their suffering and approved by Animal Ethics Committee of Kyoto Institute of Technology.

Osmotic stimulation For chronic osmotic stimulation, mice were stimulated by drinking 2% NaCl ad lib. instead of tap water for 3 and 5 days and then sacrificed for immunohistochemistry, western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). For acute osmotic stimulation, animals received an i.p. injection of hyperosmotic solution (100 ll of 3% NaCl) and then were sacrificed 1.5 and 3.0 h after the injection.

Immunohistochemistry Mice were perfused transcardially with phosphate buffer saline (PBS; pH 7.2) containing 5 U/ml heparin followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4) after deep anaesthesia with urethane. After the perfusion of PFA, the brain and pituitary gland were dissected out and postfixed in 4% PFA in 0.1 M PB (pH 7.4) at 4 °C overnight. Fixed tissues were then cryoprotected by 30% sucrose in PBS and frozen quickly in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). The sections were obtained by coronal cut on a cryostat (Leica Geosystems AG, St. Gallen, Switzerland) at a thickness of 30 lm. For single- and double-labelling immunohistochemistry, a standard immunofluorescence technique was performed on free-floating sections as described previously (17). In brief, the sections were washed with PBS and treated with 25 mM glycine in PBS for 20 min. For immunohistochemistry of Notch3, the sections were treated with 0.05% citraconic anhydride solution (Immunosaver; Nisshin EM Co., Tokyo, Japan) for 30 min at 95 °C. The sections were incubated with 5% normal goat serum in PBS containing 0.3% Triton X-100 (PBST) for 24 h at 4 °C and then incubated with the following primary antibodies in PBST for 2–3 days at 4 °C: rat antibody against DLL4 (dilution 1 : 100, clone #207811; R&D Systems, McKinley Place, MN, USA); rabbit antibody against AVP (dilution 1 : 1000; Millipore-Chemicon, Temecula, CA, USA), Notch3 (dilution 1 : 300, M-134; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and vesicular GABA transporter (VGAD; dilution 1 : 400, No. 131-003; Synaptic Systems GmbH, Goettingen, Germany); mouse antibody against OXTneurophysinn (dilution 1 : 20, PS41; Dr Gainer, NIH, Bethesda, MD, USA), AVP-neurophysin (dilution 1 : 20, PS38; Dr. Gainer, NIH) and synaptotagmin Journal of Neuroendocrinology, 2014, 26, 497–509

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(dilution 1 : 3000, mAB30; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); and guinea pig antibody against laminin (YI-2008, dilution 1 : 200) (31), glial fibrillar acidic protein (GFAP; TN-2006, dilution 1 : 400) (32) and vesicular glutamate transporter 1 (VGLUT1; dilution 1 : 5000, AB5905; Millipore-Chemicon). The antibodies were visualised by using Alexa 488- or Alexa 594-conjugated secondary goat antibody against mouse, rat, rabbit and guinea pig IgG (dilution 1 : 400; Jackson ImmunoResearch, West Grove, PA, USA) in PBST for 2 h. The coverslips were sealed with Vectashield (Vector Labs, Burlingame, CA, USA) and observations were made using confocal microscopes (LSM510; Carl Zeiss, Oberkochen, Germany; or Fluoview, FV10i; Olympus, Tokyo, Japan). For nuclear staining, sections were incubated with 40 ,6-diamidino-2-phenylindole (1 lg/ml; Dojindo, Kumamoto, Japan) or propidium iodide (40 lg/ml; Sigma-Aldrich, Tokyo, Japan). The DLL4 antibody used is the rat monoclonal antibody raised against amino acids 28–525 of mouse DLL4 and shown to be available for the immunohistochemistry and western blotting in mouse tissues (33). The Notch 3 antibody used is a rabbit polyclonal antibody raised against amino acids 2107–2240 of Notch 3 of mouse origin and reported to be useful for the immunohistochemistry and western blotting in mouse tissues (34,35). The omission of DLL4 and Notch3 antibodies resulted in no detectable immunological signals.

Western blotting Western samples were obtained from PFA-fixed tissues of adult mice in accordance with previous studies (36,37). Western analysis was performed by using three different samples and confirmed the reproducibility. Briefly, the forebrain and pituitary gland were collected immediately after the decapitation and then fixed with 4% PFA in 0.1 M PB (pH 7.4) for 30 min at 4 °C with vigorous shaking. Coronal sections were obtained by vibratome (DTK-1000; DSK, Kyoyo, Japan) at a thickness of 100 lm and the pieces, including the SON, PVN, and NH of adult mice, were carefully dissected using a fine forceps and razor under a microscope. The tissues were homogenised with a VCX T301B ultrasonic generator (Vibro Cell; Sonics & Materials Inc., Newtown, CT, USA) in a four-fold amount of PBS containing 1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, 10 lg/ml leupeptin, 10 lg/ml pepstatin and 10 lg/ml aprotinin at 4 °C. After the determination of protein concentration using a Bradford protein assay kit (Bio-Rad, Tokyo, Japan), the homogenate was dissolved in 50 mM Tris HCl (pH 7.4) containing 20% sodium dodecylsulphate and incubated at 99 °C for 20 min followed by at 60 °C for 2 h (36,37). After the addition of sample boiling buffer, the protein samples were separated on sodium dodecylsulphate polyacrylamide gel. Electroblotting was performed on a polyvinylidene difluoride (PVDF) membrane (pore size, 0.45 lm; GE Healthcare Bio-Science, Little Chalfont, UK) in the 25 mM Tris-HCl buffer solution containing 192 mM glycine and 20% methanol. The PVDF membrane was treated with 10% H2O2 for 10 min to inactivate endogenous peroxidase activity in accordance with the manufacturer’s instructions. Nonspecific binding of proteins to the membrane was incubated with 5% ECL Prime Blocking Agent (GE Healthcare Bio-Science) in 50 mM Tris-buffered saline containing 0.5% Tween-20 (TBST) overnight at 4 °C. The PVDF membrane was incubated with the rat antibody against DLL4 (dilution 1 : 700; R&D Systems), the rabbit antibody against Notch3 (dilution 1 :1 000; Santa Cruz Biotechnology) or actin (dilution 1 : 500, JLA20; Developmental Studies Hybridoma Bank) in the TBST containing 0.2% bovine serum albumin for 2 h. The membrane was incubated further with horseradish peroxidase-labelled anti-rabbit, -rat or -mouse IgG (dilution 1 : 20 000; GE Healthcare Bio-Science) in TBST containing 5% bovine serum albumin for 1 h at 37 °C. The approximate molecular weight for each protein was estimated using Precision Plus Protein WesternC Standards and Precision Protein Strep-Tactin HRP Conjugate (dilution 1 : 50 000; Bio-Rad Laboratories, Hercules, CA, USA). The peroxidase was finally activated with an enhanced chemiluminescence kit (ECL Prime Western blotting detection

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system; GE Healthcare Bio-Sciences) and the immunoreactivity was visualised employing RX-U Medical X-ray film (Fuji Film, Tokyo, Japan).

RT-PCR The experiment was performed in accordance with a previous method (32). After the decapitation of adult mice, the brain and pituitary gland were immediately dissected out and immersed in RNAlater RNA stabilisation reagent (Qiagen, Hilden, Germany). The tissue pieces containing the SON, PVN and NH were carefully isolated under the microscope. Total RNA was extracted by using SV Total RNA isolation system (Promega Corp., Madison, WI, USA) and then subjected to cDNA synthesis with oligo (dT)20 primers using PC320 thermal controller (Astec, Fukuoka, Japan) and ReverTra Ace reverse transcriptase (Toyobo, Tokyo, Japan). The cDNA was subsequently amplified by PCR with the primers: Notch3 primers (38): sense, 50 -GCA CCTGCAACCCTGTTTAT-30 , antisense, 50 -ACAGAGCCGGTTGTCAATCT-30 , 40 cycles of 95 °C for 10 s, 61 °C for 2 s and 72 °C for 60 s, amplicon 366 bp: glyceraldehydes-3-phosphate dehydrogenase primers: sense, 50 -ACCACAGTCCATGCCATC AC-30 , antisense, 50 -TCCACCACCCTGTTGCTGTA-30 , 35 cycles of 98 °C for 10 s, 63 °C for 2 s and 72 °C for 60 s, amplicon 445 bp. The omission of reverse transcriptase did not yield detectable signals.

Quantitative and statistical analysis For quantitative analysis, confocal images were obtained under the same pinhole size, brightness and contrast setting. We saved the images (1024 9 1024 pixels) as TIF files by employing LSM510 IMAGE BROWSER (Carl Zeiss) for Windows, and arranged using PHOTOSHOP, version 7.0 (Adobe Systems, San Jose, CA, USA). The AVP- and OXT-immunoreactive areas and relative intensity of Notch3 immunoreactivity were measured using WINROOF (Mitani Corp., Fukui, Japan) for which the threshold intensity was set to include the measurement profile by visual inspection and was kept constant. For quantification of the number of DLL4-immunoreactive puncta, DLL4immunoreactive puncta that did not juxtapose against AVP- or OXT-positive magnocellular somatodendrites were eliminated manually using PHOTOSHOP, version 7.0, and then the number of remaining puncta juxtaposing against AVP- or OXT-positive ones was quantified using WINROOF. Analyses of all images were performed such that the experimenter was blind to the treatment group. Statistical difference was assessed using an unpaired Student’s t-test or ANOVA with Tukey’s post-hoc test. P < 0.05 was considered statistically significant.

Results Expression of DLL4 in the HNS A low magnification view of confocal images revealed that the immunoreactivity of DLL4 was stronger in the SON of the adult mouse compared to neighbouring hypothalamic brain regions (Fig. 1A,A″). Western analysis revealed a specific band at a molecular mass of 80 kDa that corresponds to the estimated molecular mass of DLL4 (Fig. 1B). High magnification views showed that DLL4immunoreactive puncta were seen at somatodendrites of AVP(Fig. 1C,D) and OXT-positive (Fig. 1E,F) magnocellular neurones but not GFAP-positive astrocytes (Fig. 1G). Double-labelling immunohistochemistry with an axonal bouton marker synaptotagmin further revealed that the immunoreactivity of DLL4 was observed at a subpopulation of axonal boutons (Fig. 1H). Three-dimensional (3D) image analysis confirmed the presence of DLL4 immunoreactivity at © 2014 British Society for Neuroendocrinology

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Fig. 1. Confocal microscopic images showing the localisation of Delta-like ligand 4 (DLL4) in the supraoptic nucleus (SON) of the adult mouse. Low magnification views revealed that the immunoreactivity of DLL4 was stronger in the SON compared to adjacent hypothalamic brain regions (A, A″). Western analysis revealed the specific immunological signal at a molecular mass of 80 kDa (arrowhead) from SON homogenate (B). Double-labelling immunohistochemistry revealed that the immunoreactivity of DLL4 was observed, with many puncta localising at somatodendrites of arginine vasopressin (AVP) - (C, D) and oxytocin (OXT)-positive magnocellular neurones (E, F), whereas it was not seen at glial fibrillar acidic protein (GFAP)-positive astrocytes (G). Double-labelling immunohistochemistry with an axonal bouton marker synaptotagmin showed that the immunoreactivity of DLL4 was observed at some axonal boutons (H, left). Threedimensional image analysis confirmed the presence of DLL4 immunoreactivity at axonal boutons (H, right). The immunoreactivity of DLL4 was seen at some of the vesicular GABA transporter (VGAD)- (I,I″) and vesicular glutamate transporter 1 (VGLUT1)-positive (J,J″) axonal boutons. Arrowheads indicate DLL4-immunoreactive puncta. DAPI, 40 ,6-diamidino-2-phenylindole; LH, lateral hypothalamic area; oc, optic chiasm; Syt, synaptotagmin. Scale bars = 50 (A, C, E), 10 [D, F, G, I, J and H (left)], 1 lm [H (right)].

the synaptotagmin-positive axonal boutons (Fig. 1H). The immunoreactivity of DLL4 was observed at some of the VGAD- (Fig. 1I,I″) and VGLUT1-positive (Fig. 1J,J″) axonal boutons. The density of DLL4-immunoreactive puncta was significantly higher in the SON (number of DLL4-immunoreactive puncta/mm2: 2816.66  540.46, P = 0.00721 by an unpaired Student’s t-test, n = 4) compared to the lateral hypothalamic area or a hypothalamic region adjacent to the SON (30.83  2.15). Stronger immunoreactivity of DLL4 was seen at the PVN compared to adjacent hypothalamic regions (Fig. 2A,A″). High magnification © 2014 British Society for Neuroendocrinology

views showed that DLL4-immunoreactive puncta were seen at somatodendrites of AVP- (Fig. 2B,C) and OXT-positive (Fig. 2D,E) magnocellular neurones but not at GFAP-positive astrocytes (Fig. 2F). Double-labelling immunohistochemistry revealed that the immunoreactivity of DLL4 was observed at a subpopulation of synaptotagmin-positive axonal boutons in the PVN (Fig. 2G). 3D image analysis confirmed the presence of DLL4 immunoreactivity at the synaptotagmin-positive axonal boutons (Fig. 2G). The immunoreactivity of DLL4 was observed at some of the VGAD- (Fig. 2H,H″) and VGLUT1-positive (Fig. 2I,I″) axonal boutons. The density of Journal of Neuroendocrinology, 2014, 26, 497–509

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Fig. 2. Confocal microscopic images showing the localisation of Delta-like ligand 4 (DLL4) in the paraventricular nucleus (PVN) of the adult mouse. Low magnification views revealed that the immunoreactive of DLL4 was stronger at magnocellular part of the PVN than adjacent other hypothalamic regions (A, A″). The immunoreactivity of DLL4 was seen at somatodendrites of arginine vasopressin (AVP) - (B, C) and oxytocin (OXT)-positive (D, E) magnocellular neurones. The immunoreactivity of DLL4 was observed at a subpopulation of synaptotagmin-positive axonal boutons (G, left), but not at glial fibrillar acidic protein (GFAP)positive astrocytes (F). Three-dimensional image analysis confirmed the presence of DLL4 immunoreactivity at axonal boutons (G, right). The immunoreactivity of DLL4 was observed at some of the vesicular GABA transporter (VGAD)- (H, H″) and vesicular glutamate transporter 1 (VGLUT1)-positive (I, I″) axonal boutons. Arrowheads indicate DLL4-immunoreactive puncta. AHC, central part of the anterior hypothalamic area; DAPI, 40 ,6-diamidino-2-phenylindole; 3V, third ventricle; Syt, synaptotagmin. Scale bars = 50 (A, B, D), 10 [C, E, F, H, I and G (left)] and 1 lm [G (right)].

DLL4-immunoreactive puncta was significantly higher in the PVN (number of DLL4-immunoreactive puncta/mm2: 3361.55  566.50, P = 0.0125 by an unpaired Student’s t-test, n = 4) compared to the central part of the anterior hypothalamic area or a hypothalamic area adjacent to the PVN (39.80  3.58). Prominent immunoreactivity of DLL4 was observed at the NH but not the anterior and intermediate pituitary (Fig. 3A,A″). High magnification views showed that the immunoreactivity of DLL4 was observed at OXT-positive axonal terminals (Fig. 3B,B″) but scarcely seen at AVP-positive ones (Fig. 3C,C0 ). The 3D image analysis confirmed that the immunoreactivity of DLL4 was observed at OXT-positive axonal terminals (Fig. 3D). DoubleJournal of Neuroendocrinology, 2014, 26, 497–509

labelling immunohistochemistry with a vascular basement membrane marker laminin revealed that DLL4-immunoreactive puncta were likely to locate in close proximity to the vasculature of the NH (Fig. 3E,E″).

Changes in DLL4 expression by chronic osmotic stimulation To assess activity-dependent regulation of DLL4 expression, we examined the effects of chronic salt loading on the change of DLL4 immunoreactivity. Chronic salt loading by drinking 2% NaCl is known to increase the blood AVP and OXT levels and structural alterations such as synaptic reconstruction in the hypothalamic © 2014 British Society for Neuroendocrinology

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Fig. 3. Confocal microscopic images showing the localisation of Delta-like ligand 4 (DLL4) in the neurohypophysis (NH) of the adult mouse. Low magnification views showed that the immunoreactivity of DLL4 was observed in the NH (A, A″). The immunoreactivity of DLL4 was seen at oxytocin (OXT)-positive magnocellular terminals (B, B″) but not at arginine vasopressin (AVP)-positive ones (C, C0 ). Three-dimensional image analysis confirmed the occurrence of DLL4 immunoreactivity at OXT-positive axonal terminals (D). Double-labelling immunohistochemistry with a vascular basement membrane marker laminin showed that DLL4-immunoreactive puncta often situated closely to laminin-positive vasculature (E, E″). Arrowheads indicate DLL4-immunoreactive puncta. AP, anterior pituitary; IP, intermediate pituitary; PI, propidium iodide. Scale bars = 100 (A), 10 (B, C, E) and 1 lm (D).

nuclei and neurovascular contacts in the NH (1,16). Confocal images (Fig. 4) and quantitative morphometrical analysis (Fig. 5) revealed that the number of DLL4-immunoreactive puncta at AVPpositive magnocellular neurones was significantly decreased in the SON by 3-day salt loading (P = 0.0486 by ANOVA with Turkey’s test, n = 5; Figs 4A–C and 5A) and PVN by 3-day (P = 0.000192; Figs 4G, H and 5B) and 5-day (P = 0.00328; Figs 4G,I and 5B) salt loading. The chronic salt loading also decreased the number of DLL4-immunoreactive puncta at OXT-positive magnocellular neurones in the SON by 5-day salt loading (P = 0.0256; Figs 4D–F and 5A) and the PVN by 3-day salt loading (P = 0.0184; Figs 4J–L and 5B). Chronic salt loading did not significantly change the number of © 2014 British Society for Neuroendocrinology

DLL4-immunoreactive OXT-positive axonal terminals in the NH by salt loading compared to the control (P > 0.05; Figs 4M–O and 5C).

Expression of Notch3 and its proteolytic cleavage by chronic and acute osmotic stimulation A low magnification view of confocal images revealed that the immunoreactivity of Notch3 was stronger in the SON (Fig. 6A), PVN (Fig. 6B) and NH (Fig. 6C) of the adult mouse. Double-labelling immunohistochemistry revealed that the immunoreactivity of Notch3 was seen at somatodendrites of AVP- (Fig. 6D,D0 ,F,F0 ) and OXT-positive (Fig. 6H,H0 ,J,J0 ) magnocellular neurones in the SON and Journal of Neuroendocrinology, 2014, 26, 497–509

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NH Fig. 4. Confocal microscopic images showing the effects of chronic physiological stimulation, salt loading, on the immunoreactivity of Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system of adult mice. Chronic salt loading decreased the number of DLL4-immunoreactive puncta on arginine vasopressin (AVP)- and oxytocin (OXT)-positive magnocellular neurones in the supraoptoic nucleus (SON) (A–F). Chronic salt loading also decreased the number of DLL4-immunoreactive puncta AVP- and OXT-positive magnocellular neurones in the paraventricular nucleus (PVN) (G–L). In the neurohypophysis (NH), DLL4immunoreactive puncta appeared not to be changed at OXT-positive magnocellular axonal terminals by salt loading (M–O). Arrowheads indicate DLL4-immunoreactive puncta. Scale bars = 10 lm.

PVN. 3D image analysis confirmed the presence of Notch3 immunoreactivity at somata of AVP- (Fig. 6E,G) and OXT-positive (Fig. 6I,K) magnocellular neurones. Double-labelling immunohistochemistry Journal of Neuroendocrinology, 2014, 26, 497–509

showed that the immunoreactivity of Notch3 was not observed at GFAP-positive astrocytes (Fig. 6L,M). Some synaptotagmin-positive axonal boutons were seen to localise closely to Notch3-immunoreactive © 2014 British Society for Neuroendocrinology

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Fig. 5. The effects of chronic osmotic stimulation on the number of Delta-like ligand 4 (DLL4)-immunoreactive puncta at the arginine vasopressin (AVP)- and oxytocin (OXT)-positive magnocellular neurones in the supraoptoic nucleus (SON) (A) and paraventricular nucleus (PVN) (B) and at OXT-positive axonal terminal in the neurohypophysis (NH) (C) of the adult mouse. Total number of DLL4-immunoreactive puncta examined was 684 (control, 48 sections), 300 (5-day salt loading, 38 sections) and 494 (5-day salt loading, 50 sections) at AVP-positive magnocellular neurones and 118 (control, 23 sections), 147 (3-day salt loading, 18 sections) and 72 (5-day salt loading, 23 sections) at OXT-positive ones in the SON. Total number of DLL4-immunoreactive puncta examined was 557 (control, 27 sections), 316 (3-day salt loading, 31 sections) and 674 (5-day salt loading, 28 sections) at AVP-positive magnocellular neurones and 478 (control, 26 sections), 280 (3-day salt loading, 30 sections) and 542 (5-day salt loading, 33 sections) at OXT-positive ones in the PVN. Total number of DLL4-immunoreactive puncta examined was 801 (control, 13 sections), 732 (3-day salt loading, 16 sections) and 864 (5-day salt loading, 14 sections) at OXT-positive axonal terminals in the NH. Analysis of variance with Turkey’s post-hoc tests (*P < 0.05, **P < 0.01, ***P < 0.001; n = 5).

magnocellular neurones in the hypothalamic nuclei (Fig. 6N,O). In the NH, double-labelling immunohistochemistry revealed that the immuoreactivity of Notch3 was seen at GFAP-positive pituicytes (Fig. 6P). Some AVP- (Fig. 6Q) and OXT-positive (Fig. 6R) axonal terminals were localised in close proximity to Notch3-immunoreactive © 2014 British Society for Neuroendocrinology

pituicytes. 3D image analysis confirmed the presence of Notch3 immunoreactivity at GFAP-positive pituicytes (Fig. 6P). The relative intensity of Notch3 immunoreactivity was significantly higher in the SON (relative intensity of Notch3 immunoreactivity: 100.69  24.22, P = 0.00461 by an unpaired Student’s t-test, Journal of Neuroendocrinology, 2014, 26, 497–509

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Fig. 6. Confocal microscopic images showing the localisation of Notch3 in the hypothalamic-neurohypophysial system of the adult mouse. Low magnification views revealed that the immunoreactivity of Notch3 was observed at the supraoptic nucleus (SON) (A), paraventricular nucleus (PVN) (B) and neurohypophysis (NH) (C). High magnification views showed that the immunoreactivity of Notch3 was seen at arginine vasopressin (AVP)- (D, D0 , F, F0 ) and oxytocin (OXT)-positive (H, H0 , J, J0 ) magnocellular neurones in the SON and PVN. On the other hand, the immunoreactivity of Notch3 was scarcely seen at glial fibrillar acidic protein (GFAP)-positive astrocytes (L, M) and synaptotagmin-positive axonal boutons (N, O) in the SON and PVN. Three-dimensional (3D) image analysis revealed the immunoreactivity of Notch3 at AVP- (E,G) and OXT-positive (I, K) magnocellular neurones. In the NH, the immunoreactivity of Notch3 was seen at GFAP-positive pituicytes (P, left) but not AVP (Q) and OXT-positive (R) axonal terminals. 3D image analysis revealed that the immunoreactivity of Notch3 was observed at GFAP-positive astrocytes (P, right). Arrowheads indicate the immunoreactivity of Notch3. Data are expressed as the mean  SEM number of DLL4-immunoreactive puncta per AVP- or OXT-positive area (mm2). AHC, central part of the anterior hypothalamic area; AP, anterior pituitary; IP, intermediate pituitary; LH, lateral hypothalamic area; oc, optic chiasma; Syt, synaptotagmin; 3V, third ventricle. Scale bars = 50 (A, C, D, L) and 10 lm (E, N, P, Q).

n = 4) compared to the lateral hypothalamic area (22.25  12.93). The relative intensity of Notch3 immunoreactivity was significantly higher in the PVN (313.83  89.45, P = 0.00245, n = 4) compared to the central part of the anterior hypothalamic area (44.67  16.46). To assess activity-dependent regulation of Notch3 signalling, we examined proteolytic cleavage of Notch3 by western blotting. Journal of Neuroendocrinology, 2014, 26, 497–509

Drinking of 2% NaCl and the i.p. administration of 3% NaCl solution were used as chronic and acute osmotic stimulations, respectively. The binding of Notch and its ligand is known to yield the cleavage products, NTM and NICD, by presenilin protease of the c-secretase complex (39). Chronic salt loading for 5 days but not 3 days increased the levels of NTM3 at a molecular mass of 110 kDa and NICD3 at a molecular mass of 90 kDa in the SON (Fig. 7A). The © 2014 British Society for Neuroendocrinology

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Fig. 7. Increase of proteolytic cleavage and mRNA expression of Notch3 by chronic and acute osmotic stimulation. Mice were stimulated by applying 2% NaCl instead of tap water for chronic osmotic stimulation or the intraperitoneal administration of 3% NaCl for acute osmotic stimulation. Western blotting revealed that two cleaved forms of Notch3, NTM3 and NICD3, were detected at molecular mass of 110 and 90 kDa, respectively (A). The levels of NTM3 and NICD3 were prominently increased in the supraoptic nucleus (SON), paraventruicular nucleus (PVN) and neurohypophysis (NH) 1.5 and 3.0 h after the administration 3% NaCl solution. The increase of NTM3 and NICD3 levels was also observed in the SON, PVN and NH by 5-day salt loading (A). Reverse transcriptase-polymerase chain reaction analysis showed that the mRNA expression of Nocth3 was increased in the SON by 5-day salt loading and 3 h after the administration of 3% NaCl (B). The mRNA of Nocth3 was also increased in the NH by 3- and 5-day salt loading and 3 h after the administration of 3% NaCl (B). Cont, control.

levels of NTM3 and NICD3 were progressively increased in the SON 1.5 and 3.0 h after the i.p. administration of 3% NaCl solution (Fig. 7A). In the PVN, 5-day salt loading and the i.p. administration of 3% NaCl prominently increased the level of NTM3 and NICD3 (Fig. 7A). As well as the hypothalamic nuclei, the level of NTM3 and NICD3 was increased by chronic salt loading and the i.p. administration of 3% NaCl in the NH (Fig. 7A). RT-PCR analysis revealed that the mRNA level of Notch3 was increased in the SON by 5-day salt loading and in the NH by 3- and 5-day salt loading (Fig. 7B). The mRNA level of Notch3 was also increased in the SON 3 h and in the NH 1.5 h after stimulation with 3% NaCl (Fig. 7B). No apparent increase of Notch3 mRNA was observed in the PVN.

Discussion New evidence is provided by the results of the present study: (i) the expression of DLL4 was observed at axonal boutons against AVP- and OXT-containing magnocellular neurones in the SON and PVN and at OXT-containing axonal terminals in the NH; (ii) the expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the hypothalamic nuclei and at pituicytes in the NH; (iii) the expression of DLL4 in the hypothalamic nuclei was decreased by chronic osmotic stimulation; © 2014 British Society for Neuroendocrinology

and (iv) the cleavage forms of Notch3, NTM3 and NICD3 were increased in the HNS by chronic and acute osmotic stimulation. Thus, the present study is the first demonstration of activity-dependent Notch signalling in the HNS of adult mice (see Supporting information, Fig. S1). Until now, little was known about whether Notch signalling participates in synaptic plasticity in mature neurones of the adult brain, except the SGZ of the hippocampus, although numerous studies have shown that Notch signalling is involved in the maintenance and differentiation of neural stem cells and neurite outgrowth and dendritic arbour of immature neurones in developing brains and the SGZ and SVZ of adult ones (18,22,40). The transgenic mouse overexpressing NICD1 reduces the density of dendritic spine and filopodia and limits long-term potentiation in the adult visual cortex (41). Stimulation with NMDA and GABAergic receptor antagonist bicuculine leads to an increase in the level of NICD1 in the hippocampus and conditional deletion of Notch1 in the postnatal hippocampus results in the inability to learn and affects shortterm memory by disrupting both long-term potentiation and depression in adult brains (28). In the SON and PVN, the expression of Dlk1 is reported at somatodendrites of the SON and PVN in adult mice (30). In the present study, we found that DLL4 was expressed at axonal boutons against AVP- and OXT-containing magnocellular neurones in the SON and PVN and at OXT-containing Journal of Neuroendocrinology, 2014, 26, 497–509

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axonal terminals in the NH. Moreover, Notch3 was highly expressed at somatodendrites in the SON and PVN and at pituicytes in the NH. Thus, the present study indicates that DLL4 and Notch3 are possible candidates for plasticity-related master regulators in the HNS of the adult mouse. The present study revealed, in the SON and PVN, that DLL4 was expressed at axonal boutons against AVP- and OXT-containing magnocellular neurones. Moreover, it was shown that Notch3 was expressed at somatodendrites of AVP- and OXT-containing magnocellular neurones. A previous study has shown that Dlk1 is specifically localised at somatodendrites of AVP- and OXT-containing magnocellular neurones in the hypothalamic nuclei (30). In cultured hippocampal neurones, Notch1 is enriched at somatodendrites but the ligand Jagged1 is enriched at presynaptic terminals (28). The activation of Notch1 decreases the density of the mushroom spine and the number of filopodia of dendrites, as well as soma size, but does not change the size of spine head and length in adult visual cortex (42). Conditional knock out of Notch1 in the postnatal mouse hippocampus results in a decrease in mushroom spine density and an increase in thin spine density without changing the gross dendritic morphology in the hippocampal CA1 (28). Moreover, the activity-regulated protein ARC/ARG3.1 stimulates Notch1 signalling via presynaptic Jagged1 and somatodendritic Notch1 and is required for the induction of both long-term potentiation and long-term depression (28). In the present study, we found that activity-dependent Notch signalling occurred in the SON and PVN of the adult mouse. First, the expression level of DLL4 was significantly decreased by chronic salt loading. Second, the expression level of NTM3 and NICD3 was increased by chronic and acute osmotic stimulation. It is shown that Notch ligands are capable of binding with all Notch molecules (43,44). The interaction between DLL4 of endothelial cell and Notch3 of tumour cells promotes the survival and growth of tumour cells (45). The binding of Notch ligand results in a cleavage of Notch outside the membrane mediated by metalloprotease and then cleavage of the intracellular domain of Notch by c-secretase complex to release NICD (23,40). The endocytosis of Notch ligands promotes the proteolysis of Notch and the release the NICD (46,47). Notch ligand is endocytotically incorporated into the endosome of signal sending cells via ubiquitylation and epsin endocytic adaptors, with subsequent degradation and recycling (48,49). In the hypothalamic nuclei, the SON and the PVN, chronic physiological stimulation causes dynamic synaptic reconstruction that is accompanied by an increase in multiple synapse formation (1,2,50,51). It is reported that these synaptic reconstructions occur in both GABAergic and glutamatergic synapses (52,53). In the present study, the expression of DLL4 was observed at VGAD-positive inhibitory and GLUT1-positive excitatory axonal boutons. The immunohistochemistry of synaptotagmin-1 revealed that synaptotagmin-1 is localised at putative presynaptic boutons (54). Although the synaptotagmin antibody used in the present study recognises the cytoplasmic domain that is common in all 13 synaptotagmin isoforms (55), it should be emphasised that the synaptotagmin-immunoreactive puncta included putative presynaptic boutons. The cis-interaction of Notch receptor and Notch ligand controls the Notch signalling within the same cell (20). Dlk-1 and Journal of Neuroendocrinology, 2014, 26, 497–509

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-2 are able to interact with Notch and are proposed to block Notch signalling by binding to Notch and thereby preventing the binding of Notch ligand (56,57). Thus, it is possible that trans-interaction of DLL4 and Notch3 and cis-interaction of Dlk1 and Notch3 are possible Notch signalling pathway in the magnocellular neurones of adult hypothalamic nuclei. Several studies have reported that PSA-NCAM, F3 and tenascin-C are involved in the synaptic plasticity in the SON and PVN (1,6,9,10,58). Cell adhesion molecule F3 is shown to act as functional ligand for Notch in oligodendrocyte lineage cells (25). The expression of tenascin-C is reported to be regulated by Notch2 in a CBF-1/RBP Jk-dependent manner in glioma cells (26). The activation of Notch signalling induces the expression of NCAM in isolated Xenopus caps (24). Thus, it is possible that Notch signalling is responsible for controlling the expression of these cell adhesion and matrix molecules and thereby regulating synaptic reconstruction in the SON and PVN. Until now, no study has reported the expression Notch signalling molecules in the NH. In the present study, we found that DLL4 and Notch3 were expressed at axonal boutons against OXT-containing magnocelluar neurones and pituicytes, respectively. Moreover, DLL4positive axonal terminals were often localised in close proximity to neurohypophysial vasculature and pituicytes. Under unstimulated conditions, cellular processes of pituicytes intervene between the magnocellular terminals and the basal lamina of the capillary vessels, whereas pituicytes withdraw their cellular processes to allow the neurosecretary terminals of magnocellular neurones to contact directly with the vascular basement membrane in response to chronic physiological stimulation (15,16). The reconstruction of neuroglial and neurovascular structural interaction probably facilitates peptide release in the NH (1,16). Moreover, in the present study, chronic and acute osmotic stimulation significantly increased the levels of NTM3 and NICD3 in the NH. It is also demonstrated that PSA-NCAM, F3 and tenascin-C are possible candidates for neurovascular and/or neuroglial reconstruction in the NH (3,7,10,17). Notch signalling is shown to control the expression of NCAM, F3 and tenascin-C (24–26). Chronic osmotic stimulation increases the levels of plasma AVP and OXT (59) and AVP and OXT mRNA in the SON and PVN (60). Although the reason why the expression of DLL4 is limited to OXT-containing axonal terminals remains unknown, it is presumed that different Notch ligands are expressed at AVP-containing axonal terminals to discriminate from OXT-containing ones. Taken together, these results indicate that DLL4-Notch signalling is probably involved in the interaction between OXT-containing axonal terminals and pituicytes that leads to structural reconstruction in the NH.

Acknowledgements This work was supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 24500411 to S. Miyata). We are grateful to Dr H. Gainer for generous supplies of monoclonal antibodies against AVP- and OXT-neurophysin. Synaptotagmin antibody (mAB30) developed by Dr L. Reichardt and actin antibody (JLA20) developed by Dr J. J-C. Lin were obtained from the Developmental Studies Hybridoma Bank

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developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA, USA.

Received 28 November 2013, revised 24 May 2014, accepted 12 June 2014

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Supporting Information The following supplementary material is available: Fig. S1. Schematic illustration showing possible pathways of Notch signalling in the hypothalamic-neurohypophysial system.

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