Stress-induced dendritic internalization and nuclear translocation of the neurokinin-3 (NK3) receptor in vasopressinergic profiles of the rat paraventricular nucleus of the hypothalamus

brain research 1590 (2014) 31–44

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Stress-induced dendritic internalization and nuclear translocation of the neurokinin-3 (NK3) receptor in vasopressinergic profiles of the rat paraventricular nucleus of the hypothalamus Zachary Miklosa, Francis W. Flynnb, Andre´e Lessarda,n a

Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, MD 21228, USA Department of Zoology and Physiology, and Graduate Neuroscience Program, University of Wyoming, Laramie, WY 82072, USA

b

art i cle i nfo

ab st rac t

Article history:

Central neuronal circuits that relay stress information include vasopressin- (AVP) and oxytocin-

Accepted 18 September 2014

(OC) containing neurons of the paraventricular nucleus of the hypothalamus (PVN). These

Available online 2 October 2014

neurons are potentially modulated by neurokinin-3 receptors (NK3Rs) of the tachykinin family of neuropeptides. NK3Rs have been localized in PVN neurons and have showed nuclear

Keywords:

translocation following an osmotic challenge in rodents. However, their subcellular distribution

Peptide receptors

in AVP or OC neurons of the PVN and plasticity following restraint stress in rats are unknown.

Tachykinin

In the present study, densities of NK3Rs in PVN AVP- or OC-labeled somatodendritic profiles

Hypothalamus

were measured by quantitative immunoelectron microscopy in control or stressed rats.

Substance P

In resting conditions, NK3Rs were predominantly located in AVP neurons, however sparsely

Anxiety

distributed in OC neurons of the PVN. All NK3-labeled somata of the PVN in control rats showed cytoplasmic but no nuclear immunolabeling. An acute restraint stress session of 30 min significantly increased nuclear NK3R density in AVP-labeled somata but not in OC-labeled somata. These changes were accompanied by a respective decrease and increase in plasmalemmal and cytoplamic NK3R densities in AVP-labeled but not in OC-labeled dendrites. The results of this study suggest that in the rat PVN 1) NK3R distribution is conducive to modulation of systemic and/or central AVP release through PVN inputs to the posterior pituitary and/or the amygdala and 2) acute restraint stress activates (internalizes) NK3Rs on surface and evokes

Abbreviations: ACTH, receptor 1b; CRF, ME,

adenocorticotropin hormone; AVP,

corticotropin-releasing factor; GPCRs,

median eminence; NK3R,

vasopressin; Avpr 1a,

vasopressin receptor 1a; Avpr 1b,

G protein-coupled receptors; HPA,

neurokinin-3 receptor; NK3Rs,

neurokinin-3 receptors; NLS,

vasopressin

hypothalamic-pituitary-adrenal axis; nuclear localization signal;

OC, oxytocin; PB, phosphate buffer; PVN, paraventricular nucleus of the hypothalamus n Corresponding author at: Department of Psychiatry, Maryland Psychiatric Research Center, University of Maryland School of Medicine, Maple and Locust Streets, Baltimore, MD 21228, USA. Fax: þ1 410 402 6066. E-mail address: [email protected] (A. Lessard). http://dx.doi.org/10.1016/j.brainres.2014.09.043 0006-8993/& 2014 Elsevier B.V. All rights reserved.

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nuclear NK3R translocation exclusively in AVP neurons. This trafficking might contribute to neurochemical imbalances observed in neuronal circuits involved in stress-related disorders such as anxiety. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

The paraventricular nucleus of the hypothalamus (PVN) is a key component of the cardiovascular and behavioral response to stress through its unique connections to the pituitary, the autonomic nervous system as well as other stress-related areas of the central nervous system (Carrasco and Van de Kar, 2003). Indeed, the PVN represents the main site synthesizing stressrelated hormones such as vasopressin (AVP) and oxytocin (OC) that are released in circulation through the posterior pituitary of the neurohypophyseal system (Holmes et al., 1986; Wotjak et al., 2002). This stress circuitry, along with the release of the adenocorticotropin hormone (ACTH) and corticotropin-releasing factor (CRF) through the hypothalamic-pituitary-adrenal (HPA) axis and the median eminence (ME), is contributing to the neuroendocrine response to stress (Carrasco and Van de Kar, 2003). Catecholaminergic pathways from the PVN to the brainstem allow a fast sympathetic response that corresponds to the autonomic response to stress (Ziegler and Herman, 2002; Carrasco and Van de Kar, 2003). In addition, however, much less investigated AVP- and OC-containing PVN inputs to the central amygdala might rather be involved in the behavioral/emotional response to stress (Meyer-Lindenberg et al., 2011). In addition to AVP and OC, the activity of PVN neurons is modulated by numerous neuropeptides and G protein-coupled receptors (GPCRs). Among these, the neurokinin-3 receptor (NK3R) of the tachykinin family of neuropeptides has been localized in the rat PVN by autoradiography, in situ hybridization and light microscopy; where some of the labeling was observed in vasopressinergic profiles (Eguchi et al., 1996; Shughrue et al., 1996; Ding et al., 1999, 2000). Pharmacological studies conducted in rats showed that central injection of NK3R agonists in the PVN (1) activate PVN neurons (Ding et al., 2000; Smith and Flynn, 2000) and, (2) increase the release of AVP from the posterior pituitary, leading to cardiovascular (increase in blood pressure) and anti-diuretic effects (Polidori et al., 1989; Nakayama et al., 1992; Eguchi et al., 1996). Conversely, hyperosmolarity evokes internalization of PVN neurokinin-3 receptors (NK3Rs), confirming their potential role in osmotic balances through the release of AVP (Haley and Flynn, 2006). Interestingly, an osmotic challenge also evoked nuclear translocation of PVN NK3Rs (Jensen et al., 2008). The association of nuclear NK3Rs with acetylated histones in hypothalamic neurons (Flynn et al., 2011) is highlighting a new signaling pathway by which NK3Rs might change gene transcription in AVP or other phenotypes of PVN neurons. While the involvement of PVN NK3Rs in the release of AVP from the pituitary gland is well known, their potential role in the behavioral and emotional response to stress is poorly understood. In the present study, the subcellular distribution of NK3Rs in AVP- and OC- PVN neurons was examined in resting conditions, and compared to rats subjected to a 30 min session of

restraint. In control animals, NK3R immunoreactivity was predominant in AVP profiles of the PVN, however weak in OClabeled PVN profiles. The NK3-immunolabeling was mainly observed in plasmalemmal and cytoplasmic portions of somatodendritic profiles, and absent in the neuronal nuclei. Restraint stress induced a respective decrease and increase in plasmalemmal and cytoplasmic NK3R densities in AVP dendrites; a plasticity that was accompanied by a significant increase in nuclear NK3R density. The stress-evoked NK3R trafficking was not significant in non-AVP, non-OC and OC-labeled PVN profiles. The results suggest that similarly to a physiological stress such as hyperosmolarity, a mental stress in rats also evokes internalization and nuclear translocation of NK3Rs in AVP neurons of the PVN. These evidences potentially involve NK3Rs of the PVN in the pathological neurochemical imbalances leading to stress-related disorders such as anxiety.

2.

Results

2.1. Light microscopic examination of NK3 receptors in the paraventricular nucleus of the hypothalamus Single light microscopic immunodetection of NK3Rs in three normal Sprague-Dawley rats was achieved in PVN sections incubated with a polyclonal anti-sheep antiserum provided by the laboratory of Dr. Francis W. Flynn from the University of Wyoming. In a previous study also conducted in normal rats, we used a commercial polyclonal rabbit anti-NK3R antiserum in PVN sections and examined the tissue by light microscopy. A specific immunolabeling was seen in the magnocellular and parvocellular portions of the PVN, while no labeling was observed in the neuronal nuclei (Misono and Lessard, 2012). The polyclonal antisheep NK3R antiserum used in the present study produced a similar NK3R immunolabeling in rat PVN sections processed for single detection of immunoperoxidase (Fig. 1). The NK3R immunolabeling is concentrated in the magnocellular part of the PVN and minimal detection is observed in areas surrounding the PVN; suggesting a minimal background immunolabeling evoked by the NK3 antiserum. This distribution is also consistent with additional reports, one of which is using the same polyclonal sheep NK3R antiserum, showing dense NK3R immunoreactivity in the magnocellular portion of the PVN in normal rats (Flynn et al., 2011).

2.2. Electron microscopic distribution of NK3 receptors in PVN profiles with or without AVP or OC immunolabeling in control rats In sections processed for electron microscopy (NK3Rs immunogold; OC and AVP immunoperoxidase in five Sprague-Dawley

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Fig. 1 – Modified picture from a rat brain atlas (Paxinos and Watson, 2007), at 1.92 mm posterior to the Bregma (left) and light micrograph (right) showing a section of the paraventricular nucleus of the hypothalamus (PVN; delimited by the dashed line) single immunolabeled for the NK3 receptor. Immunoperoxidase labeling is prominently located in the magnocellular portion of the PVN, while areas in the vicinity of the PVN show weak immunolabeling. 3 V; 3rd ventricle. Scale: 0.5 mm.

rats), NK3 immunogold particles were prominently located in somatodendritic profiles of the rat PVN. Occasionally, NK3R immunolabeling was found on an isolated axon terminal or microglia in proximity of a dendrite or soma endowed with many NK3 immunogold particles (see an NK3-labeled axon terminal in Fig. 9B). As compared to profiles immunolabeled with OC which were often devoid of any NK3 immunogold particles, the vast majority of AVP-labeled profiles also showed NK3 immunolabeling. Within a total sample area of 17,390 mm2 that covered both magnocellular and parvocellular portions of the PVN in five normal rats (4608 mm2; 2505 mm2; 3971 mm2;

2612 mm2; 3694 mm2), only 12% of NK3-labeled somatodendritic profiles also contained immunoperoxidase reaction product for OC (n¼11 profiles double-labeled with NK3 and OC in a total of 87 NK3-labeled somatodendritic profiles). In contrast, PVN sections processed for NK3R (immunogold) and AVP (immunoperoxidase) showed a high level of co-localization (85%; n¼147 profiles double-labeled with NK3 and AVPþ in a total of 174 NK3-labeled somatodendritic profiles). Nonetheless, OC-labeled profiles (cell bodies or dendrites) containing NK3 immunogold particles, when found, expressed a similar density of NK3R as compared to AVPlabeled somatodendritic profiles (data not shown).

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2.3. NK3 receptor distribution and stress-evoked trafficking in AVP, but not OC somata of the PVN Electron microscopic examination and quantification of NK3R immunogold particles was performed in PVN AVP- or OC-soma

of control rats, and compared in rats receiving acute restraint stress (single session of 30 min in a restrainer). Ultrastructural examination of sections dual immunolabeled for NK3R and AVP revealed a high concentration of NK3 immunogold particles in cytoplasmic portions of AVP-labeled somata (Fig. 2A), and

Fig. 2 – Electron micrographs showing somatodendritic profiles in PVN sections dual immunolabeled for NK3R (immunogold) and AVP (immunoperoxidase) in control rats. (A) NK3 immunogold particles (encircled) were largely seen in cytoplasmic portions of somata immunolabeled with AVP (AVPþSoma); near organelles such as mitochondria (m) and rough endoplasmic reticulum (rer) and seldom observed in the neuronal nuclei (1 particle showed by a square) or nucleolus (ncl). Many unlabeled dendrites (ud) are located in the vicinity. (B) Many somata without AVP immunolabeling were also devoid of NK3 immunogold particles. The unlabeled soma is beside a dendrite containing immunoperoxidase reaction product for AVP (AVP d) and NK3 immunogold particles (encircled). The AVP-labeled dendrite is in contact with an unlabeled axon terminal (ut). Many unlabeled dendrites (ud) are present in the vicinity. Scale bars¼ 2.0 lm.

Fig. 3 – Electron micrographs showing somata in PVN sections dual immunolabeled for NK3R (immunogold) and OC (immunoperoxidase) in control rats. (A) Most of OC-labeled somata were devoid of NK3 immunogold particles. The OC-labeled soma is beside a dendrite containing many NK3 immunogold particles (encircled; NK3 d). (B) NK3 immunogold particles (encircled) were prominent in cytoplasmic portions of somata without immunoperoxidase labeling for OC; often near mitochondria (m) or Golgi apparatus (Go) but seldom seen in the neuronal nuclei (one immunogold particle is showed by the square). Scale bars¼2.0 lm.

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Fig. 4 – Electron micrographs showing somatodendritic profiles in PVN sections dual immunolabeled for NK3R (immunogold) and AVP (immunoperoxidase) in control (A) and stressed (B) rats. (A) In control animals, NK3 immunogold particles are almost exclusively located in cytoplasmic portions of somata with AVP-immunoperoxidase labeling (encircled) and seldom in the neuronal nuclei (square). (B) AVP-labeled somata from rats receiving an acute restraint session contained many NK3 immunogold particles in the neuronal nuclei (squares) as well as in the cytoplasm (encircled). The nuclear NK3 immunolabeling was usually at distance from the nuclear membrane, and rarely inside the nucleolus (ncl). Scale bars¼ 2.0 lm.

Fig. 5 – Electron micrographs showing somatodendritic profiles in PVN sections dual immunolabeled for NK3R (immunogold) and OC (immunoperoxidase) in control (A) and stressed (B) rats. (A) In control conditions, the NK3 immunogold particles in a soma without OC immunoperoxidase labeling are largely located in the cytosol (encircled) and rarely in the neuronal nuclei (square). (B) In rats receiving an acute restraint session, the NK3 immunogold particles were seen in the cytoplasm (encircled) and in the neuronal nuclei (squares) of a soma without OC immunoperoxidase reaction product. A dendrite (d) endowed with NK3 immunogold particles (encircled) is located in the vicinity. Scale bars ¼2.0 lm.

sparse or no immunolabeling in somata without AVP immunoperoxidase labeling (Fig. 2B). The NK3 immunogold particles in AVP somata were located at proximity to organelles such as the rough endoplasmic reticulum (rer) and mitochondria (m) and rarely on the plasma membrane (Fig. 2A). Nuclear portions of all somata, which include the nucleolus (ncl), were generally devoid of any NK3 immunogold particles (Fig. 2A). The nuclear labeling, when observed, usually consisted of one or few immunogold particles distributed randomly which suggests a

minimal background labeling from the antiserum (Fig. 2A and B). These somata dual immunolabeled for NK3R and AVP were surrounded by many unlabeled dendrites and terminals, which also suggests a minimal background produced by the NK3R antiserum (Figs. 2–4). In contrast, PVN sections processed for NK3R and OC were often single immunolabeled with OC (Fig. 3A) or NK3R (Fig. 3B). As compared to controls (Fig. 4A), PVN sections from rats receiving acute restraint stress showed many NK3 immunogold

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particles in the neuronal nuclei of AVP somata (Fig. 4B). A slight increase in nuclear NK3R was also observed in somata without OC immunoperoxidase labeling (Fig. 5), as well as NK3-labeled somata with OC or without AVP immunoperoxidase labeling (data not shown). The proportion of NK3R co-localization with OC and AVP in PVN neurons from stressed rats remained similar to control rats. Indeed, NK3-labeled profiles from stressed animals had a 14% co-localization with OC (n¼19 in a total of 127 somata; 12% in control animals) and a 77%

co-localization with AVP (n¼126 in a total of 164 soma; 85% in controls). Quantification of NK3 immunogold particles in cytoplasmic and nuclear portions of somata revealed a significant increase in nuclear density of NK3R in AVP-labeled somata of stressed rats, as compared to controls (Fig. 6). However, these changes were not significant in somata devoid of immunolabeling for OC (Fig. 6) or AVP, as well as OC-labeled somata (data not shown). In addition, the cytoplasmic NK3R density

Fig. 6 – Bar graphs showing nuclear and cytoplasmic densities of NK3 immunogold particles in PVN somata dual immunolabeled for AVP (AVP þ) or without labeling for OC (OC ). Data were obtained from at least 12 vibratome sections that covered a minimal area of 4000 lm2 each. Vertical bars represent the mean7S.E. mean in the ratio of the number of gold particles in the nucleus or cytoplasm/area of the somata (100 lm2) in profiles containing AVP-immunolabeling or devoid of OC immunolabeling. Numbers in bars represent the number of profiles measured in each category. Statistical comparisons were made between control- (N¼5 rats) and stressed rats (N¼ 7 rats). Significant differences were determined using an unpaired Student t-test. nnPo0.01.

Fig. 7 – Electron micrographs of dendrites from PVN sections dual immunolabeled for the NK3R (immunogold) and AVP (immunoperoxidase) in control (A) and stressed (B) rats. (A) Two AVP-labeled dendrites (AVP d) from a PVN section of a control rat also contain NK3 immunogold particles located in the cytosol (arrows) or in contact with the plasma membrane (encircled). One of the dendrite receives contact from an unlabeled axon terminal (ut). Many unlabeled dendrites (ud) are located in the vicinity. (B) Two AVP-labeled dendrites (AVP d) from a PVN section of a stressed rat contain NK3 immunogold particles that are prominently located in the cytosol (arrows) except from one in contact with the plasma membrane (encircled). Both dendrites receive inputs from unlabeled axon terminals (ut). Scale bars ¼0.5 lm.

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in all types of somata was not significantly altered in stressed rats, as compare to controls (Fig. 6).

2.4. NK3 receptor distribution and stress-evoked trafficking in AVP, but not OC dendrites of the PVN Electron microscopic examination and quantification of NK3R immunogold particles was performed in PVN AVP- or OCdendrites of five control rats, and compared in five rats receiving acute restraint stress (single session of 30 min in a restrainer). As observed with somata, the NK3 immunogold particles in PVN sections processed for NK3R and AVP were prominently located in AVP-labeled dendrites, and less often in non-AVP dendrites (Fig. 7A). In AVP-labeled dendrites, NK3 gold particles were evident in the cytosol, but many of them also were in contact with the plasma membrane (Fig. 7A). These plasmalemmal NK3 particles were mostly located at extrasynaptic portions of the plasma membrane of AVP and non-AVP dendritic profiles. In stressed animals, a qualitative decrease in plasmalemmal NK3 immunogold particles was seen, as compared to control animals (Fig. 7B). A quantitative analysis of NK3 gold particles was performed in all NK3-labeled dendrites examined, those of which had cross-sectional diameter ranging from 0.3 to 1.99 mm. The sample size for very large proximal dendrites (cross-sectional diameter larger than 2 mm) was too small (3% of all NK3–

labeled dendrites) to obtain accurate statistical outcome and were pooled in the analysis (n¼ 13 proximal dendrites in a total sample of 373 dendrites). Acute restraint stress induced a significant decrease in plasmalemmal NK3R density that was accompanied by a significant increase in cytoplasmic NK3R density in AVP-labeled profiles (Fig. 8). This stressevoked trafficking was also observed when measuring the percent plasmalemmal (%) NK3R, where AVP-labeled dendrites from stressed rats showed a significant decrease in percent plasmalemmal NK3R, as compared to controls (Table 1). These changes were not significant in NK3-labeled dendrites devoid of immunoperoxidase reaction product for AVP (Fig. 8; Table 1). In PVN sections processed for NK3R and OC, the NK3R immunolabeling was prominent in non-OC dendrites (Fig. 9A). While some NK3 gold particles were found in the cytosol, many of them were in contact with extrasynaptic portions of the plasma membrane (Fig. 9A). The subcellular distribution of NK3R in non-OC dendrites did not show qualitative difference in stressed rats, as compared to controls (Fig. 9B). The subcellular distribution of NK3Rs in dendrites containing OC immunolabeling was weak and also showed no apparent changes in stressed and control rats (Fig. 9C and D). Quantitative analysis of NK3R immunogold particles confirmed that in PVN dendrites processed with NK3R and OC,

Fig. 8 – Bar graphs showing the plasmalemmal and cytoplasmic densities of NK3 immunogold particles in PVN dendrites with (AVP þ) or without (AVP  ) immunoperoxidase labeling for AVP in control and stressed rats. Data were obtained from at least 9 vibratome sections that covered a minimal area of 1000 lm2. Vertical bars represent the mean7S.E. mean in the ratio of the number of gold particles in contact with the plasma membrane/ profile perimeter and the total number of gold particles/area of the profile (100 lm2). Numbers in bars represent the number of profiles measured in each category. Statistical comparisons were made between control- (N¼4 rats) and stressed- (N¼ 5 rats) rats in all profiles. Significant differences were determined using an unpaired Student t-test. nPo0.05; nnPo0.01.

Table 1 – Plasmalemmal NK3 immunogold distribution (%) in dendrites categorized by phenotype in control and stressed rats. Dendrite phenotype

% Plasmalemmal control (n ¼5 rats)

% Plasmalemmal stress (n ¼5 rats)

AVPþ AVP  OC  OCþ

31.573.5 (n ¼ 77) 24.776.9 (n ¼ 20) 22.273.5 (n ¼ 66) 27710 (n ¼10)

5.871.8 19.274.3 1772.4 2675.5

Mean number of gold particles in contact with the plasma membrane/total NK3 gold particles in PVN dendrites. Po0.01, Chi-Square test in between control and stressed animals.

nn

(n ¼81)nn (n ¼36) (n ¼88) (n ¼13)

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Fig. 9 – Electron micrographs of PVN dendrites showing immunolabeling for the NK3R (immunogold) without ((A), (B)) or with ((C), (D)) immunolabeling for OC (immunoperoxidase) in control ((A), (C)) and stressed ((B), (D)) rats. ((A), (B)) NK3 immunogold particles are prominent in the cytosol (arrows) and on the plasma membrane (encircled) in dendrites devoid of OC labeling (NK3 D). The plasmalemmal to cytoplasmic distribution of NK3 immunogold particles seem similar in sections from control (A) and stressed (B) rats. An axon terminal endowed with NK3 immunogold particles (NK3 t) is located in the vicinity. ((C) and (D)) Plasmalemmal (encircled) and cytoplasmic (arrows) NK3 immunogold particles were occasionally found in dendrites showing immunoperoxidase reaction product for OC (OC d). No qualitative difference in the subcellular distribution of NK3R in OC labeled dendrites was observed between control (C) and stressed rats (D). ut, unlabeled axon terminal; ud, unlabeled dendrite. Scale bars¼0.5 lm.

the plasmalemmal and cytoplasmic distribution and density of NK3Rs was unaltered by acute restraint stress (data not shown). Moreover, no significant changes were observed in the percent plasmalemmal (%) distribution of NK3R in OC and non-OC dendrites of control and stressed rats (Table 1).

3.

Discussion

Results from the present study are schematically described in Fig. 10. In normal conditions, NK3Rs were prominently located in AVP somatodendritic profiles of the PVN, and rarely in OC-labeled profiles. An acute restraint stress session evoked 1) a significant increase in the nuclear density of NK3Rs in AVP neurons of the PVN and 2) a respective decrease and increase of NK3R densities in plasmalemmal and cytoplasmic portions of AVP dendrites of the PVN. The stressevoked trafficking, which corresponds to a nuclear translocation and dendritic internalization of NK3Rs, was significant in AVP-labeled but not in OC-labeled somatodendritic profiles of

the PVN. These results suggest that acute restraint stress activates NK3Rs selectively in AVP neurons of the PVN; most likely leading to systemic AVP release from the posterior pituitary. Alternatively, central AVP pathways relaying emotional stress information to the amygdala might also be recruited in response to restraint stress. We believe that our results have implications in understanding the unique role of NK3Rs in the modulation of AVP release and the consequences on pathological stress.

3.1. Subcellular distribution of NK3 receptors in PVN neurons of normal rats The predominant location of NK3Rs in AVP neurons of the PVN in normal rats is consistent with previous light microscopic immunohistochemical and pharmacological studies (Polidori et al., 1989; Saigo et al., 1993; Eguchi et al., 1996; Hatae et al., 2001; Spitznagel et al., 2001). Although our samples for electron microscopy included parvocellular and magnocellular portions of the PVN, the NK3R immunolabeling

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Fig. 10 – Schematic representation of stress-induced trafficking of the NK3 receptor in vasopressin neurons of the PVN. By quantitative electron microscopy, we observed a significant trafficking of NK3Rs in vasopressin neurons of the PVN following an acute restraint stress in rats. This plasticity is not observed in non-vasopressin as well as all profiles in sections processed for oxytocin. The respective decrease and increase in plasmalemmal and cytoplamic densities of NK3Rs suggest that restraint stress internalize NK3Rs in AVP dendrites. The significant increase in nuclear NK3R density suggests a stress-evoked nuclear translocation of NK3Rs only in AVP neurons of the PVN. We hypothesize that this trafficking is contributing to the stress-induced systemic AVP release through the posterior pituitary, but also to a central activation of AVP pathways proving inputs to the amygdala.

was concentrated in the magnocellular portion innervating the posterior pituitary and the central amygdala (Ziegler and Herman, 2002; Huber et al., 2005). Consistently, central injection of selective NK3R agonists or antagonists increased or decreased systemic AVP release through the neurohypophyseal system; an effect that had consequences on osmotic balance and renal function (Yuan and Couture, 1997; Ding et al., 1999; Haley and Flynn, 2006). While the involvement of PVN NK3Rs in the neuroendocrine response to stress as well as in the water/electrolyte homeostasis is compelling, their role in modulating central circuits relaying emotional information to other cortical or subcortical areas remains poorly understood. In addition to their direct connection to the pituitary, PVN neurons expressing AVP or OC also relay stress information to other subcortical areas such as the amygdala (Neumann et al., 2000; Ziegler and Herman, 2002; Herman et al., 2005; Huber et al., 2005; Meyer-Lindenberg et al., 2011). This PVN-amygdala circuitry has been associated with social bonding, emotional stress, depression and anxiety (Nair and Young, 2006; Meyer-Lindenberg et al., 2011; Stevenson and Caldwell, 2012). In future studies, we sough to identify by tract tracing which of these AVP pathways predominantly express NK3Rs.

3.2. Stress-induced nuclear translocation of NK3 receptor in AVP PVN neurons Traditionally, NK3Rs are internalized from the surface following ligand binding, where they activate neurons through the phospholipase C signaling pathway leading to intracellular calcium release. The internalized receptor is then recycled back to the membrane or degraded in the cytoplasm by

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endopeptidases (Regoli et al., 1994; Khawaja and Rogers, 1996). Moreover, our group and others have located NK3Rs in the neuronal nuclei in the rat PVN following physiological challenges and in the ventral tegmental area (VTA) of normal rats (Howe et al., 2004; Jensen et al., 2008; Lessard et al., 2009). NK3Rs now belong to a sub-group of nuclear GPCRs, along with the apelin, angiotensin AT1 and bradykinin B2 receptors but not the neurokinin-1 or neurokinin-2 receptor (Lee et al., 2004; Gobeil et al., 2006). In the present study, NK3R immunolabeling from nonstressed animals was abundant in cytoplasmic portions of cell bodies, however sparse or absent in the neuronal nuclei. This is consistent with previous studies showing no or weak nuclear NK3R labeling in PVN neurons of normal animals (Jensen et al., 2008; Misono and Lessard, 2012). In contrast, our results showed that an acute restraint stress session increased the nuclear NK3R density in AVP neurons of the PVN. The effect was similar to the reported nuclear translocation of NK3Rs following physiological challenges (hypotension or hyperosmolarity) in the rat hypothalamus (Howe et al., 2004; Jensen et al., 2008). NK3Rs are crossing the nuclear pore through their putative nuclear localization signal motif (NLS) sequence that binds to the carrier proteins importins (Howe et al., 2004; Lee et al., 2004; Chahine and Pierce, 2009; Jensen et al., 2010). In the rat hypothalamus, microinjection of the selective NK3R agonist senktide evoked internalization and nuclear translocation of NK3Rs, both of which were prevented by prior injection of the selective the NK3R antagonist SB222200 (Howe et al., 2004; Sladek et al., 2011). The later result suggests that part of the nuclear translocation of NK3Rs in the PVN occur through internalized NK3Rs from the surface. Nevertheless, this mechanism cannot be accounted in all brain areas since it has been previously shown that VTA microinjection of SB222200 prevented an apomorphineevoked NK3R internalization but not nuclear translocation (Hether et al., 2013). Therefore, the apomorphine-evoked nuclear translocation of NK3Rs in the VTA involves trafficking of NK3Rs from the cytoplasmic reserve rather than surface receptors. This hypothesis goes along with other studies, where nuclear translocation of the fibroblast growth factor receptor-1 (FGFR-1) results from trafficking of both surface and cytosolic receptors (Maher, 1996; Stachowiak et al., 1996). Additional investigations are needed to reveal the mechanism involved in stress-evoked nuclear translocation of NK3Rs in the PVN and their functional consequences in gene transcription or behavioral activity.

3.3. Stress-induced trafficking of NK3 receptor in AVP but not OC PVN neurons: Involvement in circuits relaying emotional information Quantitative electron microscopy is a valid method to measure trafficking of receptors in specific neuronal compartments, as demonstrated many times by our team and others (Lessard and Pickel, 2005; Hara and Pickel, 2007; Lane et al., 2008; Mengual et al., 2008; Misono and Lessard, 2012). In the present study, restraint stress increased the nuclear but not the cytoplasmic NK3R density in AVP cell bodies. These differences likely reflect a general NK3R plasticity from dendrites toward the soma, where cytoplasmic portions of

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the soma would receive NK3Rs from proximal dendrites but also lose some ongoing nuclear translocation. In dendrites, the stress-evoked decrease in plasmalemmal NK3R density was accompanied by a significant increase in cytoplasmic NK3R density. Together, this plasticity suggests a stressinduced internalization of dendritic NK3Rs. This trafficking was significant in AVP profiles, but not significant in profiles expressing OC or profiles without AVP or OC immunolabeling. The possibility remains that NK3Rs in other phenotype of PVN neurons might internalize if identified by immunocytochemistry. These include CRF local neurons providing inputs to the basolateral amygdala (Swanson et al., 1983; Liposits et al., 1985). Nonetheless, this possibility is unlikely since anatomical and pharmacological studies showed CRF immunolabeling in the parvocellular subdivision of the PVN; a region rather involved in the regulation of the HPA axis. Moreover, the CRF phenotype in the PVN is associated with activation of the neurokinin-1 receptor (Jessop et al., 2000; Ebner and Singewald, 2006; Culman et al., 2010). Alternatively, NK3Rs might be localized in GABA interneurons and glutamatergic interneurons that are expressed in the PVN (Tasker and Dudek, 1993; Daftary et al., 1998; Tasker et al., 1998). Although we cannot exclude this possibility, reports that restraint stress activates prominently magnocellular neurons of the PVN (Cullinan et al., 1995) that largely express AVP and NK3Rs suggest that AVP represent most likely the phenotype that is regulated by NK3Rs in the PVN.

3.4.

Conclusion

The adaptive stress response is crucial for survival; nevertheless it becomes deleterious in pathological stress, where anxiety behaviors are triggered by exposure to non-threatening stimuli (LeDoux, 2000). The PVN plays a pivotal role in the central and endocrinal response to stress; both of which involve central and systemic release of AVP. Central AVP, acting on their AVP 1a or AVP 1b receptors (Avpr 1a; Avpr 1b) in the PVN and amygdala contribute to the regulation of social behavior and stress (Hammock and Young, 2006; Young et al., 2006; Wersinger et al., 2007; Stevenson and Caldwell, 2012). Furthermore, Avpr 1b antagonists showed promising results against anxiety in clinical trials (Griebel et al., 2002). Unfortunately, subsequent results came up disappointing (Griebel et al., 2012). On their side, the expression of NK3Rs in human hypothalamus (Chawla et al., 1997) as well as anxiolytic effects following central injection of NK3R agonists in rats and mice (Ribeiro et al., 1999; Schable et al., 2011) target NK3R-related drugs as potential new treatment against stress-related disorders. A better understanding of the neurochemical imbalance in stress-related disorders will help in optimizing the search for new drug combinations that might include NK3R- and AVP-related drugs.

4.

Experimental procedures

4.1.

Animals

Male Sprague-Dawley rats (280–350 g) were purchased 14 days prior to their experimental use (Charles River Laboratories, Kingston NY). All animals (n¼ 12) were housed two per cage

in a colony room under a 12 h light/dark cycle. Food and water were available ad libitum. Each rat was used for only one experiment. All experiments were conducted in accordance with the NIH regulations of animal care and were approved by the Institutional Animal Care and Use Committee of University of Maryland School of Medicine. To avoid the spurious effect of a stressful stimulus by manipulation of the animals, all rats were handled for 10 min daily starting one week before the experiment. On the day of the experiment, rats from the stress group spent 30 min in the restrainer while control rats remained in their cages. The 9.5  3.8 cm acrylic half-cylinder’s shape restrainer has a flat bottom and rounded ventilation holes on the bottom and top portions. The animals were anesthetized 60 min after the procedure for perfusion-fixation of their brain tissue.

4.2.

Antisera

The NK3R localization was achieved by using a sheep polyclonal antiserum generously provided by Dr. Francis W. Flynn from the University of Wyoming. This antiserum is directed against the second extracellular loop of the NK3R; targeting amino acid sequences 220–232. The antiserum was tested for specificity using techniques of siRNA, co-immunoprecipitation and Western blot (Jensen et al., 2010). The antiserum showed a band around 60–75 kDa; a molecular weight corresponding to the NK3R (Jensen et al., 2008; Flynn et al., 2011; Sladek et al., 2011). As compared to the NK3R immunolabeling produced by a commercial NK3R antiserum (Misono and Lessard, 2012), the present anti-sheep NK3R antiserum produced a similar distribution in PVN somatodendritic profiles either by light (single immunoperoxidase) or electron microscopy (immunogold or immunoperoxidase). In addition, the NK3R antiserum produced no or minimal background in the neuronal nuclei of PVN neurons and areas in the vicinity of the PVN. The AVP or OC phenotypes were distinguished by using a mouse monoclonal anti-OC antibody and a rabbit polyclonal anti-AVP antibody, both of which were commercially obtained from Chemicon-Millipore (Billerica, MA, USA). Competitive ELISA assays revealed that the OC antiserum showed no cross-reactivity to AVP analogues (Liu et al., 2002). The AVP antiserum contains less than 1% cross-reactivity with OC (Simerly and Swanson, 1987; Markakis et al., 2004). In addition, pre-incubation of both antisera with excess OC and AVP abolished immunolabeling (Dabrowska et al., 2011). Thus, both OC and AVP antisera used in this study are well characterized and shown to have high specificities for their respective antigens.

4.3. Tissue preparation and electron microscopic single and dual immunolabeling Sixty minutes after the restraint stress session, rats were deeply anesthetized by an intraperitoneal (i.p.) injection of 1000–1500 mg/kg urethane. Control rats remained in their cages for at least 60 min in a quiet room before the anesthesia and perfusion procedure. The anesthetized animals were perfused through the aortic arch with 5–10 ml of 0.9% saline, 50 ml of 3.75% acrolein in 2% paraformaldehyde and 200 ml of 2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4.

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The brains were removed from the cranium and fixed with 2% paraformaldehyde for 30 min. Rostrocaudal brain sections (40 μm) were prepared using a Vibratome (Leica Microsystemss, Bannockburn, IL). PVN sections of tissue were then placed in 1% sodium borohydride for 30 min to neutralize reactive aldehydes as described in prior studies using acrolein fixation (Leranth and Pickel, 1989). The prepared sections were processed for dual immunogold silver and peroxidase labeling of antisera before plastic embedding (Chan et al., 1990). This approach was used to enable high-resolution detection of plasma membrane and intracellular distributions of receptors that can be lost during the process of plastic embedding (Adams et al., 2002). To minimize penetration problems inherent to the preembedding methodology, the sections were cryoprotected in 25% sucrose and 3.5% glycerol in PB, and then incubated 10 min at  80 C at decreasing concentrations of the cryoprotectant solution (100%; 70%; 50%; 30% and 0% or 100% PB). The freeze-thaw method produces minute holes in the tissue allowing greater penetration of immunoreagents. Sections used for single immunodetection of the NK3 receptor were not processed for freeze-thaw; however Triton X-100 (0.25%) was added in the primary antiserum solution. PVN sections for single (light microscopy) and dual (electron microscopy) immunolabeling were incubated for 24 h at room temperature. Sections processed for electron microscopy were incubated at 4 1C for another 12 h in a solution containing sheep anti-NK3 receptor (immunogold) with (dual) or without (single) rabbit anti-AVP or mouse anti-OC antisera (peroxidase) at dilutions of 1:500 (NK3), 1:1000 (AVP) and 1:5000 (OC). After this incubation, the sections were rinsed and placed for 30 min in biotinylated secondary horse antimouse IgG (1:400, Incstar) or goat anti-rabbit (1:400, Incstar) followed by the ABC complex (ABC; Vector, Burlingame, CA) for detection of the mouse OC or rabbit AVP antibody. The bound peroxidase was identified by reaction of the sections for 6 min in 3,30 -diaminobenzidine (Aldrich Chemicals, Milwaukee, WI) and hydrogen peroxide. Sections processed for light microscopy were mounted onto gelatin-coated slides, air dried, dehydrated through alcohols and xylenes, and then mounted beneath glass coverslips with DPX mounting medium (Aldrich). The slides were examined using a Zeiss light microscope (Jena, Germany), and images were captured using an Olympus DP70 digital camera (center Valley PA). The final images were imported to PowerPoint software (Microsoft Windows XPs) for assembly and labeling composite figures. Sections processed for electron microscopy were rinsed in Tris buffer (0.1 M, pH 7.6), and placed for 2 h in a 1:50 dilution of donkey anti-sheep IgG with bound 1 nm colloidal gold (Amersham, Arlington, IL) for detection of the sheep NK3 receptor antiserum. The gold particles were fixed to the tissue by incubation of the sections in 2% glutaraldehyde in 0.01 M phosphate buffered saline for 10 min. The particles were enlarged for microscopic examination by reaction in a silver solution from the IntenS-EM kit (Ted Pella, Redding CA) for 7 min at room temperature (see Chan et al., 1990). The sections were then postfixed in 2% osmium tetroxide in 0.1 M PB, dehydrated and flat-embedded in epon (19%EM Bed-812; 36% DDSA; 44% NMA; 1% BDMA; Electron Microscopy Sciences, Fort Washington, PA) between two pieces of Aclar

41

plastic (Electron Microscopy Sciences, Fort Washington, PA). To minimize between group variations in quantification of NK3 immunogold particles, PVN sections from control and stressed rats were co-processed under identical labeling conditions. Ultrathin sections from the outer surface of each Vibratome section in areas including the magnocellular and parvocellular portions of the PVN, at 1.92 mm posterior to the bregma (Paxinos and Watson, 2007), were collected onto grids by using an ultramicrotome (Nova, Bromma, Sweden). The sections on grids were counterstained with Reynold’s lead citrate and uranyl acetate. The thin sections were examined by using a Hitachi H7000 transmission electron microscope. Images were captured using an AMT digital camera, then imported to Photoshop software (Adobe Systems, Mountain View, CA), and adjusted for sharpness only. The final images were then imported to PowerPoint software (Microsoft Windows s) for assembly and labeling composite figures.

4.4.

Data analysis

To assess the distribution of NK3 immunogold particles, data analysis was performed on ultra-thin sections exclusively obtained from the surface (1–2 μm) of the flat-embedded tissue, where there was optimal penetration of immunoreagents. The profiles containing NK3 immunoreactivity were classified as either neuronal (dendrites, axon terminals) or glial based on well-established criteria (Peters et al., 1991). Peroxidase immunoreactive profiles had an electron density considerably greater than that seen in comparable structures in the surrounding neuropil that were considered unlabeled. To measure trafficking of NK3Rs, NK3-labeled somata and dendrites with or without AVP- or OC-immunolabeling were quantitatively analyzed in PVN sections co-processed in the same antisera solutions. The data analysis was performed by an investigator that was blind to the conditions. The profile diameter, area and perimeter were measured by using Image J software (National Institute of Health, JAVA 1.60_02). One to four vibratome sections per animal were examined, and each section generated at least 50 images of magnifications ranging from 7000  to 40,000  . A total area of 31,609 mm2 of the PVN was examined in five control (4608 mm2; 2505 mm2; 3971 mm2; 2612 mm2; 3694 mm2 for a total of 17,390 mm2) or five stressed (3306 mm2; 2967 mm2; 2765 mm2; 2038 mm2, 3143 mm2 for a total of 14,219 mm2) rats. Parameters used for statistical comparisons were (1) the number of gold particles in contact with the plasma membrane/perimeter of individual profile, (2) the number of gold particle not in contact with the plasma membrane/dendrite area, (3) the number of gold particles on the nucleus/ nucleus area and (4) the number of gold particles on somata excluding nucleus/area of somata without the nucleus. Data were also analyzed as percentage (%) of plasmalemmal NK3 immunogold particles/total number of NK3 immunogold particles in AVP, OC, non-AVP and non-OC dendrites. The plasmalemmal immunolabeling is defined as NK3 immunogold particles in direct contact with the plasma membrane (no distance allowed between the immunogold particle and the plasma membrane). Results are expressed as means7s.e. mean of (n) rats. Results were analyzed for statistical significance by

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an unpaired t-test (ratios 1–4) or Chi-Square (%) using SPSS software (Windowss Lead Technologies). Only probability values (P) less than 0.05 were considered to be statistically significant.

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