Time-course study of the effects of NEI on FSH, PRL and GH secretion accumulated in the culture media (ng/ml of culture medium). Pituitary cells were treated with NEI 100 or 400×10-8M for 1–5 h. The data are shown as the mean ±S.E.M. of three independent experiments and were evaluated by the ANOVA–Fisher test.
Neuropeptide- glutamic acid isoleucine (NEI) is a peptide related to reproduction. Although it also has an important function on behavior [2-6], we will focus here on the relationship between NEI and LH.
NEI is derived from the precursor pre- prohormone named pp melanin-concentrating hormone (pp-MCH). This precursor also gives rise to melanin concentrating hormone (MCH) and to neuropeptide-glycine-glutamic acid (NGE) [7-9].
Some studies have suggested a role of NGE at the level of the hypothalamus. For instance, NGE increases the number of neurofilaments and the production of synaptophysin in rat neurons within 18 days of development [10-12].
Immunoreactivity and mRNA expression of both MCH and NEI have been observed in certain regions of the central nervous system. The first region is the diencephalon including the rostralmedial part of the zona incerta, later refer to as the incerto-hypothalamic area (Ihy) by Sita et al. ; the three subdivisions (anterior, tuberal and posterior) of the lateral hypothalamus (LHA); the area between the dorsomedial and ventromedial nuclei of the hypothalamus, which Swanson  designated as the internuclear area, the anterior periventricular nucleus; and the dorso medial aspects of the tuberomammillary complex. The second region includes the olfatory tubercle, located in the basal forebrain. The third region includes the paramedian pontine reticular formation in the pons . It is important to note that the highest concentration of MCH/NEI ir cells is found in the Ihy and the LHA. We found nearly all the cells in these two regions were immureactive for both MCH and NEI (mean +-S.E.M, 96 +-3%) (Fig.1 and 2)
Peptides such as NGE, NEI and MCH are highly conserved among vertebrates, being abundant and widely distributed in the brain, suggesting that they could be performing important physiological functions. NEI is a 13 aminoacid peptide and has an extensive distribution in the central nervous system (CNS), acting as a neurotransmitter or neuromodulator [15-17]. Neurotranmitter NEI induces grooming behavior, locomotor activity, and stimulates sexual receptivity in female rats [1-6]. NEI injections can modify the levels of noradrenaline and dopamine in specific areas of the brain , and earlier studies demonstrated that MCH injection into the preoptic area or median eminence induces luteinizing hormone (LH) secretion. Subsequent studies have shown that modified circulating hormonal levels might modulate ppMCH neurons  It has been reported that treatment with 17β-estradiol increases MCH and NEI immunoreactivity throughout the entire diencephalon of ovariectomized cynomologous monkeys . Similar effects were observed in ovariectomized rats receiving no gonadal estradiol treatment . Levels of ppMCH mRNA are only increased in the medial zone incerta (ZI) , which houses the A13 group, a collection of dopaminergic neurons that have been previously demonstrated to play a stimulating role in gonadotropin release .
2. Ovarian steroids effects
Viale et al (1999) proposed a central role of the MCH/NEI neuronal system in the regulation of reproductive functions in rats . In fact, the MCH/NEI system of immunoreactive fibers and terminals that is encountered in hypothalamic areas such as the medial preoptic area (MPOA), is well known to be involved in the control of pre-ovulatory LH surge. These authors studied the effects of the treatment with estrogen on the immunoreactivity of NEI and MCH in OVX animals, using the non-human primate (M. Fasscicularis). A slight increase of MCH-IR after 30 days post treatment with estrogen, along with a concurrent significant rise in NEI-IR was observed. A three-fold increase in MCH and NEI –Ir was seen at 72 h post estrogen treatment, compared with the amount of both peptides-ir at 48h post treatment. These facts suggest the possible involvement of these peptides in the regulation of the pre-ovulatory mid- cycle LH surge in primates .
The MCH receptors are classified into five subtypes: MC1-R to MC5-R . MCH stimulates gonadotropin-releasing hormone (GnRH) release from hypothalamic explants, and it is interesting to note that MCH affects the release of LH  in the female rat. When MCH was injected bilaterally into the rostral preoptic area (rPOA) or medial preoptic area (mPOA) of estrogen-primed ovariectomized rats, LH release was stimulated. Two MCH receptors are involved in the MCH effect. The stimulatory action of MCH in the rPOA was inhibited by administration of antagonists for either MC-1 R or MC-5R, indicating that both ones, MC-1R and MC-5 are involved in the central control of GnRH release by MCH [19-27].
3. The effect of neuropeptide EI on LH regulation
For this study we used male and female rats, aged 10-14 weeks which were bred in our laboratory and maintained with food and water ad libitum, with a cycle of 14h/ light/10h dark and a temperature controlled environment (22±2ºC). The animal procedures were consistent with the standards established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996) and the AmericanVeterinarian Guidelines of Eutanasia.
The first evidence, on the effects of NEI on LH regulation was provided by Attademo et al , using male and ovariectomized rats treated with estrogen benzoate (10 ug) and low doses of progesterone (40ug). These animals revealed the following: when male rats were treated with intraventricular injections of NEI (1ug/1ul), the peptide induced an increase of serum LH concentration throughout the entire period studied (10-90 min). At 90 min the serum LH slightly decreased, possibly signaling initiation of the recovery of normal LH serum levels. Control rats injected with artificial cerebrospinal fluid showed practically no changes on serum LH concentrations (Fig. 3). It was also possible to see the NEI effect on ovariectomized female rats treated with estrogen plus progesterone, by using a low dose of progesterone to permit the visualization of modifications in the LH surge. Again, the neuropeptide increased LH release compared with control animals (Fig.4)
The fact that the effect of NEI may be mediated by the noradrenergic system must be taken into consideration. The peptide is known to modify DA and NA in the nucleus accumbens and caudate putamen during grooming behavior and locomotion activity . As NEI behaves similarly to α-MSH, it is important to note that there is some relation between MCH-NEI and α-MSH, indicating that all three peptides are associated in a complex inter-relationship .
4. Distribution of NEI immunoreactivity
In this study, we described the anatomical substrate underlying the NEI effect of inducing LH secretion, using techniques of double and triple label immunohitochemistry, as well as dual label immunofluorescence. A group of female rats were perfused on day 15 postovariectomy; a second group received10 µg of estradiol benzoate and were perfused two days later and a third group received 10 µg of estradiol benzoate and two days later 40 µg of progesterone and were perfused 5 h after treatment. To mimic the manipulation of the animals, we used a fourth group of ovariectomized rats treated with sesame oil, and also used female intact rats at proestrus and diestrus .
Using these techniques, we were able to obtain the following results:
NEI-ir neurons were observed in the medial ZI, in the perifornix at the tuberal hypothalamic level and in the lateral hypothalamus. Fibers were distributed throughout the forebrain, including areas related to reproductive control and LH secretion. We observed a dense number of NEI-ir fibers in the medial septal nucleus, the diagonal band of Broca, the environs of OVLT, the preoptic area and in the internal layer of the median eminence (Fig. 5).
All fibers seen in these areas show varicosities and terminal-like structures. NEI and terminal-like structures were in close apposition with portal blood vessels and GnRH neurons expressing Fos (Fig. 6 A and B)
5. NEI-ir fibers innervate GnRH and AVPV neurons expresing fos
We observed an increased expression of Fos immunoreactivity in the anteroventral periventricular nucleus (AVPV) neurons of rats perfused during the afternoon of the day of proestrus, as well as in ovariectomized rats treated with estradiol benzoate plus progesterone, as described by others [30, 31]. Close to a 10% of the AVPV neurons expressing Fos receive NEI fibers in close apposition. Very little Fos immunoreactivity was observed in rats perfused in the afternoon of the diestrus day, for either of the following conditions: ovariectomized treated with estradiol benzoate or ovariectomized treated with sesame oil. In addition, all groups, showed a sparse distribution of Fos immunoreactivity in the medial zone incerta and in the LHA, whereas co-localization of NEI fibers with any of these cells was not observed (Fig. 6 D).
It has been reported that Fos protein is expressed in a portion of GnRH neurons that are active in the afternoon of the proestrus day, as well as in the GnRH neurons of ovariectomized rats treated with estradiol benzoate plus progesterone [31-33]. Based on these results, we investigated the pattern of NEI innervations in areas that expressed GnRH neurons in these animals. Most of the neurons expressing Fos were found to be in the vicinity of the organum vasculosum of the lamina terminalis (OVLT) and of the preoptic area. NEI fibers were also found in the median eminence, and NEI and GnRH varicosities presented similar distribution, thus revealing close apposition between them. Immediately before and during LH surge (on the afternoon of the proestrus day or following treatment with estrogen plus progesterone), only a portion of the GnRH neurons expressed Fos [30,31,33,34-36].
AVPV, is a nucleus that also expresses Fos in the afternoon of the proestrus day and has been widely implicated in the control of reproduction , with Fos expression indicating neuronal response . However, in rats perfused on the diestrus day or in ovariectomized rats receiving estradiol benzoate or sesame oil, we did not find Fos expression in GnRH neurons. The pattern of distribution of NEI-ir and GnRH ir in the median eminence, showed NEI fibers to be denser in the internal layer than in the external one. Nevertheless, in the external layer, NEI and GnRH varicosities presented similar distributions, revealing an apparent close apposition between them. In the present study, we labeled distinctive cell compartments by using dual and triple-label immunohistochemistry which revealed NEI-ir fibers to be innervating the Fos-positive neurons in the AVPV, as well as the GnRH neurons positive for Fos immunoreactivity. In these experiments we labeled distinct cell compartments (cytoplasm, nuclei and terminals) using various antisera, all raised in rabbit. In the control tests, the second or third antisera were omitted, and no reaction was evident. This indicates that the observed labeling of cell bodies, fibers or both was not the result of cross-reactivity of the secondary antibody. In addition, evaluation of the data under light microscopy at a high magnification revealed only a suggestion of synaptic contact (Fig 6 A-B).
6. NEI innervation of GnRH neurons
Our results indicated that NEI fibers were in close apposition with GnRH neurons expressing Fos in the afternoon of the proestrus day. Since Fos protein expression in GnRH neurons increases in parallel with rises in the plasma LH levels [27, 31, 32], it can be assumed that these neurons project to the median eminence and induce LH secretion during proestrus. Therefore, we can suggest that NEI (through projections to a subset of GnRH neurons) modulated GnRH activity and, consequently, LH surges.
The role that NEI plays in GnRH secretion has not been investigated. However, NEI varicosities in some parts of the median eminence display a pattern of distribution similar to that of GnRH, revealing a possible effect on the modulation of GnRH secretion directly at the terminals. This may represent one of the mechanisms by which intracerebroventricular administration of NEI causes an increase in LH secretion (Fig. 6 E)
Experiments using in vitro preparations or intracerebroventricular injections have shown that various neurotransmitters can regulate gonadotropin release [38-40]. One such neurotransmitters is the cocaine- and amphetamine- regulated transcript (CART) peptide, which has been shown to increase the GnRH pulse amplitude in cycling female rats and to decrease GnRH pulse intervals in prepubertal rats [41-43]. These effects can be achieved through direct CART innervation of the GnRH neurons [39, 40]. Interestingly, in the medial ZI and lateral hypothalamus, MCH/NEI neurons coexpress CART [44, 46]. In the present study, we did not explore the origins of NEI innervation of GnRH neurons or areas related to reproductive behavior. However, it is intriguing that the number of NEI-ir neurons in ovariectomized rats was increased only in the medial ZI, a brain region that projects to the AVPV and GnRH- containing areas [47,48], as well as to the circumventricular organs, probably including the median eminence [44, 45]. This result is in agreement with those of other studies in which ppMCH mRNA expression was found to be greater in the medial ZI of untreated ovariectomized rats than in that of ovariectomized rats primed with estradiol benzoate or estradiol benzoate plus progesterone.
7. In vitro studies
Taken into account the above results, we decided to investigate whether NEI could act directly at the pituitary level by modulating hormone secretion. With this purpose the effects of NEI were studied in pituitary cell cultures from female rats on the release of several pituitary hormones (GH, LH and prolactin). Furthermore, the ability of NEI to activate pituitary cells was evaluated by electron microscopy and immunocitochemistry , and finally, the ability of NEI to potentiate GnRH-induced LH release was tested. The study of the effects of physiological stimuli involved in the regulation of pituitary hormone secretion was facilitated by the availability of a system consisting of a suspension of single dispersed pituitary cells, in which the cell functions were essentially the same as in situ . For this study we used female rats in order to obtain a primary culture. The results were as follows:
NEI induced a fast release of LH in the culture cell media. In addition, there were differences in the LH levels obtained for the various time periods of hormonal stimuli assayed, which were closely associated with the doses applied. The lowest dose of NEI (100x 10-8M) induced a significant increase of LH secretion after 2h of stimulus, achieving maximum response after 4h of NEI treatment. At this time, the LH levels almost reached a five-fold increase over controls and then maintained these values (Fig.7). In spite of NEI being effective in stimulating LH secretion, none of the assayed doses were capable of significantly promoting FSH secretion from gonadotrophs. In addition, no significant increases were observed on prolactin or GH secretion, at doses ranging from 100x10-8 to 400x10-8 M NEI in primary cell cultures, thus confirming the specificity of NEI stimuli on LH secretion (Table 1). To determine whether or not NEI was able to synergize with GnRH in stimulating LH release, pituitary cells were simultaneously incubated with GnRH (0.1 or 1x10-9M) and different concentrations of NEI (1, 10 or 100x10-8M) for 3 h and then the media was collected and tested for LH by RIA (Fig. 8). Although NEI at the dose of 10x10-8M had no effect on LH secretion, GnRH 1x10-9M plus NEI 10x10-8M induced a slight but significant increase in LH concentrations (16%; p< 0.01). A combined treatment with the highest doses of both NEI and GnRH significantly stimulated the secretory response, which was more effective than that observed with the same dose of GnRH alone  (Fig.8).
When pituitary cells from female rats were cultured, different types of secretory cells were observed (Fig. 9). These were identifiable by their ultra structural characteristics, essentially by the profile of the secretory granules, which constituted a distinctive feature. The most frequent populations observed were lactotroph and somatotroph cells, which were in close contact with gonadotrph cells. In the control group, the lactotroph and somatotroph cells have numerous polymorphic and round mature secretory granules respectively. These also had high electron densities and were stored in the cytoplasm. The gonadotroph were
characterized by a conspicuous accumulation of round secretory granules of two different sizes and by electron densities in the cytoplasm. The most abundant granules were about 150nm in diameter and filled with homogenous material, whereas the others less frequent but larger in size (about 400nm) (Fig. 10 A and B).Stimulation with NEI (100 x 10-8 and 400 x 10 -8) for 2 and 4 hour, promoted several subtle structural changes, particularly in the
gonadotroph cell population. For this cell type, the most prominent changes consisted of a striking development of the rough endoplasmic reticulum (RER) and Golgi complex (Fig 10B) when compared to the control group. Many secretory granules were located to the cell membrane and presented images of exocytosis after NEI treatment (Fig. 10 C) Other pituitary cell populations, the lactotrophs, thyrotrophs and somatotrophs, did not exhibit any features indicating a significant activation of hormone release after NEI treatment.
The present results were the first demonstration of a specific and direct action of NEI in cultured pituitary cells without modifying other pituitary hormones. Moreover, the analysis of the electron microscope images taken 2 and 4h after NEI treatment was indicative of the stimulation of LH release occurring at these times.
From the present study, it is possible to conclude that NEI is effective when injected into the brain to release LH in male and female rats. The anatomical substrate underlying this effect was identified using combined methods of immunohistochemistry. A schematic representation of the proposed pathways by which NEI participates in LH secretion is depicted in Figure 11.
NEI is also capable of inducing a marked release of LH without modifying the other pituitary hormones in the pituitary cultured cells. There is an interaction between NEI and GnRH in vivo and in vitro.
First of all I wish to thank to Dr. Paul Hobson for critical reading of this manuscript. I also wish to thank Dr. Jorge Paván, Miss Laura Alazraki and Miss Marina Juarez for editing the manuscript. Thanks for the grants from CONICET; SECyT and FONCyT. MEC is member of CONICET.