Anna Lerant

Ovarian steroids differentially regulate the expression of PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study

The aims of this study were (1) to identify the possible hypothalamic targets for a short prolactin (PRL) feedback in the adult female rat by identifying DAergic neuron populations expressing PRL receptor (PRL-R); (2) to describe the effect of ovarian steroids on the expression of PRL-R and (3) to compare the distribution of both the extracellular (EC) and ligand binding (LB) domains of the PRL-R on the hypothalamic dopaminergic neurons by applying double label immunocytochemistry for the different domains of PRL-R and for tyrosine hydroxylase (TH). Five- to six-month-old female rats were ovariectomized (OVX) and implanted with either 17 beta-estradiol (E2), progesterone (P4) or received an E2 and a P4 implant (E2 + P4) at the same time. In the periventricular nucleus and in the dorsomedial portion of the middle arcuate nucleus, a dramatic increase in PRL-REC immunoreactivity was observed in E2 implanted rats. This increase was attenuated in E2 + P4 rats, but P4 treatment alone had no effect. Changes in PRL-REC expression were paralleled by changes in serum PRL levels. Interestingly, PRL-REC expression in the rostral arcuate nucleus decreased in P4 implanted rats, however, P4 did not attenuate the E2-induced increase in PRL-REC density. PRL-REC immunostaining was observed on the membrane, in the cytoplasm and in the nucleus. PRL-RLB immunoreactivity was also detectable in the TH positive neurons, but no nuclear staining was observed with this antibody. However, we found a strong PRL-RLB immunostaining in the ependymal lining of the 3rd ventricle and in the processes of tanycytes projecting to the median eminence. These data indicate that (1) all neuroendocrine DAergic cells can be targets for PRL, (2) expression of PRL-R is differentially affected by ovarian steroids in the different TH cell populations, (3) PRL-RLB domain may be involved in trafficking PRL in the median eminence.

Dopamine transporters participate in the physiological regulation of prolactin

Three populations of hypothalamic neuroendocrine dopaminergic (NEDA) neurons, arising from the arcuate and periventricular nuclei of the hypothalamus release dopamine (DA) that acts at the pituitary gland to regulate the secretion of PRL. It is generally accepted that NEDA neurons lack functional DA transporters (DATs), which are responsible for uptake of DA from the synaptic cleft into the presynaptic axon terminal. This study localized DATs to the hypothalamo-pituitary axis and evaluated the effect of DAT blockade on the hypothalamo-pituitary regulation of PRL. After 7 days of treatment with cocaine (a nonspecific amine transporter blocker) or mazindol (a specific DAT blocker), the relative abundance of PRL messenger RNA (mRNA) in the anterior lobe (AL) of OVX rats was significantly decreased, whereas the relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was significantly increased. The effect of cocaine or mazindol administration on DA turnover and serum PRL concentration was examined...

Ovarian Steroids Modulate Responsiveness to Dopamine and Expression of G-Proteins in Lactotropes

The effects of chronic ovarian steroid treatment on the secretory activity of individual lactotropes and the mechanisms modulating their responsiveness to dopamine (DA) were studied. Female rats were ovariectomized (OVX) and implanted with Silastic capsules containing progesterone (P₄), 17β), 17β-estradiol (E₂) or both E₂ (E₂+P₄). Ten days after surgery, anterior pituitaries were enzymatically dispersed and the reverse hemolytic plaque assay (RHPA) was performed to assess the release of prolactin (PRL) from individual lactotropes. RHPA was combined with immunocytochemistry (ICC) for PRL, G_αs or G_iα3/G_αo proteins. E₂ treatment alone or in combination with P₄ increased the percentage of immunoreactive lactotropes among anterior pituitary cells. Incidence of active (plaque-forming) lactotropes, however, was increased both in P₄-, and E₂-treated rats and E₂+P₄ treatment increased it even further. While P₄ treatment did not affect the frequency distribution of lactotropes, both E₂ and E₂+P₄ treatments increased the large plaque-forming lactotrope population. This increase was reflected by the significantly greater mean plaque areas of lactotropes from E₂- and E₂+P₄-treated rats compared to OVX or P₄-treated animals. The responsiveness of lactotropes to DA from P₄-treated rats did not differ from that of OVX rats: thus challenge with 1 µM DA inhibited the release of PRL, while 100 pM DA had no effect. E₂ and E₂+P₄ treatments, however, profoundly changed the lactotrope’s responsiveness: challenge with 1 µM DA had no effect and 100 pM DA resulted in moderate stimulation of PRL release in E₂+P₄ rats. Double-label ICC revealed that ovarian steroid treatments did not affect the expression of G_αs in lactotropes. The incidence of G_iα3/G_αo-immunoreactive lactotropes, however, decreased after E₂ treatment, alone or in combination with P₄. Although expression of G_αs was similar in all plaque-forming cells regardless of plaque size, lactotropes expressing G_iα3/G_αo were more likely to form small plaques in all treatment groups. These data suggest: (1) ovarian steroid treatment recruits quiescent lactotropes to release PRL; (2) E₂ treatment alone or in combination with P₄ increases the amount of PRL rleased by individual lactotropes; (3) E₂-induced alterations in the frequency distribution and lactotrope responsiveness to DA may be due in part to a decreased expression of G_iα3/G_αo.

Dopaminergic neurons in periventricular and arcuate nuclei of proestrous and ovariectomized rats: endogenous diurnal rhythm of Fos-related antigens expression.

The expression of Fos-related antigens (FRAs) in the A12 and the A14 hypothalamic dopaminergic neurons was compared throughout the day in proestrous (PRO) and ovariectomized (OVX) rats to establish the relationship between secretion of prolactin (PRL) and dopaminergic neuronal activation. Animals with intact ovaries were sacrificed at 11:00, 13:00, 15:00, 17:00 and 21:00 h on the day of proestrus and 06:00 and 09:00 h in the morning of estrus. OVX animals were sacrificed at the same time points on the 12th day after surgery. Double-label immunocytochemistry was performed by using antibodies against FRAs as markers of tonic neuronal activity and tyrosine hydroxylase to identify dopaminergic neurons. Serum PRL levels were determined by radioimmunoassay. A pattern of FRAs expression was present in the A14 and A12 neurons of PRO rats. The incidence of FRAs expressing neurons was the highest in the first half of the day when the PRL levels were low, and decreased prior to the surge of serum PRL during the afternoon. This pattern was present in the dopaminergic neurons of the periventricular nucleus (A14), and in all portions of the arcuate nucleus (A12) except the ventrolateral portion of the middle arcuate nucleus. A similar pattern of FRAs expression existed in the A14 and A12 neurons of OVX rats in spite of that there were no detectable changes in serum PRL levels. However, the amplitude of decrease in the incidence of FRAs-labeled neurons was lower in OVX than in PRO rats. These data suggested that similar to the control of hypothalamic PRL releasing factors, the activation/deactivation pattern of the A12 and the A14 neurons is governed by a seemingly endogenous rhythmic input. The incidence of this rhythm is independent of the reproductive state, but its amplitude is enhanced by ovarian steroids. The disparity between the FRAs expression and serum PRL levels in OVX rats indicated that the effect of this endogenous dopaminergic rhythm upon PRL secretion is dependent on the ovarian steroid background.

Endothelin is an autocrine regulator of prolactin secretion.

The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ET(A)-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide, [22Val]big ET1-(16-38), increased PRL secretion, whereas the ET(B) receptor-specific antagonist BQ788 was ineffective. The ET(A) antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequency distribution of PRL plaque sizes using logarithmically binned data revealed two subpopulations of lactotrophs with differential responsiveness to endogenous ETs. These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ETA-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide,[ 22Val]big ET1-(16–38), increased PRL secretion, whereas the ETB receptor-specific antagonist BQ788 was ineffective. The ETA antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequenc...

Regulation of neuropeptide Y in the rat amygdala following unilateral olfactory bulbectomy

While the mechanisms are not fully understood, olfactory bulbectomy (OBX) is a well-known rat model of depression and depression-related disorders such as anxiety and aggression. Alterations in neuropeptide Y (NPY) levels in the brain have been linked to depression and have been shown to be involved in the response to stress. This study explored the possible regulation of NPY immunoreactivity in specific regions of the amygdala 14 days after OBX in adult male Sprague-Dawley rats (n=6). Unilateral OBX and immunohistochemistry permitted comparisons of NPY in the ipsilateral amygdala with NPY in the contralateral (sham) amygdala. OBX resulted in significant increases (P<0.05) in NPY immunoreactivity in the anterior medial amygdala (threefold) and the posterior medial amygdala (2.5-fold). These regions receive projections from the accessory olfactory bulb (AOB). In contrast, the anterior and posterolateral cortical nuclei of the amygdala receive projections from the main olfactory bulb (MOB). NPY was not increased in these nuclei. These data show that not only does OBX increase NPY immunoreactivity in the amygdala, but also suggest that the AOB plays a prominent role in this regulation.

Free zinc increases at the site of injury after cortical stab wounds in mature but not immature rat brain

The accumulation of free zinc (Zn2+) appears to play a role in the neuronal degeneration that occurs after brain injury. Given that neonates respond to brain injury with increased plasticity compared to adults, this study compared the effect of age on free Zn2+ and the Zn2+-binding protein metallothionein-3 (MT-3) after injury. Unilateral cortical stab wounds were produced in 3-day-old and adult rats. Four weeks later, brains were removed for in situ visualization of free Zn2+ and measurement of MT-3 mRNA. Free Zn2+ and MT-3 mRNA accumulated after 4 weeks at the site of injury site when injury occurred in adults. However, 4 weeks after neonatal injury there was no increase in free Zn2+ or MT-3 mRNA in or around the site of injury.