Ibolya Bodnár

Nesfatin-1/NUCB2 may participate in the activation of the hypothalamic-pituitary-adrenal axis in rats

Nesfatin-1 is an anorexigenic peptide originating from nucleobinding-2 (NUCB2) protein. Nesfatin-1/NUCB2-immunoreactive neurons are present in the hypothalamic paraventricular nucleus, the center of the stress-axis, and in the medullary A1 and A2 catecholamine cell groups. The A1 and A2 cell groups mediate viscerosensory stress information toward the hypothalamic paraventricular nucleus. They contain noradrenaline, but subsets of these neurons also express prolactin-releasing peptide acting synergistically with noradrenaline in the activation of the hypothalamic paraventricular nucleus during stress. We investigated the possible role of nesfatin-1/NUCB2 in the stress response. Intracerebro-ventricular administration of nesfatin-1 elevated both plasma adrenocorticotropin and corticosterone levels, while in vitro stimulation of the hypophysis was ineffective. Single, long-duration restraint stress activated (Fos positivity) many of the nesfatin-1/NUCB2-immunoreactive neurons in the parvocellular part of the hypothalamic paraventricular nucleus, evoked nesfatin-1/NUCB2 mRNA expression in the parvocellular part of the paraventricular nucleus and in the A1, but not in the A2 cell group. Nesfatin-1/NUCB2 was shown to co-localize in a high percentage of prolactin-releasing peptide producing neurons, in both medullary catecholamine cell groups further supporting its involvement in the stress response. Finally, bilateral adrenalectomy evoked an increasing nesfatin-1/NUCB2 mRNA expression, indicating that it is under the negative feedback of adrenal steroids. These data provide the first evidence for possible participation of nesfatin-1/NUCB2 in the stress-axis regulation, both at the level of the brainstem and in the hypothalamus.

Stress‐ as Well as Suckling‐Induced Prolactin Release is Blocked by a Structural Analogue of the Putative Hypophysiotrophic Prolactin‐Releasing Factor, Salsolinol

Prolactin is secreted from the anterior lobe of the pituitary gland in response both to suckling and to stress. We recently observed that 1‐methyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline (salsolinol), produced in the neurointermediate lobe of the pituitary gland, as well as in the medial basal hypothalamus, can selectively release prolactin from the anterior pituitary. Therefore, it has been proposed that salsolinol is a putative endogenous prolactin‐releasing factor (PRF). Here, we report that one structural analogue of salsolinol, 1‐methyl‐3,4‐dihydroisoquinoline (1MeDIQ), can block salsolinol‐induced release of prolactin, but does not affect prolactin release in response to thyrotropin releasing hormone (TRH), α‐methyl‐p‐tyrosine (αMpT) (an inhibitor of tyrosine hydroxylase), domperidone (a D2 dopamine receptor antagonist), or 5‐hydroxytryptophan (5‐HTP), a precursor of serotonin). 1MeDIQ profoundly inhibited suckling‐, immobilization‐, as well as formalin‐stress induced prolactin release without any influence on corticosterone secretion. The 1MeDIQ‐induced reduction in prolactin response to immobilization stress was dose‐dependent. These results suggest that salsolinol can play a pivotal role in the regulation of prolactin release induced by either physiological (suckling) or environmental (stress) stimuli.

Neurons containing tuberoinfundibular peptide of 39 residues project to limbic, endocrine, auditory and spinal areas in rat.

Accumulating evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) may be the endogenous ligand of the parathyroid hormone 2 receptor. The vast majority of TIP39-containing neurons are localized in two regions, the subparafascicular area at the thalamic-midbrain junction, and the medial paralemniscal nucleus in the rostral pons. In contrast to the restricted localization of TIP39-containing cell bodies, TIP39-containing fibers have a widespread distribution. TIP39 neurons were lesioned electrolytically to determine the origin of TIP39-containing fibers within different parts of the rat CNS. Following bilateral lesions of the medial subparafascicular area including the subparafascicular nucleus, TIP39-immunoreactive fibers almost completely disappeared from forebrain regions including the anterior limbic cortical areas, the shell and cone portions of the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the amygdaloid nuclei, the fundus striati, the subiculum, the thalamic paraventricular nucleus, and the hypothalamic paraventricular, dorsomedial and arcuate nuclei. Unilateral lesions of the medial and the lateral subparafascicular area demonstrated that the projections are ipsilateral and that medial lesions produce higher reductions in the density of TIP39 fibers except in the amygdala and the hypothalamus. Following lesions of the medial paralemniscal nucleus, TIP39-immunoreactive fibers disappeared from the medial geniculate body, the periaqueductal gray, the deep layers of the superior colliculus, the external cortex of the inferior colliculus, the cuneiform nucleus, the nuclei of the lateral lemniscus, the lateral parabrachial nucleus, the locus coeruleus, the subcoeruleus area, the medial nucleus of the trapezoid body, the periolivary nuclei, and the spinal cord, suggesting that these regions receive TIP39-containing fibers from the medial paralemniscal nucleus, and unilateral lesions demonstrated that the projections are ipsilateral. The projections of the TIP39-containing cells in the subparafascicular area suggest their involvement in limbic and endocrine functions, while the projections of the TIP39-containing cells in the medial paralemniscal nucleus suggest their involvement in auditory and nociceptive functions.

Tuberoinfundibular Peptide of 39 Residues Is Activated during Lactation and Participates in the Suckling-Induced Prolactin Release in Rat

Tuberoinfundibular peptide of 39 residues (TIP39) and the PTH-2 receptor (PTH2R) constitute a peptide-receptor neuromodulator system. Based on the abundance of TIP39 fibers and axonal terminals as well as PTH2R-containing neurons and their processes in the hypothalamic para- and periventricular and arcuate nuclei TIP39 has been suggested to play a role in neuroendocrine regulation. We showed previously that TIP39 expression decreased dramatically by adulthood. In the present study, using in situ hybridization histochemistry, real-time RT-PCR, and immunohistochemistry, we found that TIP39 mRNA and peptide expression levels are markedly elevated in the posterior intralaminar complex of the thalamus (PIL) of lactating dams, one of the three locations of TIP39-containing cell bodies in the brain. In addition, in mother rats, these TIP39 neurons showed Fos expression in response to pup exposure. Transection of TIP39 fibers originating in the PIL resulted in an ipsilateral disappearance of TIP39 immunoreactivity throughout the mediobasal hypothalamus of mother rats, suggesting that TIP39 fibers there arise from the PIL. To elucidate the function of TIP39 activation in dams, mothers separated from their pups for 4 h on postpartum d 9 received injection of a PTH2R antagonist into the lateral ventricle 5 min before returning the pups. Blood samples were taken seven times during the experimental period through jugular cannulae. The PTH2R antagonist administered in two different concentrations markedly inhibited suckling-induced elevation of plasma prolactin levels in a dose-dependent manner. These results suggest that TIP39 neurons in the PIL may regulate suckling-induced prolactin release in rat dams.

Demonstration of autoantibody binding to muscarinic acetylcholine receptors in the salivary gland in primary Sjögren's syndrome

A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögrens syndrome (pSS) has been suggested. However, the binding of these antibodies to the receptors in the target tissues has not yet been demonstrated. In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studied with light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivo deposited antibodies in pSS salivary glands was also investigated. Light microscopic immunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membrane of acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelial cell membranes. With electron microscopy, the autoantibody binding was observed to be localised to the junctions of epithelial cell membranes with nerve endings, both in normal and pSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivary glands.

Effect of neonatal treatment with monosodium glutamate on dopaminergic and L-DOPA-ergic neurons of the medial basal hypothalamus and on prolactin and MSH secretion of rats

The effect of neonatal treatment with monosodium L-glutamate (MSG) on the dopaminergic systems of the medial basal hypothalamus has been investigated using tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) immunocytochemistry. Changes in plasma levels of prolactin (PRL) and alpha-melanocyte-stimulating hormone (MSH) have also been determined in intact and in MSG-treated rats after inhibition of TH by alpha-methyl-p-tyrosine (alpha-MpT) or without inhibition of enzyme activity. Monosodium glutamate resulted in a 40% reduction in the number of TH immunopositive tuberoinfundibular neurons, but no change in the number of AADC-positive tuberoinfundibular nerve cells, indicating that this reduction has occurred mainly in TH-positive but AADC-negative elements, i.e., in L-DOPA-ergic neurons. In contrast, MSG did not cause changes in the number of TH and AADC immunoreactive neurons of the periventriculohypophysial and tuberohypophysial dopaminergic systems, and it did not influence basal plasma PRL levels. alpha-methyl-p-tyrosine has increased plasma PRL concentrations in both control and MSG-treated rats of both sexes, but significantly higher responses were detected in females. None of the treatments had any effect on plasma MSH level. These findings suggest that MSG affects primarily L-DOPA-ergic neurons located in the ventrolateral part of the arcuate nucleus, but not dopaminergic neurons situated in the dorsomedial part of the arcuate nucleus; neither PRL nor MSH secretion is altered by MSG; a significant sex difference exists in the pituitary PRL response to inhibition of TH, and this response is not affected by MSG.

The peripheral noradrenergic terminal as possible site of action of salsolinol as prolactoliberin.

Salsolinol, an endogenous isoquinoline, induces selective prolactin release in rats [Tóth, B.E., Homicskó, K., Radnai, B., Maruyama, W., DeMaria, J.E., Vecsernyés, M., Fekete, M.I.K., Fülöp, F., Naoi, M., Freeman, M.E., Nagy, G.M., 2001. Salsolinol is a putative neurointermediate lobe prolactin releasing factor. J. Neuroendocrinol. 13, 1042-1050]. The possible role of dopaminergic and adrenergic signal transduction was investigated to learn the mechanism of this action. The effect of salsolinol (10mg/kg i.v.) was inhibited by reserpine treatment (2.5mg/kg i.p.) and reinstated by pretreatment with monoamine oxidase inhibitor (pargyline 75 mg/kg i.p.). Salsolinol did not affect the in vitro release of dopamine (DA) in the median eminence, and did not inhibit the L-DOPA induced increase of DA level in the median eminence. 1-Methyl dihydroisoquinoline (1MeDIQ) is an antagonist of salsolinol induced prolactin release and causes increase in plasma NE level [Mravec, B., Bodnár, I., Fekete, M.I.K., Nagy, G.M., Kvetnansky, R., 2004. An antagonist of prolactoliberine induces an increase in plasma catecholamine levels in the rat. Autonom. Neurosci. 115, 35-40]. Using tissue catecholamine contents as indicators of the interaction between salsolinol and 1MeDIQ we found no interaction between these two agents to explain the changes in prolactin release in the median eminence, lobes of the pituitary, superior cervical and stellate ganglion. Increasing doses of salsolinol caused a dose dependent decrease of tissue dopamine concentration and increase of NE/DA ratio in the salivary gland, atrium and spleen. These changes of DA level and NE/DA ratio run parallel in time with the increase of prolactin release. 1MeDIQ antagonized the increase of prolactin release and decrease of tissue DA content caused by salsolinol. Neither this increase of prolactin secretion nor the decrease of DA level in spleen could be demonstrated in NE transporter (NET) knock out mice. The results presented argue for the possible role of peripheral norepinephrine release as a target for salsolinol in its action releasing prolactin. The dominant role of norepinephrine transporter may be suggested.

The role of catecholamines in the prolactin release induced by salsolinol.

Salsolinol (1,2,3,4-tetrahydro-6,7-dihydroxy-1-methylisoquinoline) is an endogenous prolactin releasing agent. Its action can be inhibited by another isoquinoline, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), which has a strong norepinephrine releasing activity. Salsolinol does not alter the dopamine release in median eminence in vitro, providing evidence for the lack of interaction with presynaptic D2 dopamine receptors. At the same time, lack of norepinephrine transporter abolishes salsolinols action. Salsolinol decreases tissue level of dopamine and increases norepinephrine to dopamine ratio in organs innervated by the sympathetic nervous system indicating a possible decrease of norepinephrine release. Enzymes of catecholamine synthesis and metabolism are probably also not the site of action of salsolinol. In summary, based upon all of these observations a physiologically relevant interplay might exist between the sympatho-neuronal system and the regulation of prolactin release.

Brain Structures Mediating the Suckling Stimulus‐Induced Release of Prolactin

Suckling‐induced prolactin release is a widely studied neuroendocrine reflex, comprising a neural afferent and a humoral efferent component. The information on the brain structures involved in this reflex is fairly limited. The present studies focused on this question. The following hypothalamic interventions were made in lactating rats and the dams were tested for the suckling‐induced prolactin response: (i) unilateral or (ii) bilateral frontal cuts at the level of the anterior and posterior hypothalamus; (iii) administration of 5,7‐dihydroxytryptamine or (iv) 6‐hydroxydopamine into the hypothalamic paraventricular nucleus (PVN) to destroy serotonergic and catecholaminergic innervation of the cell group, respectively; (v) lesion of the medial subdivision of the PVN; and (vi) horizontal knife cuts below the PVN. Bilateral posterior and bilateral or unilateral anterior frontal cuts caused blockade of the suckling‐induced release of prolactin. Likewise, most dams receiving 5,7‐dihydroxytryptamine in the PVN did not respond to the suckling stimulus. Immunocytochemistry revealed that, in those rats which did not show a rise in plasma prolactin, there were almost no serotonergic fibres and terminals in the PVN, while in dams which exhibited a response, numerous serotonergic elements were evident. 6‐Hydroxydopamine treatment did not cause significant alteration in the prolactin response. Lesion of the medial, largely parvocellular subdivision of the PVN, or horizontal knife cuts below this cell group, blocked the hormone response. The findings demonstrate for the first time that: (i) interruption of the connections between the brain stem and the hypothalamus interferes with the prolactin response to the suckling stimulus; (ii) serotonergic fibres terminating in the hypothalamic PVN are involved in the mediation of the suckling stimulus; and (iii) within the PVN, neurones in the medial, largely parvocellular subdivision of the cell group take part in the transfer of the neural signal, eventually inducing prolactin release.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation.

Nursing has important physiological and psychological consequences on mothers during the postpartum period. Tuberoinfundibular peptide of 39 residues (TIP39) may contribute to its effects on prolactin release and maternal motivation. Since TIP39-containing fibers and the receptor for TIP39, the parathyroid hormone 2 receptor (PTH2 receptor) are abundant in the arcuate nucleus and the medial preoptic area, we antagonized TIP39 action locally to reveal its actions. Mediobasal hypothalamic injection of a virus encoding an antagonist of the PTH2 receptor markedly decreased basal serum prolactin levels and the suckling-induced prolactin release. In contrast, injecting this virus into the preoptic area had no effect on prolactin levels, but did dampen maternal motivation, judged by reduced time in a pup-associated cage during a place preference test. In support of an effect of TIP39 on maternal motivation, we observed that TIP39 containing fibers and terminals had the same distribution within the preoptic area as neurons expressing Fos in response to suckling. Furthermore, TIP39 terminals closely apposed the plasma membrane of 82% of Fos-ir neurons. Retrograde tracer injected into the arcuate nucleus and the medial preoptic area labeled TIP39 neurons in the posterior intralaminar complex of the thalamus (PIL), indicating that these cells but not other groups of TIP39 neurons project to these hypothalamic regions. We also found that TIP39 mRNA levels in the PIL markedly increased around parturition and remained elevated throughout the lactation period, demonstrating the availability of the peptide in postpartum mothers. Furthermore, suckling, but not pup exposure without physical contact, increased Fos expression by PIL TIP39 neurons. These results indicate that suckling activates TIP39 neurons in the PIL that affect prolactin release and maternal motivation via projections to the arcuate nucleus and the preoptic area, respectively.