Betaglycan binds inhibin and can mediate functional antagonism of activin signalling.

Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function.

Activin A regulation of gonadotropin-releasing hormone synthesis and release in vitro.

Activin is essential for the regulation of normal mammalian reproductive function at both the pituitary and gonadal levels. However, its central actions in the control of the hypothalamic-pituitary-gonadal axis remain largely unexplored. The present study aims to determine whether activin could regulate the reproductive axis at the level of the hypothalamus, through control of the GnRH neuroendocrine system. Using the GnRH-secreting GT1-7 neuronal cell line as a model system, we demonstrate expression of mRNAs encoding activin receptor types I, IB, and II. We examined the effects of activin A on GnRH protein secretion and mRNA levels in GT1-7 cells. Treatment with rh-activin A regulated both GnRH protein secretion and GnRH mRNA expression in the GT1-7 cells in a time-dependent fashion. Using transient transfection assays, we explored a potential transcriptional basis for these changes. Activin A increased reporter gene activity driven by minimal GnRH enhancer and promoter elements, suggesting that activin may regulate GnRH gene expression at the level of transcription. Lastly, activin A treatment of male rat hypothalami, in vitro, increased GnRH protein secretion. Collectively, molecular and physiological evidence support the presence of an activin system which might act at a hypothalamic site to regulate mammalian reproduction via activation of GnRH synthesis and release.

Expression of activin and follistatin in the rat hypothalamus: anatomical association with gonadotropin-releasing hormone neurons and possible role of central activin in the regulation of luteinizing hormone release.

The central role of activin in the regulation of the reproductive axis remains largely unexplored. Evidence suggests that activin may play a role in control-linggonadotropin-releasing hormone (GnRH) release. We assessed potential neuroanatomical associations between activin- and GnRH-neuronal systems via examination of the distribution of activin betaA-subunit and activin binding protein (follistatin) protein and mRNA signals relative to GnRH neurons in the adult rat brain. Activin betaA-subunit-immunostained fibers were distributed throughout the hypothalamus and GnRH-positive perikarya, and fibers were in close association with betaA-subunit-immunoreactive fibers. Follistatin mRNA-expressing cells were also identified throughout the hypothalamus with GnRH fibers often observed juxtaposed to follistatin cell bodies. Colocalization of either the betaA-subunit or follistatin within GnRH neurons was not detected. The functional significance of central activin in the regulation of the reproductive axis was also demonstrated. The intracerebroventricular infusion of rh-activin A significantly increased luteinizing hormone, but not follicule-stimulating hormone, serum levels in adult male rats. Taken together, the present results support an interaction between activin and GnRH neuronal systems in the rat hypothalamus, and suggest activin may act within the brain to regulate the reproductive axis.

Distribution of follistatin messenger ribonucleic acid in the rat brain: implications for a role in the regulation of central reproductive functions.

Follistatin (FS), which binds to the inhibin/activin beta A- or beta B-subunit is localized with and modulates the biological actions of activin in many systems. However, in contrast to the wide distribution of the activin beta-subunit proteins and messenger RNAs (mRNA) in the brain, demonstration of FS mRNA signal has been limited to the olfactory tubercle and layer II of the frontal cortex. We have hypothesized a more extensive distribution of central FS gene expression and localization in regions coinciding with inhibin/activin beta-subunits and possible activin-mediated effects. In the present study, we examined the central distribution of FS mRNA expression in the normal adult male rat. With in situ hybridization analysis, using a 33P-labeled RNA probe specific for rat FS, gene expression is shown to be widely distributed throughout the brain. Abundant FS mRNA expression is localized in several areas of the olfactory bulb as well as the frontal cortex, a few thalamic nuclei, and in septal regions. Moderate FS mRNA is observed in the caudate putamen and various hypothalamic areas including the paraventricular, ventromedial, dorsomedial, and arcuate nuclei. Several brain stem regions are also found to express FS mRNA, including the medial vestibular and solitary tract nuclei. Notably, FS mRNA, including the medial vestibular and solitary septal/diagonal band region is localized in patterns that are highly correlative with those of GnRH gene expression and hence may serve to regulate possible activin-mediated effects in these areas. FS mRNA is also expressed in areas associated with the activin-oxytocin pathway (solitary tract nucleus and paraventricular nucleus) and is therefore in a position to modulate the role of activin in the solitary tract nucleus-paraventricular nucleus pathway (afferent system mediating the milk-ejection reflex). The results suggest that FS is centrally localized in sites compatible with a role in the regulation of central reproductive functions.

Effects of haloperidol in a response-reinstatement model of heroin relapse.

The present study employed an animal model of drug relapse in which previously extinguished heroin self-administration behavior was reinstated following a single reinforced trial. Male albino rats were trained to traverse a straight-alley for a reinforcer consisting of a single IV injection of 0.06 mg/kg diacetylmorphine (heroin). Once the alley-running had been established, the heroin reinforcer was removed and the operant behavior permitted to extinguish over trials. On treatment day, animals were injected 45 min prior to testing with 0.0, 0.075, 0.10, 0.15 or 0.3 mg/kg of the dopamine receptor antagonist, haloperidol. A single trial was then conducted during which some animals continued to experience extinction conditions while others were injected with the heroin reinforcer upon entry into the goal box. The effects of these manipulations were determined during an additional single test trial conducted 24 h later when the subjects were no longer drugged. While heroin produced a reliable reinstatement in operant responding, this effect was dose-dependently prevented by pretreatment with haloperidol. These data suggest that dopamine receptor antagonism alters the reinforcing consequences of heroin administration as measured by heroin's ability to reinstate operant behavior following a prolonged period of nonreinforced responding.

Pimozide prevents the development of conditioned place preferences induced by rewarding locus coeruleus stimulation.

Electrical stimulation in the vicinity of the cell bodies of the locus coeruleus (LC) has been shown to support self-stimulation behaviors in rats. In the present study, a Conditioned Place Test, sensitive to both rewarding and aversive qualities of brain stimulation, was employed to determine (a) whether rewarding locus coeruleus stimulation would result in place preferences and (b) if so, whether dopamine receptor antagonism would affect the development of such place preferences. Animals were pretreated with pimozide (0.0, 0.5 or 1.0 mg/kg) prior to exposure to two distinctive environments only one of which was paired with locus coeruleus stimulation. Rats that received vehicle injections prior to stimulation/place pairings developed strong preferences for the stimulation-paired environment while those animals pretreated with 0.5 mg/kg pimozide showed no reliable shift in preference from baseline performance. Additionally, animals injected with the 1.0 mg/kg dose of pimozide exhibited mild place aversions to the stimulation-paired environment. It is hypothesized that dopamine neurotransmission is important for the rewarding effects of locus coeruleus stimulation without which such stimulation appears to be aversive.

Opposite effects of prefrontal cortex and nucleus accumbens infusions of flupenthixol on stimulant-induced locomotion and brain stimulation reward.

Ventral tegmental area (VTA) stimulation produced conditioned place preferences for stimulation-paired environments the magnitudes of which were dose-dependently reduced by systemic application of the dopamine antagonist, haloperidol (0.0, 0.15, 0.3 mg/kg). Bilateral microinjections of cis-flupenthixol (FLU) into the nucleus accumbens (0.0, 1.0, 5.0 or 10.0 micrograms) also resulted in reductions in the size of stimulation-induced place preferences as well as reductions in the magnitude of the hyperlocomotor response to 1.5 mg/kg (s.c.) D-amphetamine. Comparable microinjections of FLU into the medial prefrontal cortex (PFC) produced diametrically opposite effects: the size of VTA stimulation-induced place preferences was either unaffected (1.0 and 5.0 microgram groups) or slightly increased (10 micrograms group) and amphetamine-stimulated hyperlocomotion was dose-dependently potentiated. These behavioral findings suggest a dopamine-mediated modulatory role for the PFC over reward relevant elements within the nucleus accumbens.