Tatiana Fiordelisio

Expression and differential cell distribution of low-threshold Ca2+ channels in mammalian male germ cells and sperm

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca2+ influx through voltage‐gated Ca2+ (CaV) channels. Although the electrophysiological characterization of Ca2+ currents in mature sperm has proven difficult, functional studies have revealed the presence of low‐threshold (CaV3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of CaV3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three CaV3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only CaV3.1 and CaV3.2 were detected in the head, suggesting its participation in the AR. CaV3.1 and CaV3.3 were found in the principal and the midpiece of the flagella. All CaV3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of CaV3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit CaV3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high‐threshold (HVA) CaV channels caused small but significant motility alterations. Antibodies to HVA channels detected CaV1.3 and CaV2.3 in human sperm flagella.

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca2+ signaling

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimers disease and amyotrophic lateral sclerosis.

The Balance of Striatal Feedback Transmission Is Disrupted in a Model of Parkinsonism

Inhibitory connections among striatal projection neurons (SPNs) called “feedback inhibition,” have been proposed to endow the striatal microcircuit with computational capabilities, such as motor sequence selection, filtering, and the emergence of alternating network states. These properties are disrupted in models of Parkinsonism. However, the impact of feedback inhibition in the striatal network has remained under debate. Here, we test this inhibition at the microcircuit level. We used optical and electrophysiological recordings in mice and rats to demonstrate the action of striatal feedback transmission in normal and pathological conditions. Dynamic calcium imaging with single-cell resolution revealed the synchronous activation of a pool of identified SPNs by antidromic stimulation. Using bacterial artificial chromosome-transgenic mice, we demonstrate that the activated neuron pool equally possessed cells from the direct and indirect basal ganglia pathways. This pool inhibits itself because of its own GABA release when stimuli are frequent enough, demonstrating functional and significant inhibition. Blockade of GABAA receptors doubled the number of responsive neurons to the same stimulus, revealing a second postsynaptic neuron pool whose firing was being arrested by the first pool. Stronger connections arise from indirect SPNs. Dopamine deprivation impaired striatal feedback transmission disrupting the ability of a neuronal pool to arrest the firing of another neuronal pool. We demonstrate that feedback inhibition among SPNs is strong enough to control the firing of cell ensembles in the striatal microcircuit. However, to be effective, feedback inhibition should arise from synchronized pools of SPNs whose targets are other SPNs pools.

Dopamine D2-class receptor supersensitivity as reflected in Ca2+ current modulation in neostriatal neurons

The loss of dopaminergic neurons followed by dopamine (DA) depletion in the neostriatum is a hallmark of Parkinsons disease. Among other changes, DA D(2)-receptor class (D(2)R-class) supersensitivity is a result of striatal DA depletion. Pharmacological, biochemical and behavioral data have documented this phenomenon, but clear electrophysiological-functional correlates are still lacking. This work describes an electrophysiological correlate of D(2)R-class supersensitivity in DA-depleted striata after unilateral 6-hydroxydopamine (6-OHDA) lesions in the rat substantia nigra compacta (SNc). Ca2+ current modulation mediated by D(2)R-class activation reflected an altered sensitivity. Thus, while the concentration-response relationship (C-R plot) from control striata was better fit with a two sites model, the C-R plot obtained from DA-depleted striata was better fit by a three sites model, exhibited a considerable leftward shift, and presented an increased maximal response. Because Ca2+ current modulation by D(2)R-class activation is involved in the control of spiny neurons excitability and their synaptic GABA release, the present findings may help to explain several functional changes found in the striatal circuitry after dopaminergic denervation.

Nerve growth factor promotes development of glucose-induced insulin secretion in rat neonate pancreatic beta cells by modulating calcium channels.

Neonatal javascript:app(lower case beta)ß cells are functionally immature as they secrete less insulin than adults and lack of glucose response. The mechanisms that participate in the functional maturation of these cells are not known. Adult rat javascript:app(lower case beta)ß cells synthesize and secrete nerve growth factor (NGF) and express NGF receptors. NGF increases glucose-induced insulin secretion by modulating electrical activity in adult javascript:app(lower case beta)ß cells. In this work, we explored if NGF is involved in the maturation of glucose-induced insulin secretion coupling in rat neonate javascript:app(lower case beta)ß-cells.Pancreatic javascript:app(lower case beta)ß-cells were cultured for 48 h in control conditions, either with NGF or a neutralizing NGF antibody. We measured glucose-stimulated insulin secretion and whole-cell Ca2+ currents. We also analyzed both synthesis and molecular expression of Ca2+ channel subunits.Compared to controls, NGF-treated neonate javascript:app(lower case beta)ß-cells increased insulin biosynthesis and secretion in response to glucose and increased Ca2+ current density by translocation of LD-type Ca2+ channels to the plasma membrane. This type of secretion is similar to that observed in adults.NGF contributes to in vitro maturation by inducing beta cells to secrete insulin accordant with external glucose concentration, in other words promoting glucose sensitivity. Similar NGF-autocrine mechanisms could be active in vivo, because these effects were blocked by a neutralizing NGF-antibody. Understanding mechanisms that contribute to normal development of javascript:app(lower case beta)ß cell could provide insights to recognize alterations concomitant with type-2 diabetes.

Oestrogen regulates neurofilament expression in a subset of anterior pituitary cells of the adult female rat.

It is the prevailing view that the neurohypophysis derives from neural crest while the pituitarys anterior lobe is of ectodermal origin. However, it has been recently suggested that anterior pituitary cells could have in part neuro‐ectodermal origin, and thus should express specific neuronal markers. This issue was examined previously with conflicting results. The present study attempts to clarify the question of whether or not neuronal markers are expressed in the adenohypophysis. Using quantitative immunofluorescence, we have positively identified a subset of anterior pituitary cells, which express immunoreactivity for neuronal markers, including 68u2003kDa neurofilament (NF68). Interestingly, we noticed that the expression of NF68 is sexually dimorphic (i.e. neurofilament‐positive cells are more abundant in sexually mature female rats). In addition, NF68 expression in female rats increases during ontogenic development and reaches a plateau level after puberty. Thereafter, it displays plastic changes along the oestrous cycle, with the maximum of neurofilament expression at oestrus and the minimum at proestrus. NF68 immunoreactivity was examined after ovariectomy, oestradiol replacement and treatment with an specific oestrogen receptor antagonist. Bilateral ovariectomy induced a significant reduction in the number of NF68‐positive cells. This effect was completely prevented by treatment of ovariectomized rats with oestradiol. When intact female rats were treated with the anti‐oestrogen tamoxifen, a drastic decrease in NF68 expression in anterior pituitary cells was observed. Furthermore, oestradiol administration in castrated male rats increased NF68 immunoreactivity. Double‐immunolabelling experiments provided evidence that pituitary cells expressing neuronal traits correspond to subsets of lactotrophs, somatotrophs, thyrotrophs and gonadotrophs. It remains to be established if NF68 induction in the pituitary is due to direct and/or indirect effects of oestrogens. Also, the possible functional role of this subset of NF68‐positive anterior pituitary cells in the female rat remains to be examined.

GnRH-Induced Ca2+ Signaling Patterns and Gonadotropin Secretion in Pituitary Gonadotrophs. Functional Adaptations to Both Ordinary and Extraordinary Physiological Demands

Pituitary gonadotrophs are a small fraction of the anterior pituitary population, yet they synthesize gonadotropins: luteinizing (LH) and follicle-stimulating (FSH), essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca2+ rises produced do not influence secretion, which is mainly driven by Gonadotropin-Releasing Hormone (GnRH), a decapeptide synthesized in the hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation. GnRH binding to G-protein-coupled receptors triggers Ca2+ mobilization from InsP3-sensitive intracellular pools, generating the global Ca2+ elevations necessary for secretion. Ca2+ signaling responses to increasing (GnRH) vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory), to biphasic (spike-oscillatory or spike-plateau). This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca2+ signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin, and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases, and Ca2+ signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary functional demand.

Immunoreactivity to Neurofilaments in the Rodent Anterior Pituitary is Associated with the Expression of α1A Protein Subunits of Voltage-Gated Ca2+ Channels

We recently reported that rodent anterior pituitary (AP) cells (with the exception of corticotrophs and melanotrophs) express neuronal markers, including 68‐kDa neurofilaments (NF68) in an oestrogen‐dependent manner. The functional significance of neurofilament (NF) expression in the AP is unknown, but recent data in myelinated nerve fibres from NF‐null mice suggest that NFs can regulate ion channel function. Because Ca2+ influx through voltage‐gated Ca2+ channels is required for hormone secretion in AP cells, and oestrogen regulates the expression of Ca2+ channels in AP cells, the present study examined the expression of α1 subunits of voltage gated Ca2+ channels in relation to that of NF68. Using quantitative immunofluorescence, we demonstrate that α1C and α1D subunits are abundantly expressed in female AP cells, α1A subunits are moderately expressed, and α1G and α1B subunits are expressed at the lowest levels. Double‐immunostaining showed that NF68 expression is not correlated with that of α1C, α1D or α1B. Expression of α1G and NF68 appear to be mutually exclusive from each other. Moreover, α1A subunit and NF68 expression are significantly correlated and α1A immunoreactivity is sexually dimorphic (i.e. low in males and high in females) and its levels of expression vary during the oestrous cycle, similar to NF68. Finally, ω‐agatoxin IVA, a specific blocker of P/Q type Ca2+ currents that are a result of the activity of α1A subunits, inhibited to a greater extent spontaneous [Ca2+]i fluctuations in AP cells from females in oestrous and dioestrous, whereas cells from females in pro‐oestrous and males were less affected by this toxin. These results suggest a preferential participation of P/Q‐type Ca2+ channels and hence α1A subunits, in regulating spontaneous Ca2+ transients in AP cells under conditions where the proportion of NF68‐expressing cells is high. It remains to be determined whether the expression of NF68 affects that of α1A Ca2+ channel subunits or vice versa.

Prolactin Released in vitro from the Pituitary of Lactating, Pregnant, and Steroid-Treated Female or Male Rats Stimulates Prolactin Secretion from Pituitary Lactotropes of Male Rats

We have previously shown that soluble factor(s) in conditioned media (CM) from the central and peripheral regions of the anterior pituitary (AP) gland of lactating rats promoted the in vitro dose-related release of prolactin (PRL) from pituitary glands of male rats. In the present experiments we sought to determine whether CM from rats in different physiological states provoked similar effects (like those of lactating rats), and the nature of the factors, whether 23K PRL or other variants of the hormone, were responsible for these effects. Stimulatory effects were induced by CM from pregnant females and steroid-treated castrated males or females, but not from untreated castrated rats, intact males, or by a PRL standard. More potent effects occurred with CM from APs of early- than from mid- or late-lactating rats, and from rats unsuckled for 8 or 16 h than from those unsuckled for 32 h. With respect to the nature of factor(s) responsible for these effects, immunoprecipitation of PRL from the CM of lactating females and of steroid-treated, castrated males eliminated, whereas dephosphorylation or deglycosylation of CM of lactating rats greatly increased its effects upon PRL release. Also, electrophoretic analysis and Western blotting of the CM proteins under native and denaturing conditions revealed a variety of PRL variants, ranging from 14 to <90 kDa, in CM from lactating rats, and the main effects on PRL release were provoked by the 23- to 46-kDa PRL variants. These results indicate that specific effects upon male rat lactotropes may be exerted by PRL variants released from APs of lactating and non-lactating rats.

Cells of Proopiomelanocortin Lineage from the Rodent Anterior Pituitary Lack Sexually Dimorphic Expression of Neurofilaments

Lactotrophs, gonadotrophs, thyrotrophs and somatotrophs of the rat anterior pituitary (AP) express 68-kDa neurofilaments (NF68) and other neuronal markers. NF68 expression in the AP appears to be estrogen-dependent, but its significance is unknown. The aims of this work were: (1) to establish the expression pattern of NF68 immunoreactivity in the mouse AP, and (2) discover if corticotrophs and melanotrophs from both rodent species also express NF68. Primary cultures and frozen sections of AP from sexually mature mice were immunolabeled with anti-NF68 antibodies. In separate experiments, samples were immunostained for NF68 and AP hormones. Here we report that mouse lactotrophs, gonadotrophs, thyrotrophs and somatotrophs also express NF68 in a sexually dimorphic manner. The percentages of non-expressing, weakly expressing and strongly expressing cells were similar between both rodent species, although NF68+ cells were about 50% less abundant in the mouse compared to the rat pituitary. Remarkably, our study shows for the first time that rodent pituitary cells from the proopiomelanocortin lineage nearly completely lack NF68 immunoreactivity. In this regard, they differ from the rest of the AP population. Our findings establish a foundation for experiments aimed at investigating the functional significance of estrogen-dependent regulation of NF68 expression in rodent AP cells.