Endothelin increases the proliferation of rat olfactory mucosa cells.

The olfactory mucosa holds olfactory sensory neurons directly in contact with an aggressive environment. In order to maintain its integrity, it is one of the few neural zones which are continuously renewed during the whole animal life. Among several factors regulating this renewal, endothelin acts as an anti-apoptotic factor in the rat olfactory epithelium. In the present study, we explored whether endothelin could also act as a proliferative factor. Using primary culture of the olfactory mucosa, we found that an early treatment with endothelin increased its growth. Consistently, a treatment with a mixture of BQ123 and BQ788 (endothelin receptor antagonists) decreased the primary culture growth without affecting the cellular death level. We then used combined approaches of calcium imaging, reverse transcriptase-quantitative polymerase chain reaction and protein level measurements to show that endothelin was locally synthetized by the primary culture until it reached confluency. Furthermore, in vivo intranasal instillation of endothelin receptor antagonists led to a decrease of olfactory mucosa cell expressing proliferating cell nuclear antigen (PCNA), a marker of proliferation. Only short-term treatment reduced the PCNA level in the olfactory mucosa cells. When the treatment was prolonged, the PCNA level was not statistically affected but the expression level of endothelin was increased. Overall, our results show that endothelin plays a proliferative role in the olfactory mucosa and that its level is dynamically regulated. This study was approved by the Comité d'éthique en expérimentation animale COMETHEA (COMETHEA C2EA -45; protocol approval #12-058) on November 28, 2012.

Dual role of DMXL2 in olfactory information transmission and the first wave of spermatogenesis.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice.

IL-17c is involved in olfactory mucosa responses to Poly(I:C) mimicking virus presence.

At the interface of the environment and the nervous system, the olfactory mucosa (OM) is a privileged pathway for environmental toxicants and pathogens towards the central nervous system. The OM is known to produce antimicrobial and immunological components but the mechanisms of action of the immune system on the OM remain poorly explored. IL-17c is a potent mediator of respiratory epithelial innate immune responses, whose receptors are highly expressed in the OM of mice. We first characterized the presence of the IL-17c and its receptors in the OM. While IL-17c was weakly expressed in the control condition, it was strongly expressed in vivo after intranasal administration of polyinosinic-polycytidylic (Poly I:C), a Toll Like Receptor 3 agonist, mimicking a viral infection. Using calcium imaging and electrophysiological recordings, we found that IL-17c can effectively activate OM cells through the release of ATP. In the longer term, intranasal chronic instillations of IL-17c increased the cellular dynamics of the epithelium and promoted immune cells infiltrations. Finally, IL-17c decreased cell death induced by Poly(I:C) in an OM primary culture. The OM is thus a tissue highly responsive to immune mediators, proving its central role as a barrier against airway pathogens.

Endothelin impacts on olfactory processing in rats.

In the olfactory epithelium, olfactory sensitive neurons and their axons are surrounded by glia-like cells called sustentacular cells, which maintain both the structural and ionic integrity of the olfactory mucosa. We have previously found that endothelin-1 (ET-1) can uncouple sustentacular cell gap junctions in vitro similarly as carbenoxolone, a known gap junction uncoupling agent. The role of gap junctions in odorant transduction remains controversial and we explored here if ET-1 naturally produced by the olfactory mucosa could impact odorant detection. Using calcium imaging on olfactory mucosa explant, we first confirmed that ET-1 uncouples gap junctions in an olfactory mucosa preparation preserving the tissue integrity. We next measured the olfactory epithelium responses to odorant stimulation using electro-olfactogram recordings. While the amplitude of the response was not modified by application of ET-1 and carbenoxolone, its repolarizing phase was slower after both treatments. We finally examined the behavioral performances of rat pups in an orientation test based on maternal odor recognition after intranasal instillations of ET-1 or carbenoxolone. While rat pups performances were decreased after ET-1 treatment, it was unchanged after carbenoxolone treatment. Overall, our results indicate that ET-1 modulates olfactory responses at least partly through gap junction uncoupling.

Fasting induces astroglial plasticity in the olfactory bulb glomeruli of rats.

The detection of food odors by the olfactory system, which plays a key role in regulating food intake and elaborating the hedonic value of food, is reciprocally influenced by the metabolic state. Fasting increases olfactory performance, notably by increasing the activity of olfactory bulb (OB) neurons. The glutamatergic synapses between olfactory sensory neurons and mitral cells in the OB glomeruli are regulated by astrocytes, periglomerular neurons, and centrifugal afferents. We compared the expansion of astroglial processes by quantifying GFAP-labeled areas in fed and fasted rats to see whether OB glomerular astrocytes are involved in the metabolic sensing and adaptation of the olfactory system. Glomerular astroglial spreading was much greater in all OB regions of rats fasted for 17 hr than in controls. Intra-peritoneal administration of the anorexigenic peptide PYY3-36 or glucose in 17 hr-fasted rats respectively decreased their food intake or restored their glycemia, and reversed the fasting-induced astroglial spreading. Direct application of the orexigenic peptides ghrelin or NPY to OB slices increased astroglial spreading, whereas PYY3-36 resulted in astroglial retraction, in agreement with the in vivo effects of fasting and satiety on glomerular astrocytes. Thus the morphological plasticity of OB glomerular astrocytes depends on the metabolic state of the rats and is influenced by peptides that regulate food intake. This plasticity may be part of the mechanism by which the olfactory system adapts to food intake.

Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa.

Several factors modulate the first step of odour detection in the rat olfactory mucosa (OM). Among others, vasoactive peptides such as endothelin might play multifaceted roles in the different OM cells. Like their counterparts in the central nervous system, the olfactory sensory neurons are encompassed by different glial-like non-neuronal OM cells; sustentacular cells (SCs) surround their cell bodies, whereas olfactory ensheathing cells (OECs) wrap their axons. Whereas SCs maintain both the structural and ionic integrity of the OM, OECs assure protection, local blood flow control and guiding of olfactory sensory neuron axons toward the olfactory bulb. We previously showed that these non-neuronal OM cells are particularly responsive to endothelin in vitro. Here, we confirmed that the endothelin system is strongly expressed in the OM using in situ hybridization. We then further explored the effects of endothelin on SCs and OECs using electrophysiological recordings and calcium imaging approaches on both in vitro and ex vivo OM preparations. Endothelin induced both robust calcium signals and gap junction uncoupling in both types of cells. This latter effect was mimicked by carbenoxolone, a known gap junction uncoupling agent. However, although endothelin is known for its antiapoptotic effect in the OM, the uncoupling of gap junctions by carbenoxolone was not sufficient to limit the cellular death induced by serum deprivation in OM primary culture. The functional consequence of the endothelin 1-induced reduction of the gap junctional communication between OM non-neuronal cells thus remains to be elucidated.

Neuropeptide Y enhances olfactory mucosa responses to odorant in hungry rats.

Neuropeptide Y (NPY) plays an important role in regulating appetite and hunger in vertebrates. In the hypothalamus, NPY stimulates food intake under the control of the nutritional status. Previous studies have shown the presence of NPY and receptors in rodent olfactory system, and suggested a neuroproliferative role. Interestingly, NPY was also shown to directly modulate olfactory responses evoked by a food-related odorant in hungry axolotls. We have recently demonstrated that another nutritional cue, insulin, modulates the odorant responses of the rat olfactory mucosa (OM). Therefore, the aim of the present study was to investigate the potential effect of NPY on rat OM responses to odorants, in relation to the animal's nutritional state. We measured the potential NPY modulation of OM responses to odorant, using electro-olfactogram (EOG) recordings, in fed and fasted adult rats. NPY application significantly and transiently increased EOG amplitudes in fasted but not in fed rats. The effects of specific NPY-receptor agonists were similarly quantified, showing that NPY operated mainly through Y1 receptors. These receptors appeared as heterogeneously expressed by olfactory neurons in the OM, and western blot analysis showed that they were overexpressed in fasted rats. These data provide the first evidence that NPY modulates the initial events of odorant detection in the rat OM. Because this modulation depends on the nutritional status of the animal, and is ascribed to NPY, the most potent orexigenic peptide in the central nervous system, it evidences a strong supplementary physiological link between olfaction and nutritional processes.

Olfaction under metabolic influences.

Recently published work and emerging research efforts have suggested that the olfactory system is intimately linked with the endocrine systems that regulate or modify energy balance. Although much attention has been focused on the parallels between taste transduction and neuroendocrine controls of digestion due to the novel discovery of taste receptors and molecular components shared by the tongue and gut, the equivalent body of knowledge that has accumulated for the olfactory system, has largely been overlooked. During regular cycles of food intake or disorders of endocrine function, olfaction is modulated in response to changing levels of various molecules, such as ghrelin, orexins, neuropeptide Y, insulin, leptin, and cholecystokinin. In view of the worldwide health concern regarding the rising incidence of diabetes, obesity, and related metabolic disorders, we present a comprehensive review that addresses the current knowledge of hormonal modulation of olfactory perception and how disruption of hormonal signaling in the olfactory system can affect energy homeostasis.

Short-term effects of kainic acid on CA1 hippocampal interneurons differentially vulnerable to excitotoxicity.

PURPOSE: We sought to identify the inhibitory interneurons of the rat hippocampal CA1 region selectively vulnerable in the kainic acid (KA) model of temporal lobe epilepsy and to determine whether their selective vulnerability could be due to differential short-term KA effects. METHODS: We quantified vulnerable interneurons in stratum oriens-alveus (O/A) by using immunohistochemistry for glutamic acid decarboxylase (GAD), parvalbumin (PV), and somatostatin (SS) after KA injections in rats, and then compared in normal slices the effects of KA on interneurons either in O/A (vulnerable to KA) or in strata radiatum and lacunosum-moleculare (R/LM) (resistant to KA) by using whole-cell recording and calcium imaging. RESULTS: GAD-, PV- and SS-positive cells in O/A were decreased after KA treatment in P20 and P30 rats. Both short (1-min) and long (10-min) applications of KA produced similar tetrodotoxin (TTX)-insensitive membrane depolarization and decrease in input resistance in O/A and R/LM interneurons. KA responses were antagonized by CNQX and GYKI52466, suggesting AMPA receptor activation. KA also generated a similar increase in intracellular Ca2+ in O/A and R/LM interneurons, which was antagonized by CNQX and GYKI52466. CONCLUSIONS: The selective vulnerability of GAD-, PV-, and SS-immunopositive O/A interneurons in the KA model may not arise from cell-specific short-term membrane effects or calcium responses induced by KA, but from other glutamate receptor-mediated excitotoxic processes.

Differential regulation of metabotropic glutamate receptor- and AMPA receptor-mediated dendritic Ca2+ signals by presynaptic and postsynaptic activity in hippocampal interneurons.

Calcium plays a crucial role as a ubiquitous second messenger and has a key influence in many forms of synaptic plasticity in neurons. The spatiotemporal properties of dendritic Ca2+ signals in hippocampal interneurons are relatively unexplored. Here we use two-photon calcium imaging and whole-cell recordings to study properties of dendritic Ca2+ signals mediated by different glutamate receptors and their regulation by synaptic activity in oriens/alveus (O/A) interneurons of rat hippocampus. We demonstrate that O/A interneurons express Ca2+-permeable AMPA receptors (CP-AMPARs) providing fast Ca2+ signals. O/A cells can also coexpress CP-AMPARs, Ca2+-impermeable AMPARs (CI-AMPARs), and group I/II metabotropic glutamate receptors (mGluRs) (including mGluR1a), in the same cell. CI-AMPARs are often associated with mGluRs, resulting in longer-lasting Ca2+ signals than CP-AMPAR-mediated responses. Finally, CP-AMPAR- and mGluR-mediated Ca2+ signals demonstrate distinct voltage dependence and are differentially regulated by presynaptic and postsynaptic activity: weak synaptic stimulation produces Ca2+ signals mediated by CP-AMPARs, whereas stronger stimulation, or weak stimulation coupled with postsynaptic depolarization, recruits Ca2+ signals mediated by mGluRs. Our results suggest that differential activation of specific glutamate receptor-mediated Ca2+ signals within spatially restricted dendritic microdomains may serve distinct signaling functions and endow oriens/alveus interneurons with multiple forms of Ca2+-mediated synaptic plasticity. Specific activation of mGluR-mediated Ca2+ signals by coincident presynaptic and postsynaptic activity fulfills the conditions for Hebbian pairing and likely underlies their important role in long-term potentiation induction at O/A interneuron synapses.

Postsynaptic injection of calcium-independent phospholipase A2 inhibitors selectively increases AMPA receptor-mediated synaptic transmission.

The calcium-independent form of phospholipase A2 (iPLA2), an enzyme known to generate arachidonic acid (AA), was recently identified as the predominant constitutive phospholipase in the hippocampus. The present study shows that the iPLA2 inhibitor bromoenol lactone, when introduced into hippocampal CA1 pyramidal cells through a patch pipette, generated a dose-dependent increase in the amplitude of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs). The iPLA2 inhibitor by itself interfered with neither paired pulse facilitation nor N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs, suggesting that its influence on synaptic transmission is postsynaptic in origin and specific to the AMPA subtype of glutamate receptors. Comparable results were obtained with palmitoyl trifluoromethyl ketone, a second structurally distinct iPLA2 inhibitor. The ability of iPLA2 inhibitors to increase AMPA receptor-mediated currents was also reproduced by MK-866, an inhibitor recognized to interfere with the generation of 5-lipoxygenase by-products of AA. At the biochemical level, we found that AMPA, but not NMDA glutamate receptor subunits, were upregulated in rat brain sections pre-incubated with the iPLA2 inhibitors. Collectively, these results provide the first experimental evidence that constitutive iPLA2 and/or its metabolites play an important role in the postsynaptic modulation of neurotransmission in CA1 pyramidal cells of the hippocampus.

Depolarization-induced long-term depression at hippocampal mossy fiber-CA3 pyramidal neuron synapses.

Hippocampal CA3 pyramidal neurons receive two types of excitatory afferent innervation: mossy fibers (MFs) from granule cells of the dentate gyrus and recurrent collateral fibers (CFs) from other CA3 pyramidal neurons. At CF-CA3 pyramidal neuron synapses, membrane depolarization paired with low (0.33 Hz) presynaptic stimulation generated a heterogeneous response that ranged from long-term potentiation (LTP), long-term depression (LTD), to no alteration of synaptic strength. However, the same induction paradigm applied at MF-CA3 pyramidal neuron synapses consistently induced LTD. This novel form of LTD was independent of NMDARs, mGluRs, cannabinoid receptors, opioid receptors, or coincident synaptic activity, but was dependent on postsynaptic Ca2+ elevation through L-type Ca2+ channels and release from inositol 1,4,5-trisphosphate receptor-sensitive intracellular stores. Ca2+ imaging of both proximal and distal CA3 pyramidal neuron dendrites demonstrated that the depolarizing induction paradigm differentially elevated intracellular Ca2+ levels. L-type Ca2+ channel activation was observed only at the most proximal locations where mossy fibers make synapses. Depolarization-induced LTD did not occlude the conventional 1 Hz-induced LTD or vice versa, suggesting independent mechanisms underlie each form of plasticity. The paired-pulse ratio and coefficient of variation of synaptic transmission were unchanged after LTD induction, suggesting that the expression locus of LTD is postsynaptic. Moreover, peak-scaled nonstationary variance analysis indicated that depolarization-induced LTD correlated with a reduction in postsynaptic AMPA receptor numbers without a change in AMPA receptor conductance. Our results suggest that this novel form of LTD is selectively expressed at proximal dendritic locations closely associated with L-type Ca2+ channels.

GABAB receptor- and metabotropic glutamate receptor-dependent cooperative long-term potentiation of rat hippocampal GABAA synaptic transmission.

Repetitive stimulation of Schaffer collaterals induces activity-dependent changes in the strength of polysynaptic inhibitory postsynaptic potentials (IPSPs) in hippocampal CA1 pyramidal neurons that are dependent on stimulation parameters. In the present study, we investigated the effects of two stimulation patterns, theta-burst stimulation (TBS) and 100 Hz tetani, on pharmacologically isolated monosynaptic GABAergic responses in adult CA1 pyramidal cells. Tetanization with 100 Hz trains transiently depressed both early and late IPSPs, whereas TBS induced long-term potentiation (LTP) of early IPSPs that lasted at least 30 min. Mechanisms mediating this TBS-induced potentiation were examined using whole-cell recordings. The paired-pulse ratio of monosynaptic inhibitory postsynaptic currents (IPSCs) was not affected during LTP, suggesting that presynaptic changes in GABA release are not involved in the potentiation. Bath application of the GABAB receptor antagonist CGP55845 or the group I/II metabotropic glutamate receptor antagonist E4-CPG inhibited IPSC potentiation. Preventing postsynaptic G-protein activation or Ca2+ rise by postsynaptic injection of GDP-beta-S or BAPTA, respectively, abolished LTP, indicating a G-protein- and Ca2+-dependent induction in this LTP. Finally during paired-recordings, activation of individual interneurons by intracellular TBS elicited solely short-term increases in average unitary IPSCs in pyramidal cells. These results indicate that a stimulation paradigm mimicking the endogenous theta rhythm activates cooperative postsynaptic mechanisms dependent on GABABR, mGluR, G-proteins and intracellular Ca2+, which lead to a sustained potentiation of GABAA synaptic transmission in pyramidal cells. GABAergic synapses may therefore contribute to functional synaptic plasticity in adult hippocampus.

Perturbation of synaptic vesicle delivery during neurotransmitter release triggered independently of calcium influx.

Although much evidence suggests that calcium (Ca(2+)) usually triggers synaptic vesicle exocytosis and neurotransmitter release, the role of Ca(2+) in vesicle endocytosis and in the delivery of fusion-competent vesicles (i.e. mobilisation and/or priming) in nerve terminals remains unclear. To address this issue, we have studied synaptic vesicle dynamics in cultured rat neurones under conditions where neurotransmitter release is triggered independently of Ca(2+) using the secretagogue Ruthenium Red (RR). Using a prolonged stimulation protocol, we find that RR causes a rapid increase in neurotransmitter release followed by a gradually decrementing response. In contrast, when release is triggered by moderate membrane depolarisation caused by saline containing 18 mM K(+), release is sustained. These observations suggest that when release is triggered independently of a rise in Ca(2+), endocytosis or vesicle mobilisation/priming are perturbed. Using FM2-10, a fluorescent indicator of synaptic vesicle cycling, we find that neurotransmitter release triggered by RR is accompanied by both uptake and release of this dye, thereby suggesting that vesicle endocytosis is not blocked. To evaluate whether synaptic vesicle mobilisation/priming is perturbed in the absence of a rise in Ca(2+), we compared the kinetics of FM2-10 loss during prolonged stimulation. While 18 mM K(+) induced gradual and continuous dye loss, RR only induced substantial dye loss during the first minute of stimulation. In the presence of low concentrations of the Ca(2+) ionophore ionomycin, release caused by RR was prolonged. Taken together, these results provide evidence suggesting that, although a rise in intraterminal Ca(2+) is not required for endocytosis, it is essential for the continuous delivery of fusion-competent vesicles and to maintain neurotransmitter release during prolonged stimulation.

D2 receptors inhibit the secretory process downstream from calcium influx in dopaminergic neurons: implication of K+ channels.

Dopaminergic (DAergic) neurons possess D2-like somatodendritic and terminal autoreceptors that modulate cellular excitability and dopamine (DA) release. The cellular and molecular processes underlying the rapid presynaptic inhibition of DA release by D2 receptors remain unclear. Using a culture system in which isolated DAergic neurons establish self-innervating synapses ("autapses") that release both DA and glutamate, we studied the mechanism by which presynaptic D2 receptors inhibit glutamate-mediated excitatory postsynaptic currents (EPSCs). Action-potential evoked EPSCs were reversibly inhibited by quinpirole, a selective D2 receptor agonist. This inhibition was slightly reduced by the inward rectifier K(+) channel blocker barium, largely prevented by the voltage-dependent K(+) channel blocker 4-aminopyridine, and completely blocked by their combined application. The lack of a residual inhibition of EPSCs under these conditions argues against the implication of a direct inhibition of presynaptic Ca(2+) channels. To evaluate the possibility of a direct inhibition of the secretory process, spontaneous miniature EPSCs were evoked by the Ca(2+) ionophore ionomycin. Ionomycin-evoked release was insensitive to cadmium and dramatically reduced by quinpirole, providing evidence for a direct inhibition of quantal release at a step downstream to Ca(2+) influx through voltage-dependent Ca(2+) channels. Surprisingly, this effect of quinpirole on ionomycin-evoked release was blocked by 4-aminopyridine. These results suggest that D2 receptor activation decreases neurotransmitter release from DAergic neurons through a presynaptic mechanism in which K(+) channels directly inhibit the secretory process.