Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors.

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase. This complex enzyme cascade ultimately opens the light-dependent transient receptor potential channels, TRP and TRPL. PIP2, DAG, H± and 2-LG are possible channel activators, either individually or combined, but their direct participation in channel-gating remains unresolved. Molecular interaction with the channels, modification of the channels' lipid moiety and mechanical force on the channels by changes in the membrane structure derived from light-dependent changes in lipid composition are possible gating agents. In this regard, mechanical activation was suggested, based on a rapid light-dependent contraction of the photoreceptors mediated by the phototransduction cascade. Here, we further examined this possibility by applying force to inside-out patches from the microvilli membrane by changing the pressure in the pipette or pulling the membrane with a magnet through superparamagnetic nanospheres. The channels were opened by mechanical force, while mutant lacking both channels was insensitive to mechanical stimulation. Atomic Force Microscopy showed that the stiffness of an artificial phospholipid bilayer was increased by arachidonic acid and diacylglycerol whereas elaidic acid was ineffective, mirroring their relative effects in channel activity previously observed electrophysiologically. Together, the results are consistent with the notion that light-induced changes in lipid composition alter the membrane structure, generating mechanical force on the channels leading to channel opening.

Energy sources that fuel metabolic processes in protruding finger-like organelles.

Cells possess a variety of organelles with characteristic structure and subcellular localization intimately linked to their specific function. While most are intracellular and found in virtually all eukaryotic cells, there is a small group of organelles of elongated cylindrical shapes in highly specialized cells that protrude into the extracellular space, such as cilia, flagella, and microvilli. The ATP required by intracellular organelles is amply available in the cytosol, largely generated by mitochondria. However, such is not the case for cilia and flagella, whose slender structures cannot accommodate mitochondria. These organelles consume massive amounts of ATP to carry out high energy-demanding functions, such as sensory transduction or motility. ATP from the nearest mitochondria or other reactions within the cell body is severely limited by diffusion and generally insufficient to fuel the entire length of cilia and flagella. These organelles overcome this fuel restriction by local generation of ATP, using mechanisms that vary depending on the nutrients that are available in their particular external environment. Here, we review, with emphasis in mammals, the remarkable adaptations that cilia and flagella use to fuel their metabolic needs. Additionally, we discuss how a decrease in nutrients surrounding olfactory cilia might impair olfaction in COVID-19 patients.

Modulation of Frequency Preference in Heterogeneous Populations of Theta-resonant Neurons.

Neurons from several brain regions resonate in the theta frequency range (4-12 Hz), displaying a higher voltage response to oscillatory currents at a preferred 'resonant' frequency (fR). Subthreshold resonance could influence spiking and contribute to the selective entrainment of neurons during the network oscillatory activity that accompanies several cognitive processes. Neurons from different regions display resonance in specific theta subranges, suggesting a functional specialization. Further experimental work is needed to characterize this diversity and explore how frequency preference could be dynamically modulated. Theoretical studies have shown that the fine-tuning of resonance depends in a complex way on a variety of intrinsic factors and input properties, but their specific influence is difficult to dissect in cells. We performed slice electrophysiology, dynamic clamping and modelling to assess the differential frequency preference of rat entorhinal stellate neurons, hippocampal CA1 pyramidal neurons and cortical amygdala neurons, which share a hyperpolarization-activated current (Ih)-dependent resonance mechanism. We found heterogeneous resonance properties among the different types of theta-resonant neurons, as well as in each specific group. In all the neurons studied, fR inversely correlated with the effective input resistance (Rin), a measurable variable that depends on passive and active membrane features. We showed that resonance can be adjusted by manipulations mimicking naturally occurring processes, as the incorporation of a virtual constant conductance or cell depolarization, in a way that preserves the fR-Rin relationship. The modulation of frequency selectivity influences firing by shifting spike frequency and timing, which could influence neuronal communication in an active network.

Role of TRPM8 Channels in Altered Cold Sensitivity of Corneal Primary Sensory Neurons Induced by Axonal Damage.

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca2+ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold- and menthol-evoked intracellular [Ca2+] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K+ current IKD Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low- and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(+) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.SIGNIFICANCE STATEMENT We unveil a key role of TRPM8 channels in the sensory and autonomic disturbances associated with surgical damage of eye surface nerves. We studied the damage-induced functional alterations of corneal cold-sensitive neurons using confocal tracking of reinnervation, extracellular corneal nerve terminal recordings, tearing measurements in vivo, Ca2+ imaging and patch-clamp recordings of cultured corneal neurons, and mathematical modeling. Corneal nerve ablation upregulates TRPM8 mainly in canonical cold thermoreceptors, enhancing their cold and menthol sensitivity, inducing a rise in the ongoing firing activity of TRPM8(+) nerve endings and an increase in basal tearing. Our results suggest that unpleasant dryness sensations, together with augmented tearing rate after corneal nerve injury, are largely due to upregulation of TRPM8 in cold thermoreceptor neurons.

Performance Evaluation of Clinical Decision Support Systems (CDSS): Developing a Business Intelligence (BI) Dashboard.

This document describes the development of a Business Intelligence (BI) dashboard for tracking the drug-drug interaction (DDI) alerts implemented as Clinical Decision Support Systems (CDSS) in Electronic Health Records (EHR). CDSS are known for their potential to reduce medical error. The use of requirements in the development of BI dashboards is crucial to obtain successful software. In this work, the requirements were analysed using a score methodology, considering the relevance of the indicators and visualization methods. CDSS effectiveness and acceptance have been questioned, so it is fundamental to monitor their behaviour and performance. The dashboard was designed in order to satisfy the needed indicators. Using BI as a tool for monitoring the CDSS performance made it possible to operationalize the EHR content repository, maximizing the understanding in relation to the override and, by inference, to optimize the CDSS system by opening new lines of work.

Possible ATP trafficking by ATP-shuttles in the olfactory cilia and glucose transfer across the olfactory mucosa.

Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.

Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors.

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.

A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl- current when transfected into HEK293.

BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.

Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose.

The mechanisms that power the physiological events occurring in cilia, flagella, and microvilli are of fundamental importance for the functions of these important and ubicuous organelles. The olfactory epithelium is mostly populated by ciliated olfactory sensory neurons (OSNs) and surrounding sustentacular cells (SCs) with apical microvilli. The only OSN dendrite extends to the surface forming a knob projecting several chemosensory cilia of ∼50 × 0.2 µm, devoid of inner membranes embedded in a mucus layer. Upon odorant binding, odor receptors couple to G-protein activating adenylyl cyclase, producing cAMP. cAMP opens cyclic nucleotide-gated channels allowing a Ca2+ influx that opens Ca2+-activated Cl- channels, generating the receptor potential. Many enzymes are activated in chemotransduction to hydrolyze ATP. The knob contains approximately two mitochondria; assuming that the cilia ATP is 1 mm and diffuses along it at ∼10 µm in 500 ms, ATP from the knob mitochondria may not fulfill the demands of transduction over the full length of the cilium, which suggests an additional ATP source. We measured millimolar glucose in rat mucus; we detected glucose transporter GLUT3 in rat and toad (Caudiverbera caudiverbera) OSN cilia, SC microvilli, and glycolytic enzymes in rat cilia. We also found that the cilia and knob can incorporate and accumulate 2-deoxyglucose (glucose analog), but not when blocking GLUT. Glucose removal and the inhibition of glycolysis or oxidative phospholylation impaired the odor response. This evidence strongly suggests that glycolysis in the cilia and knob oxidative phosphorylation together fuel chemotransduction.SIGNIFICANCE STATEMENT How processes occurring in cilia and flagella are powered is a matter of general interest. Substantial progress has been made in unraveling the sensory transduction mechanisms, commonly occurring in such structures; however, the energy sources powering them have been scarcely explored. Accessibility to the specialized sensory organelles and their small dimensions have been limiting factors. Olfactory sensory neurons chemosensory cilia are elongated, mucus embedded, fully exposed structures particularly amenable for a multidisciplinary study of this problem, as done here. We demonstrate the occurrence and functionality of glucose uptake and glycolysis in the cilia. We support that odor transduction relies on ATP generated by oxidative phosphorylation in the dendrite and glycolytically in the cilia using glucose internalized from the mucus.

Implementation of an Outsourced Transnational Service of Clinical Decision Support System.

The aim of this study is to describe the implementation and evaluation of an outsourced Clinical Decision Support Systems (CDSS) service of drug-drug interaction (DDI) alerts in an Uruguayan outpatient healthcare network. A cross-sectional study was developed. 1.5 alerts were triggered of every 1000 prescriptions. Clinicians accepted 44% of the total alerts. In conclusion, the implementation of CDSS was achievable.

Single Ca(2+)-activated Cl(-) channel currents recorded from toad olfactory cilia.

BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.

Drosophila light-dependent channels, TRP and TRPL, reside in the light-sensitive microvilli of the photoreceptor's rhabdomere. Phospholipase C mediates TRP/TRPL opening, but the gating process remains unknown. Controversial evidence has suggested diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs, a DAG metabolite), phosphatidylinositol bisphosphate (PIP2), and H(+) as possible channel activators. We tested each of them directly in inside-out TRP-expressing patches excised from the rhabdomere, making use of mutants and pharmacology. When patches were excised in darkness TRP remained closed, while when excised under illumination it stayed constitutively active. TRP was opened by DAG and silenced by ATP, suggesting DAG-kinase (DGK) involvement. The ATP effect was abolished by inhibiting DGK and in the rdgA mutant, lacking functional DGK, implicating DGK. DAG activated TRP even in the presence of a DAG-lipase inhibitor, inconsistent with a requirement of PUFAs in opening TRP. PIP2 had no effect and acidification, pH 6.4, activated TRP irreversibly, unlike the endogenous activator. Complementary liquid-chromatography/mass-spectrometry determinations of DAG and PUFAs in membranes enriched in rhabdomere obtained from light- and dark-adapted eyes showed light-dependent increment in six DAG species and no changes in PUFAs. The results strongly support DAG as the endogenous TRP agonist, as some of its vertebrate TRPC homologs of the same channel family.

Transduction for pheromones in the main olfactory epithelium is mediated by the Ca2+ -activated channel TRPM5.

Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl- channels; in TRPM5-GFP+ OSNs, the Ca2+ -activated Cl- ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

Electrical resonance in the θ frequency range in olfactory amygdala neurons.

The cortical amygdala receives direct olfactory inputs and is thought to participate in processing and learning of biologically relevant olfactory cues. As for other brain structures implicated in learning, the principal neurons of the anterior cortical nucleus (ACo) exhibit intrinsic subthreshold membrane potential oscillations in the θ-frequency range. Here we show that nearly 50% of ACo layer II neurons also display electrical resonance, consisting of selective responsiveness to stimuli of a preferential frequency (2-6 Hz). Their impedance profile resembles an electrical band-pass filter with a peak at the preferred frequency, in contrast to the low-pass filter properties of other neurons. Most ACo resonant neurons displayed frequency preference along the whole subthreshold voltage range. We used pharmacological tools to identify the voltage-dependent conductances implicated in resonance. A hyperpolarization-activated cationic current depending on HCN channels underlies resonance at resting and hyperpolarized potentials; notably, this current also participates in resonance at depolarized subthreshold voltages. KV7/KCNQ K+ channels also contribute to resonant behavior at depolarized potentials, but not in all resonant cells. Moreover, resonance was strongly attenuated after blockade of voltage-dependent persistent Na+ channels, suggesting an amplifying role. Remarkably, resonant neurons presented a higher firing probability for stimuli of the preferred frequency. To fully understand the mechanisms underlying resonance in these neurons, we developed a comprehensive conductance-based model including the aforementioned and leak conductances, as well as Hodgkin and Huxley-type channels. The model reproduces the resonant impedance profile and our pharmacological results, allowing a quantitative evaluation of the contribution of each conductance to resonance. It also replicates selective spiking at the resonant frequency and allows a prediction of the temperature-dependent shift in resonance frequency. Our results provide a complete characterization of the resonant behavior of olfactory amygdala neurons and shed light on a putative mechanism for network activity coordination in the intact brain.

Fatty acid composition of Drosophila photoreceptor light-sensitive microvilli.

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

Ca2+-activated Cl- channels of the ClCa family express in the cilia of a subset of rat olfactory sensory neurons.

The Ca(2+)-activated Cl(-) channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca(2+) influx. Ca(2+) ions activate Cl(-) channels, generating a significant Cl(-) efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl(-) conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca(2+)-activated Cl(-) channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca(2+)-activated Cl(-) channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.

Fatty acid composition of Drosophila photoreceptor light-sensitive microvilli

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

Leukaemia inhibitory factor stimulates proliferation of olfactory neuronal progenitors via inducible nitric oxide synthase.

Neurogenesis continues in the adult brain and in the adult olfactory epithelium. The cytokine, leukaemia inhibitory factor and nitric oxide are both known to stimulate neuronal progenitor cell proliferation in the olfactory epithelium after injury. Our aim here was to determine whether these observations are independent, specifically, whether leukaemia inhibitory factor triggers neural precursor proliferation via the inducible nitric oxide synthase pathway. We evaluated the effects of leukaemia inhibitory factor on inducible form of nitric oxide synthase (iNOS) expression, and cell proliferation in olfactory epithelial cell cultures and olfactory neurosphere-derived cells. Leukaemia inhibitory factor induced expression of iNOS and increased cell proliferation. An iNOS inhibitor and an anti-leukaemia inhibitory factor receptor blocking antibody inhibited leukaemia inhibitory factor-induced cell proliferation, an effect that was reversed by a NO donor. Altogether, the results strongly suggest that leukaemia inhibitory factor induces iNOS expression, increasing nitric oxide levels, to stimulate proliferation of olfactory neural precursor cells. This finding sheds light on neuronal regeneration occurring after injury of the olfactory epithelium.

TRP, TRPL and cacophony channels mediate Ca2+ influx and exocytosis in photoreceptors axons in Drosophila.

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.

Altered voltage dependent calcium currents in a neuronal cell line derived from the cerebral cortex of a trisomy 16 fetal mouse, an animal model of Down syndrome.

Human Down syndrome (DS) is determined by the trisomy of autosome 21 and is expressed by multiple abnormalities, being mental retardation the most striking feature. The condition results in altered electrical membrane properties (EMPs) of fetal neurons, which are qualitatively identical to those of trisomy 16 fetal mice (Ts16), an animal model of the human condition. Ts16 hippocampal cultured neurons reportedly exhibit increased voltage-dependent calcium currents (I (Ca)) amplitude. Since Ts16 animals are unviable, we have established immortalized cell lines from the cerebral cortex of Ts16 (named CTb) and normal littermates (named CNh). Using the whole-cell patch-clamp technique, we have now studied I (Ca) in CTb and CNh cells. Current activation occurs at -40 mV in both cell lines (V (holding) = -80 mV). Trisomic cells exhibited a 2.4 fold increase in the maximal Ca(2+) current density compared to normal cells (CNh = -6.3 ± 0.77 pA/pF, n = 18; CTb = -16.4 ± 2.423 pA/pF; P < 0.01, n = 13). Time dependent kinetics for activation and inactivation did not differ between the two cell types. However, steady state inactivation studies revealed a 15 mV shift toward more depolarized potentials in the trisomic condition, suggesting that altered voltage dependence of inactivation may underlie the increased current density. Further, the total charge movement across the membrane is increased in CTb cells, in agreement with that expected by the potential sensitivity shift. These results indicate that CTb cells present altered Ca(2+) currents, similar to those of Ts16 primary cultured central neurons. The CTb cell line represents a model for studying DS-related impairments of EMPs.