Parallel processing in the honeybee olfactory pathway: structure, function, and evolution

Animals face highly complex and dynamic olfactory stimuli in their natural environments, which require fast and reliable olfactory processing. Parallel processing is a common principle of sensory systems supporting this task, for example in visual and auditory systems, but its role in olfaction remained unclear. Studies in the honeybee focused on a dual olfactory pathway. Two sets of projection neurons connect glomeruli in two antennal-lobe hemilobes via lateral and medial tracts in opposite sequence with the mushroom bodies and lateral horn. Comparative studies suggest that this dual-tract circuit represents a unique adaptation in Hymenoptera. Imaging studies indicate that glomeruli in both hemilobes receive redundant sensory input. Recent simultaneous multi-unit recordings from projection neurons of both tracts revealed widely overlapping response profiles strongly indicating parallel olfactory processing. Whereas lateral-tract neurons respond fast with broad (generalistic) profiles, medial-tract neurons are odorant specific and respond slower. In analogy to “what-” and “where” subsystems in visual pathways, this suggests two parallel olfactory subsystems providing “what-” (quality) and “when” (temporal) information. Temporal response properties may support across-tract coincidence coding in higher centers. Parallel olfactory processing likely enhances perception of complex odorant mixtures to decode the diverse and dynamic olfactory world of a social insect.     

Behavioral,neural and cellular components underlying olfactory learning in the honeybee

A top-down approach as applied to learning and memory in honeybees provides the opportunity of relating different levels of complexity to each other, and of analyzing the rules and mechanisms from the viewpoint of the respective next higher level. Olfactory conditioning of harnessed bees exemplifies essential elements of associative learning and, in general, forms a bridge between the systems and the cellular levels of analysis. Intracellular recordings of identified neurons during olfactory conditioning play a key role in this effort. They allow testing of the assumptions made by modern behavioral theories of associative learning and provide access to cellular and molecular studies, owing to the identification of their transmitters and the peculiarities of the connectivities. Analysis at this intermediate level of complexity is particularly profitable in the bee, because essential neural elements of the associative network are known and can be tested during ongoing learning behavior. In this respect, the honeybee offers unique properties for the building of bridges between the molecular, cellular neuronal, network and behavioral levels of associative learning.     

A combined electrophysiological and behavioural study for the assessment of activity-dependent changes in mice

Activity-dependent adaptive changes in the nervous system involve structural and functional changes in the cortical circuitry. In this work the cortical function was studied by repeated recording of the somatosensory and motor potentials evoked by whisker deflections after altered sensory-motor experience in adult mice. The latencies of motor and somatosensory evoked potentials were found to shorten, while their amplitudes decreased, after a behavioural challenge involving the vibrissal apparatus. Sensory deprivation achieved by whisker trimming resulted in a partial reversal of the changes observed after increased activity. The derived parameters imply that cortical information processing speeds up as a result of experience, while decreased activity has the opposite effect. The methods used throughout the experiment were minimally invasive, and thus proved to be sufficient for the long-term follow-up of cortical functions.     

A direct comparison of electrophysiological and behavioural taste responses in the blowfly

Behavioural and electrophysiological taste responses in theblowfly (Calliphora vicina) are measured in successive experiments,using the same flies twice. Inter-individual variations in behaviourare at least partly due to differences in the functioning ofthe tarsal taste hairs; flies with low behavioural thresholdvalues have taste hairs firing with higher rates. The percentageof taste hairs responding with spike trains is not important.Behavioural responses are predominantly influenced by the ‘best’firing hair. The most effective of the parameters describingspike trains seems to bef_t, the firing rate after an ‘infinite’length of stimulation time.     

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.     

A dynamical feedback model for adaptation in the olfactory transduction pathway

Olfactory transduction exhibits two distinct types of adaptation, which we denote multipulse and step adaptation. In terms of measured transduction current, multipulse adaptation appears as a decrease in the amplitude of the second of two consecutive responses when the olfactory neuron is stimulated with two brief pulses. Step adaptation occurs in response to a sustained steplike stimulation and is characterized by a return to a steady-state current amplitude close to the prestimulus value, after a transient peak. In this article, we formulate a dynamical model of the olfactory transduction pathway, which includes the kinetics of the CNG channels, the concentration of Ca ions flowing through them, and the Ca-complexes responsible for the regulation. Based on this model, a common dynamical explanation for the two types of adaptation is suggested. We show that both forms of adaptation can be well described using different time constants for the kinetics of Ca ions (faster) and the kinetics of the feedback mechanisms (slower). The model is validated on experimental data collected in voltage-clamp conditions using different techniques and animal species.     

Endocytic pathway for surfactant protein A in human macrophages: binding, clathrin-mediated uptake, and trafficking through the endolysosomal pathway

In the noninflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (MPhi) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human MPhi are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MPhi. SP-A binding was inhibited by EGTA, indicating calcium dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human MPhi into discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine MPhi over time, the trafficking pathway of SP-A through MPhi after uptake has not been reported and is of potential biological importance. We examined trafficking of SP-A in human MPhi by electron and confocal microscopy and show for the first time that SP-A is endocytosed by primary human MPhi through clathrin-coated pits and colocalizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human MPhi, is endocytosed by a receptor-mediated, clathrin-dependent process, and trafficks through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the MPhi cell surface and intracellular compartments that play important roles in SP-A modulation of lung MPhi biology.     

A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07‐07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07‐07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07‐07059 only marginally inhibits mitochondrial respiration and function. V07‐07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07‐07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)‐diacetate N‐succinimidyl ester, V07‐07059 proved superior regarding toxicity and photostability.

     

Physiological properties and response modulations of mushroom body feedback neurons during olfactory learning in the honeybee, Apis mellifera

Mushroom bodies are central brain structures and essentially involved in insect olfactory learning. Within the mushroom bodies γ-aminobutyric acid (GABA)-immunoreactive feedback neurons are the most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was investigated by analyzing modulations of odor responses of feedback neurons during olfactory learning in vivo. In the honeybee, Apis mellifera, feedback neurons were intracellularly recorded at their neurites. They produced complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials indicate that their synaptic input region lies within the lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of different neurons to the same odor were highly variable. Response modulations were determined by comparing odor responses of feedback neurons before and after one-trial olfactory conditioning or sensitisation. Shortly after pairing an odor stimulus with a sucrose reward, odor-induced spike activity of feedback neurons decreased. Repeated odor stimulations alone, equally spaced as in the conditioning experiment, did not affect the odor-induced excitation. A single sensitisation trial also did not alter odor responses. These findings indicate that the level of odor-induced inhibition within the mushroom bodies is specifically modulated by experience. Accepted: 9 September 1999     

A multimodal metabolomics approach using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used ¹⁵N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the ¹⁵N-labeled and non-labeled MS/MS spectra.     

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: a robust approach to an elusive problem

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.     

中华蜜蜂嗅觉受体AcerOrco的表达及定位分析

【目的】通过对中华蜜蜂Apis cerana cerana嗅觉受体AcerOrco的表达及蛋白定位分析,阐明AcerOrco的表达特性,以期为进一步探索其功能提供理论依据。【方法】分别通过qRT-PCR技术和Western blot技术对中华蜜蜂气味受体AcerOrco mRNA及蛋白在内勤蜂和采集蜂5个组织部位(触角、头、胸、腹和足)中的相对表达量进行分析,采用免疫组化技术对AcerOrco蛋白的表达进行定位分析。【结果】定量结果显示,AcerOrco转录本在内勤蜂和采集蜂各组织中均有表达,其中触角中的表达量最高;该基因在采集蜂各组织中的表达量普遍高于内勤蜂。AcerOrco受体蛋白在内勤蜂和采集蜂各组织中也均有表达,在触角和头部的表达量明显高于其他组织。定位结果显示,在内勤蜂触角中,AcerOrco主要在毛形感器中表达,板形感器中表达不明显;在采集蜂触角的板形感器和毛形感器中均有表达,但毛形感器中的表达更多一些。【结论】获得了AcerOrco mRNA及其蛋白在内勤蜂和采集蜂各组织中的表达特性,并将这一蛋白表达定位于工蜂触角的毛形感器和板形感器中。     

Quality coding by neural populations in the early olfactory pathway: analysis using information theory and lessons for artificial olfactory systems

In this article, we analyze the ability of the early olfactory system to detect and discriminate different odors by means of information theory measurements applied to olfactory bulb activity images. We have studied the role that the diversity and number of receptor neuron types play in encoding chemical information. Our results show that the olfactory receptors of the biological system are low correlated and present good coverage of the input space. The coding capacity of ensembles of olfactory receptors with the same receptive range is maximized when the receptors cover half of the odor input space - a configuration that corresponds to receptors that are not particularly selective. However, the ensemble's performance slightly increases when mixing uncorrelated receptors of different receptive ranges. Our results confirm that the low correlation between sensors could be more significant than the sensor selectivity for general purpose chemo-sensory systems, whether these are biological or biomimetic.     

中华蜜蜂Orco嗅觉受体基因的克隆、表达及亚细胞定位

【目的】克隆鉴定中华蜜蜂Apis cerana cerana的Orco嗅觉受体基因, 并对其在工蜂触角上进行免疫荧光定位。【方法】利用RT-PCR技术克隆中华蜜蜂Orco基因, 并对其编码的氨基酸序列进行生物信息学分析, 使用Real-time PCR技术鉴定其在中华蜜蜂不同发育时期及不同组织的表达谱; 利用免疫荧光定位技术在中华蜜蜂工蜂触角中对Orco进行亚细胞定位。【结果】获得中华蜜蜂Orco基因的全长cDNA序列, 命名为AcerOrco (GenBank登录号： JF968610.1), 其全长为1 434 bp, 编码477个氨基酸, 预测其含7个跨膜结构以及4个位于细胞膜外的亲水区。表达谱分析显示, AcerOrco在卵、 幼虫和蛹期呈低丰度表达, 1日龄及内勤蜂时期主要在触角和足中表达, 且在1日龄的触角中表达量最高; 采集蜂时期的触角、 头(去除触角)、 胸、 腹和翅中均有较高丰度的表达。亚细胞定位结果显示, AcerOrco不仅在采集蜂触角鞭节上大量表达（尤其在触角鞭节第1亚节中表达量较高）, 而且常成对出现, 并且发现AcerOrco可能主要在触角毛形感器的外部神经元(outer dendrite, OD)以及板形感器的树突神经元中表达。【结论】成功克隆了AcerOrco基因全长, 获得了其表达谱, 且将其定位于工蜂采集蜂的触角感器神经元上, 最终推测AcerOrco与中蜂嗅觉发育和触角感器功能密切相关。     

A unified approach for quantifying, testing and correcting population stratification in case-control association studies

The HapMap project has given case-control association studies a unique opportunity to uncover the genetic basis of complex diseases. However, persistent issues in such studies remain the proper quantification of, testing for, and correction for population stratification (PS). In this paper, we present the first unified paradigm that addresses all three fundamental issues within one statistical framework. Our unified approach makes use of an omnibus quantity (delta), which can be estimated in a case-control study from suitable null loci. We show how this estimated value can be used to quantify PS, to statistically test for PS, and to correct for PS, all in the context of case-control studies. Moreover, we provide guidelines for interpreting values of delta in association studies (e.g., at alpha = 0.05, a delta of size 0.416 is small, a delta of size 0.653 is medium, and a delta of size 1.115 is large). A novel feature of our testing procedure is its ability to test for either strictly any PS or only 'practically important' PS. We also performed simulations to compare our correction procedure with Genomic Control (GC). Our results show that, unlike GC, it maintains good Type I error rates and power across all levels of PS.     

Folate receptor-targeted multimodal fluorescence mesosilica nanoparticles for imaging,delivery palladium complex and in vitro G-quadruplex DNA interaction

New folic acid-conjugated mesoporous silica nanoparticles were synthesized. The effect of calcination at 400°C on the fluorescence characteristics of mesoporous silica nanoparticles were studied in this work. The formed carbon dots (CDs) from calcination were used as the source of fluorescence. 3-Aminopropyltriethoxysilane was then used to amine-functionalized the fluorescent surface of mesoporous silica nanoparticles. The amine fluorescence mesoporous silica nanoparticles (amine-FMSNs) were coupled with folic acid (FA) as the target ligand (FA-amine-FMSNs). A palladium complex was also synthesized and encapsulated in the FA-amine-FMSNs yielded fluorescent property with therapeutic effect. The in vitro release of an entrapped palladium complex from FA-amine-FMSNs was studied under physiological conditions. According to the cell viability assay on HeLa (positive FR) and Hep-G2 (negative FR) cells, the targeted delivery system inhibited the growth of positive FR with higher selectivity compared with negative FR cells. Also, the emission CDs were used for fluorescence microscopic imaging. To confirm anti-cancer activity of the palladium complex, the interaction between palladium complex and G-quadruplex DNA were investigated with multi-spectroscopic methods and molecular modeling. The molecular docking studies showed a partial intercalation mode with a 4.27 × 10⁵ M^?1 binding constant.