Neuronal Response Latencies Encode First Odor Identity Information across Subjects.

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.

Morphofunctional experience-dependent plasticity in the honeybee brain.

Repeated or prolonged exposure to an odorant without any positive or negative reinforcement produces experience-dependent plasticity, which results in habituation and latent inhibition. In the honeybee (Apis mellifera), it has been demonstrated that, even if the absolute neural representation of an odor in the primary olfactory center, the antennal lobe (AL), is not changed by repeated presentations, its relative representation with respect to unfamiliar stimuli is modified. In particular, the representation of a stimulus composed of a 50:50 mixture of a familiar and a novel odorant becomes more similar to that of the novel stimulus after repeated stimulus preexposure. In a calcium-imaging study, we found that the same functional effect develops following prolonged odor exposure. By analyzing the brains of the animals subjected to this procedure, we found that such functional changes are accompanied by morphological changes in the AL (i.e., a decrease in volume in specific glomeruli). The AL glomeruli that exhibited structural plasticity also modified their functional responses to the three stimuli (familiar odor, novel odor, binary mixture). We suggest a model in which rebalancing inhibition within the AL glomeruli may be sufficient to elicit structural and functional correlates of experience-dependent plasticity.

Spatially resolved time-frequency analysis of odour coding in the insect antennal lobe.

Antennal lobes constitute the first neurophils in the insect brain involved in coding and processing of olfactory information. With their stereotyped functional and anatomical organization, they provide an accessible model with which to investigate information processing of an external stimulus in a neural network in vivo. Here, by combining functional calcium imaging with time-frequency analysis, we have been able to monitor the oscillatory components of neural activity upon olfactory stimulation. The aim of this study is to investigate the presence of stimulus-induced oscillatory patterns in the honeybee antennal lobe, and to analyse the distribution of those patterns across the antennal lobe glomeruli. Fast two-photon calcium imaging reveals the presence of low-frequency oscillations, the intensity of which is perturbed by an incoming stimulus. Moreover, analysis of the spatial arrangement of this activity indicates that it is not homogeneous throughout the antennal lobe. On the contrary, each glomerulus displays an odorant-specific time-frequency profile, and acts as a functional unit of the oscillatory activity. The presented approach allows simultaneous recording of complex activity patterns across several nodes of the antennal lobe, providing the means to better understand the network dynamics regulating olfactory coding and leading to perception.

Macromolecule loading into spherical, elliptical, star-like and cubic calcium carbonate carriers.

We fabricated calcium carbonate particles with spherical, elliptical, star-like and cubical morphologies by varying relative salt concentrations and adding ethylene glycol as a solvent to slow down the rate of particle formation. The loading capacity of particles of different isotropic (spherical and cubical) and anisotropic (elliptical and star-like) geometries is investigated, and the surface area of such carriers is analysed. Potential applications of such drug delivery carriers are highlighted.

A multimodal approach for tracing lateralisation along the olfactory pathway in the honeybee through electrophysiological recordings, morpho-functional imaging, and behavioural studies.

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our laboratories, aimed at tracing asymmetries at different stages along the honeybee's (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy was used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation.

BACKGROUND: To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. METHODS: To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. RESULTS AND CONCLUSIONS: By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. GENERAL SIGNIFICANCE: The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Isolation of the left atrial surface from cardiac multi-detector CT images based on marker controlled watershed segmentation.

The delineation of left atrium (LA) and pulmonary veins (PVs) anatomy from high resolution images holds importance for atrial fibrillation (AF) investigation and treatment. In this study, a semiautomatic segmentation procedure for LA and PVs inner surface from contrast enhanced CT data was developed. The procedure consists of a three dimensional marker controlled watershed segmentation applied to the external morphological gradient, followed by variable threshold surface extraction from the original intensity image. A preliminary anisotropic non-linear filtering was implemented to improve the S/N ratio of CT images. The performance of segmentation was evaluated on cardiac CT scans of 12 AF patients both qualitatively and quantitatively. The qualitative evaluation by expert radiologist assessed the segmentation as overall successful in all patients and capable of extracting both the LA body and the connected vascular trees. The quantitative validation, by computing discrepancy measures with respect to a manually segmented gold standard, indicated an average of about 90% of voxels correctly classified and an average border mismatch lower than 1.5 voxels (1.2 mm). The accurate extraction of the inner LA-PVs walls provided by this method, along with the minimal required human intervention, should facilitate the use of anatomical atrial models for the non-pharmacological treatment of AF.

A stochastic approach for automatic registration and fusion of left atrial electroanatomic maps with 3D CT anatomical images.

The integration of electroanatomic maps with highly resolved computed tomography cardiac images plays an important role in the successful planning of the ablation procedure of arrhythmias. In this paper, we present and validate a fully-automated strategy for the registration and fusion of sparse, atrial endocardial electroanatomic maps (CARTO maps) with detailed left atrial (LA) anatomical reconstructions segmented from a pre-procedural MDCT scan. Registration is accomplished by a parameterized geometric transformation of the CARTO points and by a stochastic search of the best parameter set which minimizes the misalignment between transformed CARTO points and the LA surface. The subsequent fusion of electrophysiological information on the registered CT atrium is obtained through radial basis function interpolation. The algorithm is validated by simulation and by real data from 14 patients referred to CT imaging prior to the ablation procedure. Results are presented, which show the validity of the algorithmic scheme as well as the accuracy and reproducibility of the integration process. The obtained results encourage the application of the integration method in post-intervention ablation assessment and basic AF research and suggest the development for real-time applications in catheter guiding during ablation intervention.

Causal cross-spectral analysis of heart rate and blood pressure variability for describing the impairment of the cardiovascular control in neurally mediated syncope.

A causal approach to the calculation of coherence and transfer function between systolic pressure (SP) and RR interval variability was applied in eight patients and eight control subjects during prolonged tilt test for investigating the impairment of cardiovascular control related to neurally mediated syncope. The causal analysis showed a depressed baroreflex regulation in resting patients, with reduced gain and increased latency from SP to RR, and a drop of the baroreflex coupling immediately before syncope. These findings, which were not elicited by traditional cross-spectral analysis, strongly suggest the use of the causal approach for the study of syncope mechanisms.

Efficiency in endotoxin removal by a reprocessing protocol for electrophysiology catheters based on hydrogen peroxide plasma sterilization.

Electrophysiology and ablation cardiac catheters, which come in contact with blood during clinical use, are required to be non-pyrogenic (<20 endotoxin units (EU)/device). This study aimed to quantify the residual endotoxin load in reprocessed devices as a mandatory step to guarantee safe reuse. We monitored the pyrogenic status of the device (n=61) in three fundamental steps of the reprocessing protocol: after clinical use, after decontamination-cleaning treatments and after complete reprocessing, including sterilization by hydrogen peroxide gas plasma. Finally, a depyrogenation test was produced for evaluating the depyrogenation efficiency of the sole hydrogen peroxide sterilization treatment. Results showed that standard clinical use did not represent a source for endotoxin contamination, while the use of tap water and manual cleaning processing could increase the pyrogenic load in a significant way. The introduction of the sterilization by hydrogen peroxide gas plasma resulted in effective reduction of the endotoxin contamination and in safe reprocessing of 15 of 15 clinically used catheters. In addition, tests conducted on in vitro spiked catheters showed that initial pyrogenic loads of 40, 80, 200EU/device were reduced to less than 11EU/device. Depyrogenation testing demonstrated efficiency in endotoxin reduction of more than 62 times (1.8log). These results show the determining role of hydrogen peroxide gas-plasma sterilization in the reduction of pyrogenic load on medical devices. Considering actual hygienic requirements at single-use device reprocessing, hydrogen peroxide gas-plasma sterilization can be considered as an efficient treatment at non-lumen cardiac electrophysiology catheter reprocessing.

Neonicotinoid-induced impairment of odour coding in the honeybee.

Exposure to neonicotinoid pesticides is considered one of the possible causes of honeybee (Apis mellifera) population decline. At sublethal doses, these chemicals have been shown to negatively affect a number of behaviours, including performance of olfactory learning and memory, due to their interference with acetylcholine signalling in the mushroom bodies. Here we provide evidence that neonicotinoids can affect odour coding upstream of the mushroom bodies, in the first odour processing centres of the honeybee brain, i.e. the antennal lobes (ALs). In particular, we investigated the effects of imidacloprid, the most common neonicotinoid, in the AL glomeruli via in vivo two-photon calcium imaging combined with pulsed odour stimulation. Following acute imidacloprid treatment, odour-evoked calcium response amplitude in single glomeruli decreases, and at the network level the representations of different odours are no longer separated. This demonstrates that, under neonicotinoid influence, olfactory information might reach the mushroom bodies in a form that is already incorrect. Thus, some of the impairments in olfactory learning and memory caused by neonicotinoids could, in fact, arise from the disruption in odor coding and olfactory discrimination ability of the honey bees.

Differential Odour Coding of Isotopomers in the Honeybee Brain.

The shape recognition model of olfaction maintains that odorant reception probes physicochemical properties such as size, shape, electric charge, and hydrophobicity of the ligand. Recently, insects were shown to distinguish common from deuterated isotopomers of the same odorant, suggesting the involvement of other molecular properties to odorant reception. Via two-photon functional microscopy we investigated how common and deuterated isoforms of natural odorants are coded within the honeybee brain. Our results provide evidence that (i) different isotopomers generate different neuronal activation maps, (ii) isotopomer sensitivity is a general mechanism common to multiple odorant receptors, and (iii) isotopomer specificity is highly consistent across individuals. This indicates that honeybee's olfactory system discriminates between isotopomers of the same odorant, suggesting that other features, such as molecular vibrations, may contribute to odour signal transduction.

Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope.

We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology.

Quantification of synchronization during atrial fibrillation by Shannon entropy: validation in patients and computer model of atrial arrhythmias.

Atrial fibrillation (AF), a cardiac arrhythmia classically described as completely desynchronized, is now known to show a certain amount of synchronized electrical activity. In the present work a new method for quantifying the level of synchronization of the electrical activity recorded in pairs of atrial sites during atrial fibrillation is presented. A synchronization index (Sy) was defined by quantifying the degree of complexity of the distribution of the time delays between sites by Shannon entropy estimation. The capability of Sy to discriminate different AF types in patients was assessed on a database of 60 pairs of endocardial recordings from a multipolar basket catheter. The analysis showed a progressive and significant decrease of Sy with increasing AF complexity classes as defined by Wells (AF type I Sy = 0.73 +/- 0.07, type II Sy = 0.56 +/- 0.07, type III Sy = 0.36 +/- 0.04, p < 0.001). The extension of Sy calculation to the whole right atrium showed the existence of spatial heterogeneities in the synchronization level. Moreover, experiments simulated by a computer model of atrial arrhythmias showed that propagation patterns with different complexity could be the basis of different synchronization levels found in patients. In conclusion the quantification of synchronization by Shannon entropy estimation of time delay dispersion may facilitate the identification of different propagation patterns associated with AF, thus enhancing our understanding of AF mechanisms and helping in its treatment.

Photodamage in deep tissue two-photon optical biopsy of human skin.

Photodamage, induced by femtosecond laser radiation, was studied in thick samples of human skin tissue (healthy skin and neoplastic lesions). Photobleaching, photoionization, and thermomechanical damage effects were characterized comparatively. The laser power dependence of the damage rates allowed to connect macroscopic effects to underlying molecular processes. Optical effects were correlated to histopathological changes. Tissue alterations were found only from thermomechanical cavitation and limited to superficial layers of the epidermis. From the depth-dependencies of all damage thresholds a depth-dependent power-compensation scheme was defined allowing for damage-free deep tissue optical biopsy. Damage-induced luminescence pattern for different excitation powers and a corresponding threshold analysis.

A method for quantifying atrial fibrillation organization based on wave-morphology similarity.

A new method for quantifying the organization of single bipolar electrograms recorded in the human atria during atrial fibrillation (AF) is presented. The algorithm relies on the comparison between pairs of local activation waves (LAWs) to estimate their morphological similarity, and returns a regularity index (rho) which measures the extent of repetitiveness over time of the detected activations. The database consisted of endocardial data from a multipolar basket catheter during AF and intraatrial recordings during atrial flutter. The index showed maximum regularity (rho = 1) for all atrial flutter episodes and decreased significantly when increasing AF complexity as defined by Wells (type I: rho = 0.75 +/- 0.23; type II: rho = 0.35 +/- 0.11; type III: rho = 0.15 +/- 0.08; P < 0.01). The ability to distinguish different AF episodes was assessed by designing a classification scheme based on a minimum distance analysis, obtaining an accuracy of 85.5%. The algorithm was able to discriminate among AF types even in presence of few depolarizations as no significant rho changes were observed by reducing the signal length down to include five LAWs. Finally, the capability to detect transient instances of AF complexity and to map the local regularity over the atrial surface was addressed by the dynamic and multisite evaluation of rho, suggesting that our algorithm could improve the understanding of AF mechanisms and become useful for its clinical treatment.

Anticancer drug delivery system based on calcium carbonate particles loaded with a photosensitizer.

In photodynamic therapy (PDT), photosensitizers are required to arrive in high concentrations at selective targets like cancer cells avoiding toxicity in healthy tissue. In this work, we propose the application of porous calcium carbonate carriers in the form of polycrystalline vaterite for this task. We investigated the loading efficiency for the photosensitizer Photosens in vaterite micro- and nanocarriers. A possible release mechanism depending on the surrounding pH was studied, showing a fast degradation of the carriers in buffers below pH7. These results hold out the prospect of a novel PDT drug delivery system. Variation of particle size or additional coatings allow custom-design of workload release curves. An intrinsic cancer-sensitivity can be expected from the pH-dependent release in the acidic microenvironment of cancer tissue.

Tailored intracellular delivery via a crystal phase transition in 400 nm vaterite particles.

Porous vaterite containers of 400 nm size are studied with respect to intracellular drug delivery applications. A generic crystal phase transition from vaterite to calcite serves as a novel payload release mechanism, which reveals a delayed burst-release. This will permit control of the pharmacokinetics allowing for applications like preventive drug administration or scheduled application of pharmaceuticals during long term therapy. Experiments with two types of payloads, providing different molecular weights and zeta-potentials, demonstrate a flexible way of tailoring the payload delivery time via the molecular properties of the cargo. A dual in vitro cellular uptake experiment with human ovarian carcinoma cells ES2 and human fibroblasts MRC5 shows no cytotoxicity, no influence on cell viability, and fast penetration of substance-loaded containers into cells. Flow cytometry analysis proves high uptake rates and 3D microscopy analysis reveals the intracellular distribution.

Searching for anatomical correlates of olfactory lateralization in the honeybee antennal lobes: a morphological and behavioural study.

The honeybee, Apis mellifera L. (Hymenoptera: Apidae), has recently become a model for studying brain asymmetry among invertebrates. A strong lateralization favouring the right antenna was discovered in odour learning and short-term memory recall experiments, and a lateral shift favouring the left antenna for long-term memory recall. Corresponding morphological asymmetries have been found in the distribution of olfactory sensilla between the antennae and confirmed by electrophysiological odour response measurements in isolated right and left antennae. The aim of this study was to investigate whether a morphological asymmetry can be observed in the volume of the primary olfactory centres of the central nervous system, the antennal lobes (ALs). Precise volume measurements of a subset of their functional units, the glomeruli, were performed in both sides of the brain, exploiting the advantages of two-photon microscopy. This novel method allowed minimal invasive acquisition of volume images of the ALs, avoiding artefacts from brain extraction and dehydration. The study was completed by a series of behavioural experiments in which response asymmetry in odour recall following proboscis extension reflex conditioning was assessed for odours, chosen to stimulate strong activity in the same glomeruli as in the morphological study. The volumetric measurements found no evidence of lateralization in the investigated glomeruli within the experimental limits. Instead, in the behavioural experiments, a striking odour dependence of the lateralization was observed. The results are discussed on the basis of recent neurophysiological and ethological experiments in A. mellifera.