An objective, automated and robust scoring using fluorescence optical imaging to evaluate changes in micro-vascularisation indicating early arthritis.

Fluorescence optical imaging technique (FOI) is a well-established and valid method for visualization of changes in micro vascularization at different organ systems. As increased vascularization is an early feature of joint inflammation, FOI is a promising method to assess arthritis of the hands. But usability of the method is limited to the assessors experience as the measurement of FOI is semi-quantitative using an individual grading system such as the fluorescence optical imaging activity score (FOIAS). The goal of the study was to automatically and thus, objectively analyze the measured fluorescence intensity generated by FOI to evaluate the amount of inflammation of each of the subject's joints focusing on the distinction between normal joint status or arthritis in psoriatic arthritis patients compared to healthy volunteers. Due to the heterogeneity of the pathophysiological perfusion of the hands, a method to overcome the absoluteness of the data by extracting heatmaps out of the image stacks is developed. To calculate a heatmap for one patient, firstly the time series for each pixel is extracted, which is then represented by a feature value. Secondly, all feature values are clustered. The calculated cluster values represent the relativity between the different pixels and enable a comparison of multiple patients. As a metric to quantify the conspicuousness of a joint a score is calculated based on the extracted cluster values. These steps are repeated for a total number of three features. With this method a tendency towards a classification into unaffected and inflamed joints can be achieved. However, further research is necessary to transform the tendency into a robust classification model.

Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach.

Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the 'added buffer' approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

Quantitative estimation of calcium dynamics from ratiometric measurements: a direct, nonratioing method.

Measuring variations of intracellular free calcium concentration through the changes in fluorescence of a calcium-sensitive dye is a ubiquitous technique in neuroscience. Despite its popularity, confidence intervals (CIs) on the estimated parameters of calcium dynamics models are seldom given. To address this issue, we have developed a two-stage model for ratiometric measurements obtained with a charge-coupled device (CCD) camera. Its first element embeds a parametric calcium dynamics model into a fluorescence intensity model and its second element probabilistically describes the fluorescence measurements by a CCD camera. Using Monte Carlo simulations, we first show that the classical ratiometric transformation gives reliable CIs for time constants only and not baseline calcium concentration nor influx. We then introduce a direct method, which consists of fitting directly and simultaneously the fluorescence transients at both wavelengths, without any data ratioing. This approach uses a probabilistic description of the camera, leading to the construction of meaningful CIs for the calcium parameters. Moreover, using approaches inspired by constrained linear regression, we can take into account the finite precision on calibrated parameters (such as the dye dissociation constant in the cell). These key features are illustrated on simulated data using Monte Carlo simulations. Moreover, we illustrate the strength of the direct method on experimental recordings from insect olfactory interneurons. In particular, we show how to handle a time-dependent buffer concentration, thereby considerably improving our goodness of fit. The direct method was implemented in the open-source software R and is freely distributed in the CalciOMatic package.

Cholinergic currents in leg motoneurons of Carausius morosus.

We used patch-clamp recordings and fast optical Ca(2+) imaging to characterize an acetylcholine-induced current (I(ACh)) in leg motoneurons of the stick insect Carausius morosus. Our long-term goal is to better understand the synaptic and integrative properties of the leg sensory-motor system, which has served extremely successfully as a model to study basic principles of walking and locomotion on the network level. The experiments were performed under biophysically controlled conditions on freshly dissociated leg motoneurons to avoid secondary effects from the network. To allow for unequivocal identification, the leg motoneurons were backfilled with a fluorescent label through the main leg nerve prior to cell dissociation. In 87% of the motoneurons, I(ACh) consisted of a fast-desensitizing (I(ACh1)) and a slow-desensitizing component (I(ACh2)), both of which were concentration dependent, with EC(50) values of 3.7 x 10(-5) and 2.0 x 10(-5) M, respectively. Ca(2+) imaging revealed that a considerable portion of I(ACh) ( approximately 18%) is carried by Ca(2+), suggesting that I(ACh), besides mediating fast synaptic transmission, could also induce Ca(2+)-dependent processes. Using specific nicotinic and muscarinic acetylcholine receptor ligands, we showed that I(ACh) was exclusively mediated by nicotinic acetylcholine receptors. Distinct concentration-response relations of I(ACh1) and I(ACh2) for these ligands indicated that they are mediated by different types of nicotinic acetylcholine receptors.

Energy imbalance alters Ca2+ handling and excitability of POMC neurons.

Satiety-signaling, pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus play a pivotal role in the regulation of energy homeostasis. Recent studies reported altered mitochondrial dynamics and decreased mitochondria- endoplasmic reticulum contacts in POMC neurons during diet-induced obesity. Since mitochondria play a crucial role in Ca2+ signaling, we investigated whether obesity alters Ca2+ handling of these neurons in mice. In diet-induced obesity, cellular Ca2+ handling properties including mitochondrial Ca2+ uptake capacity are impaired, and an increased resting level of free intracellular Ca2+ is accompanied by a marked decrease in neuronal excitability. Experimentally increasing or decreasing intracellular Ca2+ concentrations reproduced electrophysiological properties observed in diet-induced obesity. Taken together, we provide the first direct evidence for a diet-dependent deterioration of Ca2+ homeostasis in POMC neurons during obesity development resulting in impaired function of these critical energy homeostasis-regulating neurons.

Estimating background-subtracted fluorescence transients in calcium imaging experiments: a quantitative approach.

Calcium imaging has become a routine technique in neuroscience for subcellular to network level investigations. The fast progresses in the development of new indicators and imaging techniques call for dedicated reliable analysis methods. In particular, efficient and quantitative background fluorescence subtraction routines would be beneficial to most of the calcium imaging research field. A background-subtracted fluorescence transients estimation method that does not require any independent background measurement is therefore developed. This method is based on a fluorescence model fitted to single-trial data using a classical nonlinear regression approach. The model includes an appropriate probabilistic description of the acquisition system's noise leading to accurate confidence intervals on all quantities of interest (background fluorescence, normalized background-subtracted fluorescence time course) when background fluorescence is homogeneous. An automatic procedure detecting background inhomogeneities inside the region of interest is also developed and is shown to be efficient on simulated data. The implementation and performances of the proposed method on experimental recordings from the mouse hypothalamus are presented in details. This method, which applies to both single-cell and bulk-stained tissues recordings, should help improving the statistical comparison of fluorescence calcium signals between experiments and studies.

Differences of Ca2+ handling properties in identified central olfactory neurons of the antennal lobe.

Information processing in neurons depends on highly localized Ca2+ signals. The spatial and temporal dynamics of these signals are determined by a variety of cellular parameters including the calcium influx, calcium buffering and calcium extrusion. Our long-term goal is to better understand how intracellular Ca2+ dynamics are controlled and contribute to information processing in defined interneurons of the insect olfactory system. The latter has served as an excellent model to study general mechanisms of olfaction. Using patch-clamp recordings and fast optical imaging in combination with the 'added buffer approach', we analyzed the Ca2+ handling properties of different identified neuron types in Periplaneta americana's olfactory system. Our focus was on two types of local interneurons (LNs) with significant differences in intrinsic electrophysiological properties: (1) spiking LNs that generate 'normal' Na+ driven action potentials and (2) non-spiking LNs that do not express voltage-activated Na+ channels. We found that the distinct electrophysiological properties from different types of central olfactory interneurons are strongly correlated with their cell specific calcium handling properties: non-spiking LNs, in which Ca2+ is the only cation that enters the cell to contribute to membrane depolarization, had the highest endogenous Ca2+ binding ratio and Ca2+ extrusion rate.