Cyclists' perceived safety on intersections and roundabouts - A qualitative bicycle simulator study.

INTRODUCTION: Although cycling provides both individual and societal benefits, the mode share in Germany remains at a relatively low level. One reason described in literature is the lack of perceived safety due to the cycling infrastructure, especially at junctions. The study addresses the influence of junction design on cyclists' perceived safety. METHOD: Three intersections (BS: Berlin Standard, PI: protected intersection, CbC: cycle lanes between car lanes) and one roundabout were modeled in a virtual environment. Using a bicycle simulator, n = 46 participants cycled through each junction design, followed by a qualitative interview. We conducted a structured content analysis on the interview transcripts. RESULTS: Regarding the quality of statements, PI provides the highest level of perceived safety whereas CbC provides the lowest level. Both roundabout and BS provide medium to low perceived safety. Specific design features, such as continuous cycling infrastructure, physical separation and elements enhancing cyclists' visibility improve participants' perceived safety. On the other hand, curbs, bends, and elements obstructing visibility decrease perceived safety. Our findings also point towards a difference between overextending and manageable interactions between cars and cyclists. While manageable interactions raise attention to an appropriate extent, overextending interactions diminish the quality of the cycling experience so that some cyclists rather violate rules instead of using the designated cycling infrastructure. Furthermore, three factors influence participants' perception of infrastructure design: comprehensibility, comfort, and perceived safety. CONCLUSIONS: To provide a cycling friendly infrastructure, planners should consider cyclists' perceived safety as well as comfort and comprehensibility. Furthermore, in contrast to isolated segments, a continuous high-quality cycling infrastructure network should be implemented. Lastly, infrastructure might focus on manageable interactions rather than cause overextending interactions. PRACTICAL APPLICATION: The findings should be considered in future cycling infrastructure planning. Planners may test and modify temporary solutions to find appropriate designs for each junction.

Teaching Diagnostic Reasoning to Medical Students: a four-step approach.

Diagnosis is fundamental to clinical medicine, and diagnostic errors are a serious public health problem. However, there is little consensus regarding the best approach to teaching diagnostic reasoning in medical schools. One approach ("pattern recognition") uses learned associations between patient symptoms and signs and human disorders to help experienced clinicians solve problems rapidly and efficiently. However, this approach may be ineffective when used by students with little clinical experience. Here we describe a four-step analytical approach to diagnosis that can be used by medical students before beginning clinical training. This approach complements the pattern recognition approach used by experts and can be used by students, residents in training, and attending physicians when confronting complex cases. The analytical approach also highlights critical basic science concept areas that support diagnostic reasoning and therefore warrant emphasis in medical school curricula. We propose introducing the analytical approach to medical students early in their training, coordinated with basic science instruction. Once students master relevant basic science concepts, they can use the analytical approach to diagnose disorders affecting one or more physiological systems, as a foundation for future clinical training.

Chronic impairment of ERK signaling in glutamatergic neurons of the forebrain does not affect spatial memory retention and LTP in the same manner as acute blockade of the ERK pathway.

The ERK/MAPK signaling pathway has been extensively studied in the context of learning and memory. Defects in this pathway underlie genetic diseases associated with intellectual disability, including impaired learning and memory. Numerous studies have investigated the impact of acute ERK/MAPK inhibition on long-term potentiation and spatial memory. However, genetic knockouts of the ERKs have not been utilized to determine whether developmental perturbations of ERK/MAPK signaling affect LTP and memory formation in postnatal life. In this study, two different ERK2 conditional knockout mice were generated that restrict loss of ERK2 to excitatory neurons in the forebrain, but at different time-points (embryonically and post-natally). We found that embryonic loss of ERK2 had minimal effect on spatial memory retention and novel object recognition, while loss of ERK2 post-natally had more pronounced effects in these behaviors. Loss of ERK2 in both models showed intact LTP compared to control animals, while loss of both ERK1 and ERK2 impaired late phase LTP. These findings indicate that ERK2 is not necessary for LTP and spatial memory retention and provide new insights into the functional deficits associated with the chronic impairment of ERK signaling.

Combined computational and experimental approaches to understanding the Ca(2+) regulatory network in neurons.

Ca(2+) is a ubiquitous signaling ion that regulates a variety of neuronal functions by binding to and altering the state of effector proteins. Spatial relationships and temporal dynamics of Ca(2+) elevations determine many cellular responses of neurons to chemical and electrical stimulation. There is a wealth of information regarding the properties and distribution of Ca(2+) channels, pumps, exchangers, and buffers that participate in Ca(2+) regulation. At the same time, new imaging techniques permit characterization of evoked Ca(2+) signals with increasing spatial and temporal resolution. However, understanding the mechanistic link between functional properties of Ca(2+) handling proteins and the stimulus-evoked Ca(2+) signals they orchestrate requires consideration of the way Ca(2+) handling mechanisms operate together as a system in native cells. A wide array of biophysical modeling approaches is available for studying this problem and can be used in a variety of ways. Models can be useful to explain the behavior of complex systems, to evaluate the role of individual Ca(2+) handling mechanisms, to extract valuable parameters, and to generate predictions that can be validated experimentally. In this review, we discuss recent advances in understanding the underlying mechanisms of Ca(2+) signaling in neurons via mathematical modeling. We emphasize the value of developing realistic models based on experimentally validated descriptions of Ca(2+) transport and buffering that can be tested and refined through new experiments to develop increasingly accurate biophysical descriptions of Ca(2+) signaling in neurons.

Enhanced synaptic inhibition disrupts the efferent code of cerebellar Purkinje neurons in leaner Cav2.1 Ca 2+ channel mutant mice.

Cerebellar Purkinje cells (PCs) encode afferent information in the rate and temporal structure of their spike trains. Both spontaneous firing in these neurons and its modulation by synaptic inputs depend on Ca(2+) current carried by Ca(v)2.1 (P/Q) type channels. Previous studies have described how loss-of-function Ca(v)2.1 mutations affect intrinsic excitability and excitatory transmission in PCs. This study examines the effects of the leaner mutation on fast GABAergic transmission and its modulation of spontaneous firing in PCs. The leaner mutation enhances spontaneous synaptic inhibition of PCs, leading to transitory reductions in PC firing rate and increased spike rate variability. Enhanced inhibition is paralleled by an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) measured under voltage clamp. These differences are abolished by tetrodotoxin, implicating effects of the mutation on spike-induced GABA release. Elevated sIPSC frequency in leaner PCs is not accompanied by increased mean firing rate in molecular layer interneurons, but IPSCs evoked in PCs by direct stimulation of these neurons exhibit larger amplitude, slower decay rate, and a higher burst probability compared to wild-type PCs. Ca(2+) release from internal stores appears to be required for enhanced inhibition since differences in sIPSC frequency and amplitude in leaner and wild-type PCs are abolished by thapsigargin, an ER Ca(2+) pump inhibitor. These findings represent the first account of the functional consequences of a loss-of-function P/Q channel mutation on PC firing properties through altered GABAergic transmission. Gain in synaptic inhibition shown here would compromise the fidelity of information coding in these neurons and may contribute to impaired cerebellar function resulting from loss-of function mutations in the Ca(V)2.1 channel gene.

Impact of the leaner P/Q-type Ca2+ channel mutation on excitatory synaptic transmission in cerebellar Purkinje cells.

Loss-of-function mutations in the gene encoding P/Q-type Ca(2+) channels cause cerebellar ataxia in mice and humans, but the underlying mechanism(s) are unknown. These Ca(2+) channels play important roles in regulating both synaptic transmission and intrinsic membrane properties, and defects in either could contribute to ataxia. Our previous work described changes in intrinsic properties and excitability of cerebellar Purkinje cells (PCs) resulting from the leaner mutation, which is known to reduce whole-cell Ca(2+) currents in PCs and cause severe ataxia. Here we describe the impact of this mutation on excitatory synaptic transmission from parallel and climbing fibres (PFs, CFs) to PCs in acute cerebellar slices. We found that in leaner PCs, PF-evoked excitatory postsynaptic currents (PF-EPSCs) are approximately 50% smaller, and CF-evoked EPSCs are approximately 80% larger, than in wild-type (WT) mice. To investigate whether reduced presynaptic Ca(2+) entry plays a role in attenuating PF-EPSCs in leaner mice, we examined paired-pulse facilitation (PPF). We found that PPF is enhanced in leaner, suggesting that reduced presynaptic Ca(2+) entry reduces neurotransmitter release at these synapses. Short-term plasticity was unchanged at CF-PC synapses, suggesting that CF-EPSCs are larger in leaner PCs because of increased synapse number or postsynaptic sensitivity, rather than enhanced presynaptic Ca(2+) entry. To investigate the functional impact of the observed EPSC changes, we also compared excitatory postsynaptic potentials (EPSPs) elicited by PF and CF stimulation in WT and leaner PCs. Importantly, we found that despite pronounced changes in PF- and CF-EPSCs, evoked EPSPs in leaner mice are very similar to those observed in WT animals. These results suggest that changes in synaptic currents and intrinsic properties of PCs produced by the leaner mutation together maintain PC responsiveness to excitatory synaptic inputs. They also implicate other consequences of the leaner mutation as causes of abnormal cerebellar motor control in mutant mice.

The leaner P/Q-type calcium channel mutation renders cerebellar Purkinje neurons hyper-excitable and eliminates Ca2+-Na+ spike bursts.

The leaner mouse mutation of the Cacna1a gene leads to a reduction in P-type Ca2+ current, the dominant Ca2+ current in Purkinje cells (PCs). Here, we compare the electro-responsiveness and structure of PCs from age-matched leaner and wild-type (WT) mice in pharmacological isolation from synaptic inputs in cerebellar slices. We report that compared with WT, leaner PCs exhibit lower current threshold for Na+ spike firing, larger subthreshold membrane depolarization, rapid adaptation followed by complete block of Na+ spikes upon strong depolarization, and fail to generate Ca2+-Na+ spike bursts. The Na+ spike waveforms in leaner PCs have slower kinetics, reduced spike amplitude and afterhyperpolarization. We show that a deficit in the P-type Ca2+ current caused by the leaner mutation accounts for most but not all of the changes in mutant PC electro-responsiveness. The selective P-type Ca2+ channel blocker, omega-agatoxin-IVA, eliminated differences in subthreshold membrane depolarization, adaptation of Na+ spikes upon strong current-pulse stimuli, Na+ spike waveforms and Ca2+-Na+ burst activity. In contrast, a lower current threshold for eliciting repetitive Na+ spikes in leaner PCs was still observed after blockade of the P-type Ca2+ current, suggesting secondary effects of the mutation that render PCs hyper-excitable. Higher input resistance, reduced whole-cell capacitance and smaller dendritic size accompanied the enhanced excitability in leaner PCs, indicative of developmental retardation in these cells caused by P/Q-type Ca2+ channel malfunction. Our data indicate that a deficit in P-type Ca2+ current leads to complex functional and structural changes in PCs, impairing their intrinsic and integrative properties.

Calcium dynamics: analyzing the Ca2+ regulatory network in intact cells.

Calcium signaling is critical for all cells. As a free ion (Ca(2+)), calcium links many physiological stimuli to their intracellular effectors by interacting with binding proteins whose occupancy determines the cellular effect of stimulation. Because binding site occupancy depends on the history of Ca(2+) concentration ([Ca(2+)]), Ca(2+) dynamics are critical. Calcium dynamics depend on the functional interplay between Ca(2+) transport and buffering systems whose activities depend nonlinearly on [Ca(2+)]. Thus, understanding Ca(2+) dynamics requires detailed information about these Ca(2+) handling systems and their regulation in intact cells. However, effective methods for measuring and characterizing intracellular Ca(2+) handling have not been available until recently. Using concepts relating voltage-gated ion-channel activity to membrane potential dynamics, we developed such methods to analyze Ca(2+) fluxes in intact cells. Here we describe this approach and applications to understanding depolarization-induced Ca(2+) responses in sympathetic neurons.

Depolarization-induced calcium responses in sympathetic neurons: relative contributions from Ca2+ entry, extrusion, ER/mitochondrial Ca2+ uptake and release, and Ca2+ buffering.

Many models have been developed to account for stimulus-evoked [Ca(2+)] responses, but few address how responses elicited in specific cell types are defined by the Ca(2+) transport and buffering systems that operate in the same cells. In this study, we extend previous modeling studies by linking the time course of stimulus-evoked [Ca(2+)] responses to the underlying Ca(2+) transport and buffering systems. Depolarization-evoked [Ca(2+)](i) responses were studied in sympathetic neurons under voltage clamp, asking how response kinetics are defined by the Ca(2+) handling systems expressed in these cells. We investigated five cases of increasing complexity, comparing observed and calculated responses deduced from measured Ca(2+) handling properties. In Case 1, [Ca(2+)](i) responses were elicited by small Ca(2+) currents while Ca(2+) transport by internal stores was inhibited, leaving plasma membrane Ca(2+) extrusion intact. In Case 2, responses to the same stimuli were measured while mitochondrial Ca(2+) uptake was active. In Case 3, responses were elicited as in Case 2 but with larger Ca(2+) currents that produce larger and faster [Ca(2+)](i) elevations. Case 4 included the mitochondrial Na/Ca exchanger. Finally, Case 5 included ER Ca(2+) uptake and release pathways. We found that [Ca(2+)](i) responses elicited by weak stimuli (Cases 1 and 2) could be quantitatively reconstructed using a spatially uniform model incorporating the measured properties of Ca(2+) entry, removal, and buffering. Responses to strong depolarization (Case 3) could not be described by this model, but were consistent with a diffusion model incorporating the same Ca(2+) transport and buffering descriptions, as long as endogenous buffers have low mobility, leading to steep radial [Ca(2+)](i) gradients and spatially nonuniform Ca(2+) loading by mitochondria. When extended to include mitochondrial Ca(2+) release (Case 4) and ER Ca(2+) transport (Case 5), the diffusion model could also account for previous measurements of stimulus-evoked changes in total mitochondrial and ER Ca concentration.

The amplitude distribution of release events through a fusion pore.

Neurotransmitters, hormones, or dyes may be released from vesicles via a fusion pore, rather than by full fusion of the vesicle with the plasma membrane. If the lifetime of the fusion pore is comparable to the time required for the substance to exit the vesicle, only a fraction of the total vesicle content may be released during a single pore opening. Assuming 1), fusion pore lifetimes are exponentially distributed (tauP), as expected for simple single channel openings, and 2), vesicle contents are lost through the fusion pore with an exponential time course (tauD), we derive an analytical expression for the probability density function of the fraction of vesicle content released (F): dP/dF=A (1-F)(A-1), where A=tauD/tauP. If A>1, the maximum of the distribution is at F=0; if A<1, the maximum is at F=1; if A=1, the distribution is perfectly flat. Thus, the distribution never has a peak in the middle (0

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics.

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca2+-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+]I-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.

Differential regulation of ER Ca2+ uptake and release rates accounts for multiple modes of Ca2+-induced Ca2+ release.

The ER is a central element in Ca(2+) signaling, both as a modulator of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and as a locus of Ca(2+)-regulated events. During surface membrane depolarization in excitable cells, the ER may either accumulate or release net Ca(2+), but the conditions of stimulation that determine which form of net Ca(2+) transport occurs are not well understood. The direction of net ER Ca(2+) transport depends on the relative rates of Ca(2+) uptake and release via distinct pathways that are differentially regulated by Ca(2+), so we investigated these rates and their sensitivity to Ca(2+) using sympathetic neurons as model cells. The rate of Ca(2+) uptake by SERCAs (J(SERCA)), measured as the t-BuBHQ-sensitive component of the total cytoplasmic Ca(2+) flux, increased monotonically with [Ca(2+)](i). Measurement of the rate of Ca(2+) release (J(Release)) during t-BuBHQ-induced [Ca(2+)](i) transients made it possible to characterize the Ca(2+) permeability of the ER ((~)P(ER)), describing the activity of all Ca(2+)-permeable channels that contribute to passive ER Ca(2+) release, including ryanodine-sensitive Ca(2+) release channels (RyRs) that are responsible for CICR. Simulations based on experimentally determined descriptions of J(SERCA), and of Ca(2+) extrusion across the plasma membrane (J(pm)) accounted for our previous finding that during weak depolarization, the ER accumulates Ca(2+), but at a rate that is attenuated by activation of a CICR pathway operating in parallel with SERCAs to regulate net ER Ca(2+) transport. Caffeine greatly increased the [Ca(2+)] sensitivity of ((~)P(ER)), accounting for the effects of caffeine on depolarization-evoked [Ca(2+)](i) elevations and caffeine-induced [Ca(2+)](i) oscillations. Extending the rate descriptions of J(SERCA), ((~)P(ER)), and J(pm) to higher [Ca(2+)](i) levels shows how the interplay between Ca(2+) transport systems with different Ca(2+) sensitivities accounts for the different modes of CICR over different ranges of [Ca(2+)](i) during stimulation.