Detlev Schild

Müller cells are living optical fibers in the vertebrate retina

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.

Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model Used

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results. In this paper, we derive a modified FCS model that takes into account the confinement of the detection volume. Using this model, we have carried out the first FCS measurements in dendrites of cultured neurons. We further derive, for the case of confined diffusion, the limits within which the standard two- and three-dimensional diffusion models give reliable results.

Olfactory coding with patterns of response latencies.

The encoding of odors by spatiotemporal patterns of mitral/tufted (M/T) cells in the vertebrate olfactory bulb has been discussed controversially. Motivated by temporal constraints from behavioral studies, we investigated the information contained in odor-evoked first-spike latencies. Using simultaneous recordings of dozens of M/T cells with a high temporal resolution and quantitative ensemble correlation techniques, we show that latency patterns, and in particular latency rank patterns, are highly odor specific and reproducible. They reliably predict the odor identity as well as the odor concentration on a single-trial basis and on short timescales-in fact, more reliably than patterns of firing rates. Furthermore, we show that latency ranks exhibit a better reproducibility at the level of M/T cells than in olfactory receptor neurons. Our results suggest that the latency patterns of M/T cells contain all the information higher brain centers need to identify odors and their concentrations.

Whole-cell currents in olfactory receptor cells of Xenopus laevis.

SummaryOlfactory mucosae of Xenopus laevis were dissociated without enzymatic treatment and the isolated olfactory neurones were studied with the tight-seal whole-cell recording configuration of the patch clamp technique. In the voltage clamp, five current components could be distinguished: a fast, TTX-sensitive Na+-current, a small and slow inward current carried by Ca2+ ions, a Ca2+ dependent K+-current, a K+-current which activates rapidly at voltages more positive than-20 mV and quickly inactivates, and a slowly activating and very slowly inactivating K+-current. Some of the characteristics of the whole-cell currents herein reported contradict previous findings while others verify them, thereby allowing a tentative interpretation of their physiological role in the transduction process.

Multidrug resistance transporters in the olfactory receptor neurons of Xenopus laevis tadpoles

Olfactory receptor neurons (ORNs) are the only class of neurons that is directly exposed to the environment. Therefore, they need to deal with xenobiotic and potentially cytotoxic substances. Here we show for the first time that ORNs possess transporter systems that expel xenobiotics across the plasma membrane. Using calcein and calcium‐indicator dyes as xenobiotics, we demonstrate that ORNs appear to express the multidrug resistance P‐glycoprotein (MDR1) and multidrug resistance‐associated proteins (MRP). This endows ORNs with the ability to transport a large number of substrates including calcium‐indicator dyes and calcein across their plasma membranes. Conversely, blocking P‐glycoprotein and MRP increases the net uptake of these dyes.

Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red

The intracellular calcium concentration [Ca2+]i in olfactory receptor neurones of Xenopus laevis was imaged with high spatial and temporal resolution. A new method using a mixture of the calcium indicator dyes Fluo-3 and Fura-Red was employed. The fluorescence patterns in two wavelength bands were measured on the emission side of a confocal laser scanning microscope, and the ratio R of the fluorescence intensities was taken as an estimate of [Ca2+]i. When the neurones were depolarized by elevating the extracellular potassium concentration [K+]o they showed one of three types of responses: a fast increase in [Ca2+]i, a slow increase in [Ca2+]i, or no change in [Ca2+]i. The fast increase in [Ca2+]i took place in the soma compartment. For at least 4 s after the onset of depolarization the calcium distribution in the dendrite remained essentially unchanged. To study the fast increase with high time resolution, line scan images were taken. The neurones were depolarized for brief periods applying a solution containing high [K+] onto the soma from an application pipette. The fast increase in [Ca2+]i began with a delay of about 200 ms and went from the resting concentration to about 110 nM above resting concentration. Following the depolarization, recovery from elevated [Ca2+]i to resting levels had a time constant of about 15 s. The slow response seemed to depend on the removal of [Na+] from the bath rather than on the elevated [K+] in the bath. The response was also observed with Cd2+, Ni2+, and Co2+ (1.5 mM each) in the bath.(ABSTRACT TRUNCATED AT 250 WORDS)

Small Conductance Potassium Channels Cause an Activity-Dependent Spike Frequency Adaptation and Make the Transfer Function of Neurons Logarithmic

We made a computational model of a single neuron to study the effect of the small conductance (SK) Ca2+-dependent K+ channel on spike frequency adaptation. The model neuron comprised a Na+ conductance, a Ca2+ conductance, and two Ca2+-independent K+ conductances, as well as a small and a large (BK) Ca2+-activated K+ conductance, a Ca2+ pump, and mechanisms for Ca2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca2+ concentration ([Ca2+]i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca2+]i (. Pflügers Arch. 422:223-232; Köhler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709-1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechners law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.

Cannabinoid action in the olfactory epithelium

The perception of odors is influenced by a variety of neuromodulators, and there is growing evidence that modulation already takes place in the olfactory epithelium. Here we report on cannabinergic actions in the olfactory epithelium of Xenopus laevis tadpoles. First we show that CB1 receptor-specific antagonists AM251, AM281, and LY320135 modulate odor-evoked calcium changes in olfactory receptor neurons. Second, we localize CB1-like immunoreactivity on dendrites of olfactory receptor neurons. Finally, we describe the cannabinergic influence on odor-induced spike-associated currents in individual olfactory receptor neurons. Here we demonstrate that the cannabinergic system has a profound impact on peripheral odor processing and discuss its possible function.

Sodium/calcium exchanger in olfactory receptor neurones of Xenopus laevis.

Ca2+ ions enter neurones through various types of calcium and cation channels. The mechanisms by which Ca2+ ions are spatially buffered and expelled from neurones have been studied considerable less. Using calcium imaging in conjunction with the patch clamp technique, we investigated the Na/Ca exchanger in olfactory neurones and found evidence for its localization on the dendrite. It is suggested that this tends to decouple increases in [Ca2+]i occurring in the transduction compartments of the cell from processes in the soma.

Important Contribution of α-Neurexins to Ca2+-Triggered Exocytosis of Secretory Granules

α-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three α-neurexin genes show a severe reduction of synaptic release. Although analyses of α-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+channels, it remained unclear whether α-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult α-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that α-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for α-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.