Islets of Langerhans: dye coupling among immunocytochemically distinct cell types.

Cells in isolated rat islets of Langerhans were microinjected with Lucifer Yellow CH in a medium containing 16.7 millimolar glucose. Dye was rapidly transferred from the injected cell to neighboring islet cells without specificity with regard to the immunocytochemical identity of either the donor or the recipient cells. The transfer of dye between the islet cells (types A, B, and D) demonstrates homologous and heterologous cell coupling in a system where the normal proportions and relationships of the cell types are maintained.

Prolactin enhances cell-to-cell communication among beta-cells in pancreatic islets.

To determine the role of prolactin in increasing junctional communication among islet beta-cells, we studied dye coupling in pancreatic islets exposed to elevated levels of prolactin in vivo and in vitro. Islets were isolated from rats immediately after lactation or from rats bearing mammosomatotropic tumors (MtTW15), conditions involving high levels of prolactin (either 5-fold or 1000-fold control levels, respectively). When beta-cells were microinjected with the gap junction permeant dye Lucifer yellow CH, the mean number of dye-coupled cells per injection was approximately 10-fold greater than in islets from virgin control rats. As a more direct test of the effects of prolactin on beta-cell coupling, islets isolated from virgin rats were treated for 90 min with 500 ng/ml rat prolactin in the presence of low glucose (2.8 mM) and were microinjected with dye. The mean number of dye-coupled cells per injection increased by 6.7-fold over controls with low glucose, demonstrating a direct effect of prolactin on beta-cell coupling. In vitro treatment with high glucose (16.7 mM) resulted in a 2.7-fold increase in dye-coupled cells per injection. We discuss the possible relationship between the effects of glucose and of prolactin on coupling.

In vivo modulation of gap junctions and dye coupling between B-cells of the intact pancreatic islet.

The extent of gap junctions and dye coupling between insulin-producing B-cells was analyzed on islets of Langerhans isolated from adult rats treated for one day with glibenclamide, an insulin secretagogue, or diazoxide, a blocker of insulin release, or a combination of the two drugs. Glibenclamide treatment was associated with a marked depletion of the islet insulin content, an effect which was blocked by pretreatment of the rats with diazoxide. Diazoxide alone caused a marked increase in the plasma glucose level, and a decrease in the level of circulating insulin and in the hormone content of the B-cells. Quantitative analysis showed that (1) under control conditions, B-cells are connected by minute gap junctions (as evaluated on freeze-fracture replicas) and show a nonuniform and apparently restricted dye coupling (as determined by microinjection of the low-molecular-weight fluorescent probe Lucifer Yellow CH); (2) each of the three treatments tested significantly increased the relative and absolute gap junction area of the B-cells and the number of detectable, dye coupled B-cells per microinjection. After treatment with glibenclamide alone or with diazoxide plus glibenclamide, a 1.5-1.8-fold increase in gap junction area and a 2.7-3.7-fold increase in the number of dye-coupled B-cells were observed. In contrast, following treatment with diazoxide alone, gap junctions and dye coupling were found increased 1.8 and 8.7 times, respectively, as compared with control values.

Immunocytochemical identification of dye-injected cells.

Lucifer Yellow CH may be injected into pancreatic islet cells and visualized in Epon sections of the embedded tissue both before and after plastic removal and immunocyto-chemical staining. The dye retains its fluorescence, clearly marking the injected cell and adjacent dye-coupled cells, but does not interfere with the indirect immunofluorescent staining patterns that are characteristic of the islet cells

CSlo encodes calcium-activated potassium channels in the chick's cochlea.

Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila 'slowpoke' gene. By homology screening with mouse Sla cDNA, we have isolated a full-length clone (cSlo1) from a chick's cochlear cDNA library, rSlol had greater than 90% identity with mouse Slo at the amino acid level, and was even better matched to a human brain Slo at the amino and carboxy termini. cSlol had none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection of HIEK 293 cells demonstrated that cSlol encoded a potassium channel whose conductance averaged 224 pS at +60 mV in symmetrical 140 mM K. Macroscopic currents through cSlol channels were blocked by scorpion toxin or tetraethyl ammonium, and were voltage and calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells.

Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations.

There is now convincing evidence that excessive accumulation of the excitatory amino acid glutamate (GLU) in the extracellular space is toxic to central mammalian neurons. However, the role of different GLU receptors in producing this toxicity has not been adequately ascertained. There is also no adequate information about the correlation of free intracellular calcium concentration with eventual excitotoxic death. We have used cultured rat hippocampal neurons to address these issues. Approximately 75% of our neurons died after a 20-min GLU exposure. The potent kainate/quisqualate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not significantly ameliorate the GLU toxicity, while the selective noncompetitive N-methyl-D-aspartate (NMDA) antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) blocked the GLU toxicity for periods of at least 2 hr. Interestingly, kainate was very toxic to the hippocampal neurons, but this toxicity was markedly attenuated by MK-801. These results suggest that the major toxicity of GLU is mediated by NMDA receptors and that under some conditions kainate toxicity reflects nonspecific opening of NMDA channels. The intracellular calcium concentrations in these neurons at the end of exposure to GLU and kainate (in the presence and absence of different antagonists) correlated poorly with eventual survival. Antagonists that limited the rise in calcium were still ineffective in preventing death. These results confirm earlier observations that stressed the importance of NMDA receptors in mediating GLU toxicity. However, they indicate that the relationship between toxicity and neuronal calcium concentration may be very complicated. An unexpected finding of these experiments was that MK-801, unlike competitive antagonists of GLU, elevated intracellular calcium.

Generation of large-diameter diffractive elements with laser pattern generation.

We describe the design and construction of a high-precision laser writing machine for the direct generation of large-diameter rotationally symmetric diffractive optics with continuous profiles in photoresist. The photoresist profile can be used as a replication master surface or etched into a silica substrate. Machine design methodology, as well as qualification of performance, is provided. Test results for an f/2 100-mm clear-aperture diffractive lens directly etched into a silica substrate are presented. Diffraction efficiency as a function of zone spacing and wave-front performance are given.

Release sites and calcium channels in hair cells of the chick's cochlea.

Rapid transmitter release at synapses depends on the close proximity of voltage-gated calcium channels (VGCCs). In mechanosensory hair cells of the vertebrate inner ear, dihydropyridine-sensitive VGCCs may be preferentially expressed at release sites to support transmitter release. In support of this hypothesis we have found that whole-cell current through VGCCs covaried with afferent innervation density among hair cells of the chick's basilar papilla (the avian analog of the mammalian Organ of Corti). The size as well as number of presynaptic dense bodies (PDBs) around which transmitter vesicles cluster varied systematically among equivalent populations of hair cells examined with electron microscopy. The total number of VGCCs was correlated with total release area (PDB cross-sectional area x the number of PDBs) among neurally located (tall) hair cells. Abneural, short hair cells with little or no afferent contact expressed a low number of VGCCs independent of release area. The implication is that hair cells augment calcium channel expression by adding release sites and by making release sites larger. This suggests further that aspects of hair cell excitability, such as electrical tuning, could depend on the synaptic architecture of each cell.

Single-point diamond turning and replication of visible and near-infrared diffractive optical elements.

High-fidelity diffractive surfaces have been generated with single-point diamond-turning techniques. A key to the success of this technique is the ability to shape the diamond tool tip to provide the optimum phase-relief profile, given manufacturing constraints. Replication technology is used to transfer the phase-relief surface into a thin epoxy or photopolymer layer on a glass substrate. Diffraction efficiency results for a wide range of zone widths are presented to provide the reader with a baseline of expected performance for replicated visible and near-infrared diffractive optical elements. In addition, a new method for analyzing diffractive surface structures is presented. The ray-trace algorithm quickly provides accurate results of predicted diffraction efficiency for arbitrary zone profiles, which is extremely valuable in predicting manufacturing errors.