Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 μm diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca²⁺ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABA_A and GABA_C receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release ^14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal ²¹. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion ¹¹. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level ¹⁹. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced ^{20, 23, 18, 9}. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.     

Longevity and fecundity of Japanese beetle (Popillia japonica) on foliage grown under elevated carbon dioxide

Atmospheric levels of carbon dioxide (CO(2)) have been increasing steadily over the last century. Plants grown under elevated CO(2) experience physiological changes that influence their suitability as food. Previous studies have found increased insect herbivory on plants grown under elevated CO(2). To determine effects of consuming foliage of soybean (Glycine max) grown under elevated CO(2) on adult survivorship and fecundity, Japanese beetles (Popillia japonica Newman) were fed for the duration of their adult lives leaves grown under elevated CO(2) (550 mumol/mol), under ambient atmosphere (370 mumol/mol), or grown under ambient atmosphere but supplemented with a solution of sugars. To determine effects of a diet of foliage grown under elevated ozone (O(3)), another anthropogenic gaseous pollutant, beetles in the laboratory were fed soybean leaves grown under elevated CO(2), elevated O(3), or a combination of both elevated gases. Leaf tissue was also analyzed for longevity-enhancing antioxidants, because increases in dietary antioxidants can increase lifespan. Lifespan of Japanese beetles was prolonged by 8-25% when fed foliage developed under elevated CO(2), but consuming foliage that had taken up sugars to approximately the same level as foliage grown under elevated CO(2) had no effect on fecundity or longevity. Females consuming elevated CO(2) foliage laid approximately twice as many eggs as females fed foliage grown under ambient conditions. Consuming foliage grown under elevated O(3) had no effect on fecundity. No significant differences in total antioxidant content of foliage from ambient and elevated CO(2) conditions were detected. Although the precise mechanism is unclear, by altering components of leaf chemistry other than sugar content, elevated CO(2) may increase populations of Japanese beetles and their impact on crop productivity.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.