A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs

Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit a1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses

The new concrete

The New Concrete is a long-overdue survey of the rise of concrete poetry in the digital age. The accessibility of digital text and image manipulation, modern print techniques and the rise of self-publishing have invigorated a movement that first emerged in an explosion of literary creativity during the 1950s and 1960s. This new volume is a highly illustrated overview of contemporary artists and poets working at the intersection of visual art and literature, producing some of the most engaging and challenging work in either medium. Featuring an introductory essay by renowned American poet Kenneth Goldsmith and edited by celebrated poets Victoria Bean and Chris McCabe, The New Concrete is an indispensable introduction to the breadth of concrete poetry being produced today. Edited by Victoria Bean and Chris McCabe, with an essay by Kenneth Goldsmit