The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:

Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.

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Synapses are important functional units that determine how information flows through the brain. Understanding their biophysical properties and the molecules that underpin them is an important goal of cellular neuroscience. Thus, it is of interest to develop protocols that allow easy measurement of synaptic parameters in model systems that permit molecular manipulations. Here, we used a sensitive and high-time resolution optical approach that allowed us to characterize two functional parameters critical to presynaptic efficacy: vesicle fusion probability (Pv) and readily-releasable pool size (RRP). We implemented two different approaches to determine the RRP size that were in broad agreement: depletion of the RRP by high-frequency stimulation and saturation of the calcium sensor during single action potential stimuli. Our methods are based on reporters that provide a robust, quantitative, purely presynaptic readout and present a new avenue to study molecules that affect synaptic vesicle exocytosis.

The strength of individual synaptic contacts is considered a key modulator of information flow across circuits. Presynaptically the strength can be parsed into two key parameters: the size of the readily releasable pool (RRP) and the probability that a vesicle in that pool will undergo exocytosis when an action potential fires (Pv). How these variables are controlled and the degree to which they vary across individual nerve terminals is crucial to understand synaptic plasticity within neural circuits. Here we report robust measurements of these parameters in rat hippocampal neurons and their variability across populations of individual synapses. We explore the diversity of presynaptic Ca2+ channel repertoires and evaluate their effect on synaptic strength at single boutons. Finally, we study the degree to which synapses can be differentially modified by a known potentiator of presynaptic function, forskolin. Our experiments revealed that both Pv and RRP spanned a large range, even for synapses made by the same axon, demonstrating that presynaptic efficacy is governed locally at the single synapse level. Synapses varied greatly in their dependence on N or P/Q type Ca2+ channels for neurotransmission, but there was no association between specific channel repertoires and synaptic efficacy. Increasing cAMP concentration using forskolin enhanced synaptic transmission in a Ca2+-independent manner that was inversely related with a synapse's initial Pv, and independent of its RRP size. We propose a simple model based on the relationship between Pv and calcium entry that can account for the variable potentiation of synapses based on initial probability of vesicle fusion.

The last decade has seen a proliferation of tissue clearing methods that render large biological samples transparent and allow unprecedented three-dimensional views of enormous volumes of tissue. For a scientist wondering whether these methods will be useful to address their research problems, it can be bewildering to sort through the ever-increasing number of papers introducing new clearing methods. Here, I provide a concise summary for the novice describing what tissue clearing is, which research problems it can be applied to, how to decide on a clearing method, and where the field is headed in the future.

SUMMARY Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotidebinding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.

Consolidation of long-term memory requires the activation of several transduction pathways that lead to post-translational modifications of synaptic proteins and to regulation of gene expression, both of which promote stabilization of specific changes in the activated circuits. In search of the molecular mechanisms involved in such processes, we used the context-signal associative learning paradigm of the crab Chasmagnathus. In this model, we studied the role of some molecular mechanisms, namely cAMP-dependent protein kinase (PKA), extracellular-signal-regulated kinase (ERK), the nuclear factor kappa B (NF-kappaB) transcription factor, and the role of synaptic proteins such as amyloid beta precursor protein, with the object of describing key mechanisms involved in memory processing. In this article we review the most salient results obtained over a decade of research in this memory model.