Biologic tissues are generally opaque due to optical properties that result in scattering and absorption of light. Preparation of tissues for optical microscopy often involves sectioning to a thickness of 50-100 µm, the practical limits of light penetration and recovery. A researcher who wishes to image a whole tissue must acquire potentially hundreds of individual sections before rendering them into a three-dimensional volume. Clearing removes strongly light-scattering and light-absorbing components of a tissue and equalizes the refractive index of the imaging medium to that of the tissue. After clearing, the maximum depth of imaging is often defined by the microscope optics rather than the tissue. Such visibility enables the interrogation of whole tissues and even animals without the need to section. Researchers can study a biological process in the context of its three-dimensional environment, identify rare events in large volumes of tissues, and trace cells and cell-cell interactions over large distances. This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley & Sons, Inc.

Abnormal hippocampal granule cells are present in patients with temporal lobe epilepsy, and are a prominent feature of most animal models of the disease. These abnormal cells are hypothesized to contribute to epileptogenesis. Isolating the specific effects of abnormal granule cells on hippocampal physiology, however, has been difficult in traditional temporal lobe epilepsy models. While epilepsy induction in these models consistently produces abnormal granule cells, the causative insults also induce widespread cell death among hippocampal, cortical and subcortical structures. Recently, we demonstrated that introducing morphologically abnormal granule cells into an otherwise normal mouse brain – by selectively deleting the mTOR pathway inhibitor PTEN from postnatally-generated granule cells – produced hippocampal and cortical seizures. Here, we conducted acute slice field potential recordings to assess the impact of these cells on hippocampal function. PTEN deletion from a subset of granule cells reproduced aberrant responses present in traditional epilepsy models, including enhanced excitatory post-synaptic potentials (fEPSPs) and multiple, rather than single, population spikes in response to perforant path stimulation. These findings provide new evidence that abnormal granule cells initiate a process of epileptogenesis – in the absence of widespread cell death – which culminates in an abnormal dentate network similar to other models of temporal lobe epilepsy. Findings are consistent with the hypothesis that accumulation of abnormal granule cells is a common mechanism of temporal lobe epileptogenesis.

Healthy pregnancy depends on proper placentation—including proliferation, differentiation, and invasion of trophoblast cells—which, if impaired, causes placental ischemia resulting in intrauterine growth restriction and preeclampsia. Mechanisms regulating trophoblast invasion, however, are unknown. We report that reduction of Inverted formin 2 (INF2) alters intracellular trafficking and significantly impairs invasion in a model of human extravillous trophoblasts. Furthermore, global loss of Inf2 in mice recapitulates maternal and fetal phenotypes of placental insufficiency. Inf2−/− dams have reduced spiral artery numbers and late gestational hypertension with resolution following delivery. Inf2−/− fetuses are growth restricted and demonstrate changes in umbilical artery Doppler consistent with poor placental perfusion and fetal distress. Loss of Inf2 increases fetal vascular density in the placenta and dysregulates trophoblast expression of angiogenic factors. Our data support a critical regulatory role for INF2 in trophoblast invasion—a necessary process for placentation—representing a possible future target for improving placentation and fetal outcomes.

Development of the mammalian forebrain requires a significant contribution from tubulin proteins to physically facilitate both the large number of mitoses in the neurogenic brain (in the form of mitotic spindles) as well as support cellular scaffolds to guide radial migration (radial glial neuroblasts). Recent studies have identified a number of mutations in human tubulin genes affecting the forebrain, including TUBB2B. We previously identified a mouse mutation in Tubb2b and we show here that mice heterozygous for this missense mutation in Tubb2b have significant cognitive defects in spatial learning and memory. We further showed reduced hippocampal long-term potentiation consistent with these defects. In addition to the behavioral and physiological deficits, we show here abnormal hippocampal morphology. Taken together, these phenotypes suggest that heterozygous mutations in tubulin genes result in cognitive deficits not previously appreciated. This has implications for design and interpretation of genetic testing for humans with intellectual disability disorders.

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Small-molecule aptamers are composed of RNA or DNA sequences with intricate secondary and tertiary structure allowing them to perform many functions, including ligand binding and catalytic events.

Healthy pregnancy depends on proper placentation-including proliferation, differentiation, and invasion of trophoblast cells-which, if impaired, causes placental ischemia resulting in intrauterine growth restriction and preeclampsia. Mechanisms regulating trophoblast invasion, however, are unknown. We report that reduction of Inverted formin 2 (INF2) alters intracellular trafficking and significantly impairs invasion in a model of human extravillous trophoblasts. Furthermore, global loss of Inf2 in mice recapitulates maternal and fetal phenotypes of placental insufficiency. Inf2 À/À dams have reduced spiral artery numbers and late gestational hypertension with resolution following delivery. Inf2 À/À fetuses are growth restricted and demonstrate changes in umbilical artery Doppler consistent with poor placental perfusion and fetal distress. Loss of Inf2 increases fetal vascular density in the placenta and dysregulates trophoblast expression of angiogenic factors. Our data support a critical regulatory role for INF2 in trophoblast invasion-a necessary process for placentation-representing a possible future target for improving placentation and fetal outcomes.