Characterization of the functional MRI response temporal linearity via optical control of neocortical pyramidal neurons.

The blood oxygenation level-dependent (BOLD) signal serves as the basis for human functional MRI (fMRI). Knowledge of the properties of the BOLD signal, such as how linear its response is to sensory stimuli, is essential for the design and interpretation of fMRI experiments. Here, we combined the cell-type and site-specific causal control provided by optogenetics and fMRI (opto-fMRI) in mice to test the linearity of BOLD signals driven by locally induced excitatory activity. We employed high-resolution mouse fMRI at 9.4 tesla to measure the BOLD response, and extracellular electrophysiological recordings to measure the effects of stimulation on single unit, multiunit, and local field potential activity. Optically driven stimulation of layer V neocortical pyramidal neurons resulted in a positive local BOLD response at the stimulated site. Consistent with a linear transform model, this locally driven BOLD response summated in response to closely spaced trains of stimulation. These properties were equivalent to responses generated through the multisynaptic method of driving neocortical activity by tactile sensory stimulation, and paralleled changes in electrophysiological measures. These results illustrate the potential of the opto-fMRI method and reinforce the critical assumption of human functional neuroimaging that--to first approximation--the BOLD response tracks local neural activity levels.

Hemodynamic molecular imaging of tumor-associated enzyme activity in the living brain.

Molecular imaging could have great utility for detecting, classifying, and guiding treatment of brain disorders, but existing probes offer limited capability for assessing relevant physiological parameters. Here, we describe a potent approach for noninvasive mapping of cancer-associated enzyme activity using a molecular sensor that acts on the vasculature, providing a diagnostic readout via local changes in hemodynamic image contrast. The sensor is targeted at the fibroblast activation protein (FAP), an extracellular dipeptidase and clinically relevant biomarker of brain tumor biology. Optimal FAP sensor variants were identified by screening a series of prototypes for responsiveness in a cell-based bioassay. The best variant was then applied for quantitative neuroimaging of FAP activity in rats, where it reveals nanomolar-scale FAP expression by xenografted cells. The activated probe also induces robust hemodynamic contrast in nonhuman primate brain. This work thus demonstrates a potentially translatable strategy for ultrasensitive functional imaging of molecular targets in neuromedicine.

CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.

Target-responsive vasoactive probes for ultrasensitive molecular imaging.

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.

Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits.

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8+/- mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8+/- animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.

Molecular imaging with engineered physiology.

In vivo imaging techniques are powerful tools for evaluating biological systems. Relating image signals to precise molecular phenomena can be challenging, however, due to limitations of the existing optical, magnetic and radioactive imaging probe mechanisms. Here we demonstrate a concept for molecular imaging which bypasses the need for conventional imaging agents by perturbing the endogenous multimodal contrast provided by the vasculature. Variants of the calcitonin gene-related peptide artificially activate vasodilation pathways in rat brain and induce contrast changes that are readily measured by optical and magnetic resonance imaging. CGRP-based agents induce effects at nanomolar concentrations in deep tissue and can be engineered into switchable analyte-dependent forms and genetically encoded reporters suitable for molecular imaging or cell tracking. Such artificially engineered physiological changes, therefore, provide a highly versatile means for sensitive analysis of molecular events in living organisms.

Young adult born neurons enhance hippocampal dependent performance via influences on bilateral networks.

Adult neurogenesis supports performance in many hippocampal dependent tasks. Considering the small number of adult-born neurons generated at any given time, it is surprising that this sparse population of cells can substantially influence behavior. Recent studies have demonstrated that heightened excitability and plasticity may be critical for the contribution of young adult-born cells for certain tasks. What is not well understood is how these unique biophysical and synaptic properties may translate to networks that support behavioral function. Here we employed a location discrimination task in mice while using optogenetics to transiently silence adult-born neurons at different ages. We discovered that adult-born neurons promote location discrimination during early stages of development but only if they undergo maturation during task acquisition. Silencing of young adult-born neurons also produced changes extending to the contralateral hippocampus, detectable by both electrophysiology and fMRI measurements, suggesting young neurons may modulate location discrimination through influences on bilateral hippocampal networks.

Modelling of Hampshire Adult Services--gearing up for future demands.

This paper describes a study to investigate future demand from older people for social care services in Hampshire, UK. The study used system dynamics to explore the significant challenges of an ageing population in the context of budget limitations. The results show that as anticipated, the numbers requiring care will increase considerably over the next 5 years. The effects of two possible interventions to reduce the impact of this are explored.