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Background Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. Methods We demonstrated blood oxygenation level–dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level–dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. Results The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. Conclusions This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

Increased corticotroping releasing factor (CRF) contributes to brain circuit abnormalities associated with stress-related disorders including posttraumatic stress disorder. However, the causal relationship between CRF hypersignaling and circuit abnormalities associated with stress disorders is unclear. We hypothesized that increased CRF exposure induces changes in limbic circuit morphology and functions. An inducible, forebrain-specific overexpression of CRF (CRFOE) transgenic mouse line was used to longitudinally investigate its chronic effects on behaviors and microstructural integrity of several brain regions. Behavioral and diffusion tensor imaging studies were performed before treatment, after 3-4 wks of treatment, and again 3 mo after treatment ended to assess recovery. CRFOE was associated with increased perseverative movements only after 3 wks of treatment, as well as reduced fractional anisotropy at 3 wks in the medial prefrontal cortex and increased fractional anisotropy in the ventral hippocampus at 3 mo compared to the control group. In the dorsal hippocampus, mean diffusivity was lower in CRFOE mice both during and after treatment ended. Our data suggest differential response and recovery patterns of cortical and hippocampal subregions in response to CRFOE. Overall these findings support a causal relationship between CRF hypersignaling and microstructural changes in brain regions relevant to stress disorders.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1–9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11–13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis. Metabolomics data from germ-free and specific-pathogen-free mice reveal effects of the microbiome on host chemistry, identifying conjugations of bile acids that are also enriched in patients with inflammatory bowel disease or cystic fibrosis.

Focused ultrasound can deliver energy safely and non-invasively into tissues at depths of centimetres. Here we show that the genetics and cellular functions of chimeric antigen receptor T cells (CAR-T cells) within tumours can be reversibly controlled by the heat generated by short pulses of focused ultrasound via a CAR cassette under the control of a promoter for the heat-shock protein. In mice with subcutaneous tumours, locally injected T cells with the inducible CAR and activated via focused ultrasound guided by magnetic resonance imaging mitigated on-target off-tumour activity and enhanced the suppression of tumour growth, compared with the performance of non-inducible CAR-T cells. Acoustogenetic control of the activation of engineered T cells may facilitate the design of safer cell therapies. The activity of engineered T cells within tumours can be controlled via the heat generated by pulses of focused ultrasound by modifying the cells to express a chimeric antigen receptor under the control of a promoter for the heat-shock protein.

A mosaic of cross-phyla chemical interactions occurs between all metazoans and their microbiomes. In humans, the gut harbors the heaviest microbial load, but many organs, particularly those with a mucosal surface, associate with highly adapted and evolved microbial consortia1. The microbial residents within these organ systems are increasingly well characterized, yielding a good understanding of human microbiome composition, but we have yet to elucidate the full chemical impact the microbiome exerts on an animal and the breadth of the chemical diversity it contributes2. A number of molecular families are known to be shaped by the microbiome including short-chain fatty acids, indoles, aromatic amino acid metabolites, complex polysaccharides, and host lipids; such as sphingolipids and bile acids3–11. These metabolites profoundly affect host physiology and are being explored for their roles in both health and disease. Considering the diversity of the human microbiome, numbering over 40,000 operational taxonomic units12, a plethora of molecular diversity remains to be discovered. Here, we use unique mass spectrometry informatics approaches and data mapping onto a murine 3D-model13–15to provide an untargeted assessment of the chemical diversity between germ-free (GF) and colonized mice (specific-pathogen free, SPF), and report the finding of novel bile acids produced by the microbiome in both mice and humans that have evaded characterization despite 170 years of research on bile acid chemistry16.

Methamphetamine (METH) is an addictive psychostimulant inducing neurotoxicity. Human magnetic resonance imaging and diffusion tensor imaging (DTI) of METH-dependent participants find various structural abnormities. Animal studies demonstrate immunohistochemical changes in multiple cellular pathways after METH exposure. Here, we characterized the long-term effects of METH on brain microstructure in mice exposed to an escalating METH binge regimen using in vivo DTI, a methodology directly translatable across species. Results revealed four patterns of differential fractional anisotropy (FA) and mean diffusivity (MD) response when comparing METH-exposed (n=14) to saline-treated mice (n=13). Compared to the saline group, METH-exposed mice demonstrated: 1) decreased FA with no change in MD [corpus callosum (posterior forceps), internal capsule (left), thalamus (medial aspects), midbrain], 2) increased MD with no change in FA [posterior isocortical regions, caudate-putamen, hypothalamus, cerebral peduncle, internal capsule (right)], 3) increased FA with decreased MD [frontal isocortex, corpus callosum (genu)], and 4) increased FA with no change or increased MD [hippocampi, amygdala, lateral thalamus]. MD was negatively associated with calbindin-1 in hippocampi and positively with dopamine transporter in caudate-putamen. These findings highlight distributed and differential METH effects within the brain suggesting several distinct mechanisms. Such mechanisms likely change brain tissue differentially dependent upon neural location.

The hippocampus comprises two neural signals—place cells and θ oscillations—that contribute to facets of spatial navigation. Although their complementary relationship has been well established in rodents, their respective contributions in the primate brain during free navigation remains unclear. Here, we recorded neural activity in the hippocampus of freely moving marmosets as they naturally explored a spatial environment to more explicitly investigate this issue. We report place cells in marmoset hippocampus during free navigation that exhibit remarkable parallels to analogous neurons in other mammalian species. Although θ oscillations were prevalent in the marmoset hippocampus, the patterns of activity were notably different than in other taxa. This local field potential oscillation occurred in short bouts (approximately .4 s)—rather than continuously—and was neither significantly modulated by locomotion nor consistently coupled to place-cell activity. These findings suggest that the relationship between place-cell activity and θ oscillations in primate hippocampus during free navigation differs substantially from rodents and paint an intriguing comparative picture regarding the neural basis of spatial navigation across mammals., A neurophysiology study of marmoset monkey hippocampus during free navigation identified place cells analogous to those seen in other species, but the limited interaction observed between the activity of these neurons and theta oscillations suggests notable divergence from rodent models.

The diffusion-weighted magnetic resonance imaging (DWI) technique enables quantification of water mobility for probing microstructural properties of biological tissue and has become an effective tool for collecting information about the underlying pathology of cancerous tissue. Measurements using multiple b-values have indicated biexponential signal attenuation, ascribed to “fast” (high ADC) and “slow” (low ADC) diffusion components. In this empirical study, we investigate the properties of the diffusion time (Δ)-dependent components of the diffusion-weighted (DW) signal in a constant b-value experiment. A xenograft gliobastoma mouse was imaged using Δ = 11 ms, 20 ms, 40 ms, 60 ms, and b = 500–4000 s/mm2 in intervals of 500 s/mm2. Data were corrected for EPI distortions, and the Δ-dependence on the DW-signal was measured within three regions of interest [intermediate- and high-density tumor regions and normal-appearing brain (NAB) tissue regions]. In this study, we verify the assumption that the slow decaying component of the DW-signal is non-Gaussian and dependent on Δ, consistent with restricted diffusion of the intracellular space. As the DW-signal is a function of Δ and is specific to restricted diffusion, manipulating Δ at constant b-value (cb) provides a complementary and direct approach for separating the restricted from the hindered diffusion component. We found that Δ-dependence is specific to the tumor tissue signal. Based on an extended biexponential model, we verified the interpretation of the diffusion time-dependent contrast and successfully estimated the intracellular restricted ADC, signal volume fraction, and cell size within each ROI. Copyright © 2016 Hope, White, Kuperman, Chao, Yamin, Bartch, Schenker-Ahmed, Rakow-Penner, Bussell, Nomura, Kesari, Bjørnerud and Dale. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Alpha-synuclein (alphaS) is a lipid-binding synaptic protein of unknown function that is found in an aggregated amyloid fibril form in the intraneuronal Lewy body deposits that are a defining characteristic of Parkinson's disease (PD). Although intrinsically unstructured when free in solution, alphaS adopts a highly helical conformation in association with lipid membranes or membrane mimetic detergent micelles. Two mutations in the alphaS gene have been linked to early onset autosomal dominant hereditary forms of PD, and have been shown to affect the aggregation kinetics of the protein in vitro. We have used high-resolution NMR spectroscopy, circular dichroism, and limited proteolysis to investigate the effects of these PD-linked mutations on the helical structure adopted by alphaS in the lipid or detergent micelle-bound form. We show that neither the A53T nor the A30P mutation has a significant effect on the structure of the folded protein, although the A30P mutation may cause a minor perturbation in the helical structure around the site of the mutation. The A30P, but not the A53T, mutation also appears to decrease the affinity of the protein for lipid surfaces, possibly by perturbing the nascent helical structure of the free protein. The potential implications of these results for the role of alphaS in PD are discussed.

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinson's disease-related protein alpha-synuclein in lipid and detergent micelle environments. We show that the lipid or micelle surface-bound portion of the molecule adopts a continuously helical structure with a single break. Modeling alphaS as an ideal alpha-helix reveals a hydrophobic surface that winds around the helix axis in a right-handed fashion. This feature is typical of 11-mer repeat containing sequences that adopt right-handed coiled coil conformations. In order to bind a flat or convex lipid surface, however, an unbroken helical alphaS structure would need to adopt an unusual, slightly unwound, alpha11/3 helix conformation (three complete turns per 11 residues). The break we observe in the alphaS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical alpha-helical structure. However, a quantitative analysis suggests the possibility that the alpha11/3 conformation may in fact exist in lipid-bound alphaS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of alpha-synuclein and generalize this argument to include the 11-mer repeat-containing apolipoproteins, which also require the ability to release readily from lipid surfaces. A search of protein sequence databases confirms that synuclein-like 11-mer repeats are present in other proteins that bind lipids reversibly and predicts such a role for a number of hypothetical proteins of unknown function.

alpha-Synuclein (alpha S) is a pre-synaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease (PD). Two autosomal dominant missense mutations in the alpha S gene are associated with early onset PD. Because alpha S is found in an aggregated fibrillar form in the Lewy body deposits characteristic of Parkinson's patients, aggregation of the protein is believed to be related to its involvement in the disease process. The wild type (WT) and early onset mutants A30P and A53T display diverse in vitro aggregation kinetics even though the gross physicochemical and morphological properties of the mutants are highly similar. We used high resolution solution NMR spectroscopy to compare the structural and dynamic properties of the A53T and A30P mutants with those of WT alpha S in the free state. We found that the A30P mutation disrupts a region of residual helical structure that exists in the WT protein, whereas the A53T mutation results in a slight enhancement of a small region around the site of mutation with a preference for extended conformations. Based on these results and on the anticipated effects of these mutations on elements of secondary structure, we proposed a model of how these two PD-linked mutations influence alpha S fibril formation that is consistent with the documented differences in the fibrillization kinetics of the two mutants.

α-Synuclein (αS) is a presynaptic terminal protein that is believed to play an important role in the pathogenesis of Parkinson’s disease (PD). We have used NMR spectroscopy to characterize the conformational properties of αS in solution as a free monomer and when bound to lipid vesicles and lipid-mimetic detergent micelles. Free wild-type αS is largely unfolded in solution, but exhibits a region with a preference for helical conformations that may be important in the aggregation of αS into fibrils. The N-terminal region of αS binds to synthetic lipid vesicles and detergent micelles in vitro and adopts a highly helical conformation, consistent with predictions based on sequence analysis. The C-terminal part of the protein does not associate with either vesicles or micelles, remaining free and unfolded. These results suggest that one function of αS may be to tether as of yet unidentified partners to lipid surfaces via interactions with its C-terminal tail

The protein alpha-Synuclein (aS) is a synaptic vesicle-associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane-associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane-binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle-bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane-binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine-free sixth 11-residue repeat, is observed and may be functionally important. There are no long-range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle-bound region are relatively uniform with a slight decrease in flexibility observed toward the C-terminal end. These results clarify the topological features of aS bound to membrane-mimicking detergent micelles, with implications for aS function and pathology.

Alpha-synuclein (alphaS) is a lipid-binding synaptic protein of unknown function that is found in an aggregated amyloid fibril form in the intraneuronal Lewy body deposits that are a defining characteristic of Parkinson's disease (PD). Although intrinsically unstructured when free in solution, alphaS adopts a highly helical conformation in association with lipid membranes or membrane mimetic detergent micelles. Two mutations in the alphaS gene have been linked to early onset autosomal dominant hereditary forms of PD, and have been shown to affect the aggregation kinetics of the protein in vitro. We have used high-resolution NMR spectroscopy, circular dichroism, and limited proteolysis to investigate the effects of these PD-linked mutations on the helical structure adopted by alphaS in the lipid or detergent micelle-bound form. We show that neither the A53T nor the A30P mutation has a significant effect on the structure of the folded protein, although the A30P mutation may cause a minor perturbation in the helical structure around the site of the mutation. The A30P, but not the A53T, mutation also appears to decrease the affinity of the protein for lipid surfaces, possibly by perturbing the nascent helical structure of the free protein. The potential implications of these results for the role of alphaS in PD are discussed.