Hybrid fiber optic-fMRI for multimodal cell-specific recording and manipulation of neural activity in rodents.

Significance: Multiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods. Aim: In this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation. Approach: We focus on optical techniques as these allow, in conjunction with genetically encoded proteins (e.g. calcium indicators or optical signal transducers), unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities. Results: A variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest-such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature. Conclusion: We conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity.

Whole-brain opto-fMRI map of mouse VTA dopaminergic activation reflects structural projections with small but significant deviations.

Ascending dopaminergic projections from neurons located in the Ventral Tegmental Area (VTA) are key to the etiology, dysfunction, and control of motivation, learning, and addiction. Due to the evolutionary conservation of this nucleus and the extensive use of mice as disease models, establishing an assay for VTA dopaminergic signaling in the mouse brain is crucial for the translational investigation of motivational control as well as of neuronal function phenotypes for diseases and interventions. In this article we use optogenetic stimulation directed at VTA dopaminergic neurons in combination with functional Magnetic Resonance Imaging (fMRI), a method widely used in human deep brain imaging. We present a comprehensive assay producing the first whole-brain opto-fMRI map of dopaminergic activation in the mouse, and show that VTA dopaminergic system function is consistent with its structural VTA projections, diverging only in a few key aspects. While the activation map predominantly highlights target areas according to their relative projection densities (e.g., strong activation of the nucleus accumbens and low activation of the hippocampus), it also includes areas for which a structural connection is not well established (such as the dorsomedial striatum). We further detail the variability of the assay with regard to multiple experimental parameters, including stimulation protocol and implant position, and provide evidence-based recommendations for assay reuse, publishing both reference results and a reference analysis workflow implementation.

An optimized registration workflow and standard geometric space for small animal brain imaging.

The reliability of scientific results critically depends on reproducible and transparent data processing. Cross-subject and cross-study comparability of imaging data in general, and magnetic resonance imaging (MRI) data in particular, is contingent on the quality of registration to a standard reference space. In small animal MRI this is not adequately provided by currently used processing workflows, which utilize high-level scripts optimized for human data, and adapt animal data to fit the scripts, rather than vice-versa. In this fully reproducible article we showcase a generic workflow optimized for the mouse brain, alongside a standard reference space suited to harmonize data between analysis and operation. We introduce four separate metrics for automated quality control (QC), and a visualization method to aid operator inspection. Benchmarking this workflow against common legacy practices reveals that it performs more consistently, better preserves variance across subjects while minimizing variance across sessions, and improves both volume and smoothness conservation RMSE approximately 2-fold. We propose this open source workflow and the QC metrics as a new standard for small animal MRI registration, ensuring workflow robustness, data comparability, and region assignment validity, all of which are indispensable prerequisites for the comparability of scientific results across experiments and centers.

An Automated Open-Source Workflow for Standards-Compliant Integration of Small Animal Magnetic Resonance Imaging Data.

Large-scale research integration is contingent on seamless access to data in standardized formats. Standards enable researchers to understand external experiment structures, pool results, and apply homogeneous preprocessing and analysis workflows. Particularly, they facilitate these features without the need for numerous potentially confounding compatibility add-ons. In small animal magnetic resonance imaging, an overwhelming proportion of data is acquired via the ParaVision software of the Bruker Corporation. The original data structure is predominantly transparent, but fundamentally incompatible with modern pipelines. Additionally, it sources metadata from free-field operator input, which diverges strongly between laboratories and researchers. In this article we present an open-source workflow which automatically converts and reposits data from the ParaVision structure into the widely supported and openly documented Brain Imaging Data Structure (BIDS). Complementing this workflow we also present operator guidelines for appropriate ParaVision data input, and a programmatic walk-through detailing how preexisting scans with uninterpretable metadata records can easily be made compliant after the acquisition.

An in vivo wound healing model for the characterization of the angiogenic process and its modulation by pharmacological interventions.

Angiogenesis during wound healing is essential for tissue repair and also affected during cancer treatment by anti-angiogenic drugs. Here, we introduce a minimally invasive wound healing model in the mouse ear to assess angiogenesis with high spatiotemporal resolution in a longitudinal manner in vivo using two-photon microscopy in mice expressing GCaMP2 in arterial endothelial cells. The development of vascular sprouts occurred in a highly orchestrated manner within a time window of 8 days following wounding. Novel sprouts developed exclusively from the distal stump of the transsected arteries, growing towards the proximal arterial stump. This was in line with the incidence of Ca2+ transients in the arterial endothelial cells, most probably a result of VEGF stimulation, which were more numerous on the distal part. Functional analysis revealed perfusion across the wound site via arterial sprouts developed between days 6 and 8 following the incision. At day 8, proximal and distal arteries were structurally and functionally connected, though only 2/3 of all sprouts detected were actually perfused. Treatment with the FDA approved drug, sunitinib, the preclinical drug AZD4547, as well as with the combination of the two agents had significant effects on both structural and functional readouts of neo-angiogenesis. The simplicity and high reproducibility of the model makes it an attractive tool for elucidating migratory activity, phenotype and functionality of endothelial cells during angiogenesis and for evaluating specific anti-angiogenic drug interventions.

Selective amotivation deficits following chronic psychosocial stress in mice.

Amotivation is a major symptom of several psychiatric disorders. However, which specific motivations are most affected in various illnesses is not well understood. In major depressive disorder (MDD), anecdotal evidence suggests the motivation to explore may be especially affected, but direct evidence from either patients or animal models is lacking. To investigate the potential for, and nature of, exploratory drive deficits in MDD, we subjected mice to a chronic social defeat (CSD) manipulation that gives rise to a MDD-like behavioural ensemble, and performed a behavioural battery to examine bodyweight homeostasis, ambulation, anxiety, exploratory behaviour motivated by either novelty or fear, and short-term memory. Consistent with previous reports, we found a disruption of bodyweight homeostasis and reduced ambulation following CSD treatment, but we found no evidence for anxiogenic effects or impairments in short-term memory. Surprisingly, we also observed profoundly delayed and diminished exploration of novel, safe space following CSD, while exploration motivated by fear remained intact. These results extend our knowledge of the behavioural phenotypes in mice resulting from CSD by homing in on specific motivational drives. In MDD patients, reduced exploration could compound disease symptomatology by preventing engagement in what could be rewarding exploration experiences, and targeting deficits in the motivation to explore may represent a novel avenue for treatment.