This is the last year of this Z01 due to the departure of Afonso Silva to the University of Pittsburgh. All Staff have left and therefore this is the last year of this project in the intramural NINDS, NIH. Dr. Koretsky serves as the Acting Section Head for a little bit of FY19 after Dr. Silva left NIH. This project been studying the spatiotemporal characteristics of the hemodynamic response function (HRF) to focal brain activity in marmosets (Callithrix jacchus), a small New World non-human primate that we believe is especially suitable to bridge the translational animal-to-human gap, and one in which transgenic techniques are becoming available to allow us to interrogate neurons and blood vessels in interesting and novel ways. We have trained marmosets to be imaged in the awake state and we are able to routinely obtain functional maps while the animal undergoes somatosensory, auditory, or visual stimulation. THis project continues to make excellent progress. It has mapped the BOLD fMRI response to somatosensory stimulation in awake marmosets. We used a dual-echo gradient-recalled echo planar imaging (GR-EPI) sequence to separate the deoxyhemoglobin-related response (absolute T2* differences) from the deoxyhemoglobin-unrelated response (relative S0 changes). We employed a spatial saturation pulse to saturate incoming arterial spins and reduce inflow effects. Functional GR-EPI images were obtained from a single coronal slice with two different echo times (13.5 and 40.5 ms) and TR = 0.2 s. BOLD, T2*, and S0 images were calculated and their functional responses were detected in both hemispheres of primary somatosensory cortex, from which five laminar regions (L1+2, L3, L4, L5, and L6) were derived. The spatiotemporal distribution of the BOLD response across the cortical layers was heterogeneous, with the middle layers having the highest BOLD amplitudes and shortest onset times. T2* also showed a similar trend. However, functional S0 changes were detected only in L1+2, with a fast onset time. Because inflow effects were minimized, the source of S0 functional changes in L1+2 could be attributed to a reduction of cerebrospinal fluid volume fraction due to the functional increase in cerebral blood volume and to unmodeled T2* changes in the extra- and intra-venous compartments. This work was published in Neuroimage (2018). We also investigated the spatiotemporal evolution of the BOLD and CBV HRF in conscious, awake marmosets using a block design paradigm in which the stimulus duration increased in pseudo-random order from a single pulse up to 256 electrical pulses (4 s). For CBV measurements, 30 mg/kg of ultrasmall superparamagnetic ironoxide particles (USPIO) injected intravenously, were used. Robust BOLD and CBV HRFs were obtained in the primary somatosensory cortex (S1), secondary somatosensory cortex (S2) and caudate at all stimulus conditions. In particular, BOLD and CBV responses to a single 333-s-long stimulus were reliably measured, and the CBV HRF presented shorter onset time and time to peak than the BOLD HRF. Both the size of the regions of activation and the peak amplitude of the HRFs grew quickly with increasing stimulus duration, and saturated for stimulus durations greater than 1 s. Onset times in S1 and S2 were faster than in caudate. Finally, the fine spatiotemporal features of the HRF in awake marmosets were similar to those obtained in humans, indicating that the continued refinement of awake non-human primate models is essential to maximize the applicability of animal functional MRI studies to the investigation of human brain function. In addition to using somatosensory stimulation, we also used auditory stimulation to test whether different areas of the auditory cortex could be identified using fMRI in marmosets. We used two types of stimulation, band pass noise and pure tones, to parse apart the auditory core from surrounding secondary belt fields. In contrast to most auditory fMRI experiments in primates, we employed a continuous sampling paradigm to rapidly collect data with little deleterious effects. We found robust bilateral auditory cortex activation in two marmosets and unilateral activation in a third utilizing this preparation. Furthermore, we confirmedresults previously reported in electrophysiology experiments, such as the tonotopic organization of the auditory core and regions activating preferentially to complex over simple stimuli. Overall, these data establish a key preparation for future research to investigate various functional properties of marmoset auditory cortex. Because of the extensive work using marmosets, it was necessary for us to develop an atlas of the marmoset cortex to facilitate identification of regions of interest. We constructed a 3D digital atlas based on high-resolution ex-vivo MRI images, including magnetization transfer ratio (a T1-like contrast), T2w images, and multi-shell diffusion MRI. Based on the multi-modal MRI images, we manually delineated 54 cortical areas and 16 subcortical regions on one hemisphere of the brain (the core version). The 54 cortical areas were merged into 13 larger cortical regions according to their locations to yield a coarse version of the atlas, and also parcellated into 106 sub-regions using a connectivity-based parcellation method to produce a refined atlas. Finally, we compared the new atlas set with existing histology atlases and demonstrated its applications in connectome studies, and in resting state and stimulus-based fMRI. The atlas set has been integrated into the widely-distributed neuroimaging data analysis software AFNI and SUMA, providing a readily usable multi-modal template space with multi-level anatomical labels (including labels from the Paxinos atlas) that can facilitate various neuroimaging studies of marmosets. This atlas is now shared worldwide. Last but not least, owing to our great success in generating transgenic marmosets expressing genetically encoded calcium indicators such as GCaMP5G/6s, which we believe will constitute a much-improved animal model for studying neurovascular coupling in relevant conditions, we started to develop and apply gene-editing techniques such as CRISPR/cas9 to generate a marmoset model of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). We constructed guide RNAs targeting the most common CADASIL-causing Notch3 mutations and we were able to already obtain 3 live births of mutant marmosets. We are currently working on improving our methods to produce single point mutations that are more precise. At the same time, we are developing the MRI and fMRI methods to study stroke in the marmosets.

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Yen, Cecil Chern-Chyi; Papoti, Daniel; Silva, Afonso C (2018) Investigating the spatiotemporal characteristics of the deoxyhemoglobin-related and deoxyhemoglobin-unrelated functional hemodynamic response across cortical layers in awake marmosets. Neuroimage 164:121-130
Liu, Cirong; Ye, Frank Q; Yen, Cecil Chern-Chyi et al. (2018) A digital 3D atlas of the marmoset brain based on multi-modal MRI. Neuroimage 169:106-116
Toarmino, Camille R; Yen, Cecil C C; Papoti, Daniel et al. (2017) Functional magnetic resonance imaging of auditory cortical fields in awake marmosets. Neuroimage 162:86-92
Silva, Afonso C (2017) Anatomical and functional neuroimaging in awake, behaving marmosets. Dev Neurobiol 77:373-389
Papoti, Daniel; Yen, Cecil Chern-Chyi; Hung, Chia-Chun et al. (2017) Design and implementation of embedded 8-channel receive-only arrays for whole-brain MRI and fMRI of conscious awake marmosets. Magn Reson Med 78:387-398
Park, Jung Eun; Zhang, Xian Feng; Choi, Sang-Ho et al. (2016) Generation of transgenic marmosets expressing genetically encoded calcium indicators. Sci Rep 6:34931
Yu, Xin; He, Yi; Wang, Maosen et al. (2016) Sensory and optogenetically driven single-vessel fMRI. Nat Methods 13:337-40
Belcher, Annabelle M; Yen, Cecil Chern-Chyi; Notardonato, Lucia et al. (2016) Functional Connectivity Hubs and Networks in the Awake Marmoset Brain. Front Integr Neurosci 10:9
Miller, Cory T; Freiwald, Winrich A; Leopold, David A et al. (2016) Marmosets: A Neuroscientific Model of Human Social Behavior. Neuron 90:219-33
Guy, Joseph R; Sati, Pascal; Leibovitch, Emily et al. (2016) Custom fit 3D-printed brain holders for comparison of histology with MRI in marmosets. J Neurosci Methods 257:55-63

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