The Subcortical Atlas of the Marmoset ("SAM") monkey based on high-resolution MRI and histology.

A comprehensive three-dimensional digital brain atlas of cortical and subcortical regions based on MRI and histology has a broad array of applications in anatomical, functional, and clinical studies. We first generated a Subcortical Atlas of the Marmoset, called the "SAM," from 251 delineated subcortical regions (e.g. thalamic subregions, etc.) derived from high-resolution Mean Apparent Propagator-MRI, T2W, and magnetization transfer ratio images ex vivo. We then confirmed the location and borders of these segmented regions in the MRI data using matched histological sections with multiple stains obtained from the same specimen. Finally, we estimated and confirmed the atlas-based areal boundaries of subcortical regions by registering this ex vivo atlas template to in vivo T1- or T2W MRI datasets of different age groups (single vs. multisubject population-based marmoset control adults) using a novel pipeline developed within Analysis of Functional NeuroImages software. Tracing and validating these important deep brain structures in 3D will improve neurosurgical planning, anatomical tract tracer injections, navigation of deep brain stimulation probes, functional MRI and brain connectivity studies, and our understanding of brain structure-function relationships. This new ex vivo template and atlas are available as volumes in standard NIFTI and GIFTI file formats and are intended for use as a reference standard for marmoset brain research.

The Subcortical Atlas of the Marmoset ("SAM") monkey based on high-resolution MRI and histology.

A comprehensive three-dimensional digital brain atlas of cortical and subcortical regions based on MRI and histology has a broad array of applications for anatomical, functional, and clinical studies. We first generated a Subcortical Atlas of the Marmoset, called the "SAM," from 251 delineated subcortical regions (e.g., thalamic subregions, etc.) derived from the high-resolution MAP-MRI, T2W, and MTR images ex vivo. We then confirmed the location and borders of these segmented regions in MRI data using matched histological sections with multiple stains obtained from the same specimen. Finally, we estimated and confirmed the atlas-based areal boundaries of subcortical regions by registering this ex vivo atlas template to in vivo T1- or T2W MRI datasets of different age groups (single vs. multisubject population-based marmoset control adults) using a novel pipeline developed within AFNI. Tracing and validating these important deep brain structures in 3D improves neurosurgical planning, anatomical tract tracer injections, navigation of deep brain stimulation probes, fMRI and brain connectivity studies, and our understanding of brain structure-function relationships. This new ex vivo template and atlas are available as volumes in standard NIFTI and GIFTI file formats and are intended for use as a reference standard for marmoset brain research.

Multimodal anatomical mapping of subcortical regions in marmoset monkeys using high-resolution MRI and matched histology with multiple stains.

Subcortical nuclei and other deep brain structures play essential roles in regulating the central and peripheral nervous systems. However, many of these nuclei and their subregions are challenging to identify and delineate in conventional MRI due to their small size, hidden location, and often subtle contrasts compared to neighboring regions. To address these limitations, we scanned the whole brain of the marmoset monkeys in ex vivo using a clinically feasible diffusion MRI method, called the mean apparent propagator (MAP)-MRI, along with T2W and MTR (T1-like contrast) images acquired at 7 Tesla. Additionally, we registered these multimodal MRI volumes to the high-resolution images of matched whole-brain histology sections with seven different stains obtained from the same brain specimens. At high spatial resolution, the microstructural parameters and fiber orientation distribution functions derived with MAP-MRI can distinguish the subregions of many subcortical and deep brain structures, including fiber tracts of different sizes and orientations. The good correlation with multiple but distinct histological stains from the same brain serves as a thorough validation of the structures identified with MAP-MRI and other MRI parameters. Moreover, the anatomical details of deep brain structures found in the volumes of MAP-MRI parameters are not visible in conventional T1W or T2W images. The high-resolution mapping using novel MRI contrasts, combined and correlated with histology, can elucidate structures that were previously invisible radiologically. Thus, this multimodal approach offers a roadmap toward identifying salient brain areas in vivo in future neuroradiological studies. It also provides a useful anatomical standard reference for the region definition of subcortical targets and the generation of a 3D digital template atlas for the marmoset brain research (Saleem et al., 2023). Additionally, we conducted a cross-species comparison between marmoset and macaque monkeys using results from our previous studies (Saleem et al., 2021). We found that the two species had distinct patterns of iron distribution in subregions of the basal ganglia, red nucleus, and deep cerebellar nuclei, confirmed with T2W MRI and histology.

Multimodal anatomical mapping of subcortical regions in Marmoset monkeys using high-resolution MRI and matched histology with multiple stains.

Subcortical nuclei and other deep brain structures play essential roles in regulating the central and peripheral nervous systems. However, many of these nuclei and their subregions are challenging to identify and delineate in conventional MRI due to their small size, hidden location, and often subtle contrasts compared to neighboring regions. To address these limitations, we scanned the whole brain of the marmoset monkeys in ex vivo using a clinically feasible diffusion MRI method, called the mean apparent propagator (MAP)-MRI, along with T2W and MTR (T1-like contrast) images acquired at 7 Tesla. Additionally, we registered these multimodal MRI volumes to the high-resolution images of matched whole-brain histology sections with seven different stains obtained from the same brain specimens. At high spatial resolution, the microstructural parameters and fiber orientation distribution functions derived with MAP-MRI can distinguish the subregions of many subcortical and deep brain structures, including fiber tracts of different sizes and orientations. The good correlation with multiple but distinct histological stains from the same brain serves as a thorough validation of the structures identified with MAP-MRI and other MRI parameters. Moreover, the anatomical details of deep brain structures found in the volumes of MAP-MRI parameters are not visible in conventional T1W or T2W images. The high-resolution mapping using novel MRI contrasts, combined and correlated with histology, can elucidate structures that were previously invisible radiologically. Thus, this multimodal approach offers a roadmap toward identifying salient brain areas in vivo in future neuroradiological studies. It also provides a useful anatomical standard reference for the region definition of subcortical targets and the generation of a 3D digital template atlas for the marmoset brain research (Saleem et al., 2023). Additionally, we conducted a cross-species comparison between marmoset and macaque monkeys using results from our previous studies (Saleem et al., 2021). We found that the two species had distinct patterns of iron distribution in subregions of the basal ganglia, red nucleus, and deep cerebellar nuclei, confirmed with T2W MRI and histology.

Pore diameter mapping using double pulsed-field gradient MRI and its validation using a novel glass capillary array phantom.

Double pulsed-field gradient (d-PFG) MRI can provide quantitative maps of microstructural quantities and features within porous media and tissues. We propose and describe a novel MRI phantom, consisting of wafers of highly ordered glass capillary arrays (GCA), and its use to validate and calibrate a d-PFG MRI method to measure and map the local pore diameter. Specifically, we employ d-PFG Spin-Echo Filtered MRI in conjunction with a recently introduced theoretical framework, to estimate a mean pore diameter in each voxel within the imaging volume. This simulation scheme accounts for all diffusion and imaging gradients within the diffusion weighted MRI (DWI) sequence, and admits the violation of the short gradient pulse approximation. These diameter maps agree well with pore sizes measured using both optical microscopy and single PFG diffusion diffraction NMR spectroscopy using the same phantom. Pixel-by-pixel analysis shows that the local pore diameter can be mapped precisely and accurately within a specimen using d-PFG MRI.

Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo.

Glial cell Ca2+ signals play a key role in glial-neuronal and glial-glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100beta promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100beta in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100beta, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.

Functional reactivity of cerebral capillaries.

The spatiotemporal evolution of cerebral microcirculatory adjustments to functional brain stimulation is the fundamental determinant of the functional specificity of hemodynamically weighted neuroimaging signals. Very little data, however, exist on the functional reactivity of capillaries, the vessels most proximal to the activated neuronal population. Here, we used two-photon laser scanning microscopy, in combination with intracranial electrophysiology and intravital video microscopy, to explore the changes in cortical hemodynamics, at the level of individual capillaries, in response to steady-state forepaw stimulation in an anesthetized rodent model. Overall, the microcirculatory response to functional stimulation was characterized by a pronounced decrease in vascular transit times (20%+/-8%), a dilatation of the capillary bed (10.9%+/-1.2%), and significant increases in red blood cell speed (33.0%+/-7.7%) and flux (19.5%+/-6.2%). Capillaries dilated more than the medium-caliber vessels, indicating a decreased heterogeneity in vessel volumes and increased blood flow-carrying capacity during neuronal activation relative to baseline. Capillary dilatation accounted for an estimated approximately 18% of the total change in the focal cerebral blood volume. In support of a capacity for focal redistribution of microvascular flow and volume, significant, though less frequent, local stimulation-induced decreases in capillary volume and erythrocyte speed and flux also occurred. The present findings provide further evidence of a strong functional reactivity of cerebral capillaries and underscore the importance of changes in the capillary geometry in the hemodynamic response to neuronal activation.

Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier.

During action potential conduction, the axonal specializations at the node, together with the adjacent paranodal terminations of the myelin sheath, interact with glial processes that invest the nodal gap. The nature of the mutual signals between axons and myelinating glia, however, are not well understood. Here we have characterized the distribution of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the axoglial apparatus by immunohistochemistry, using known myelin domain-specific markers. While IP(3)R1 is not expressed in the Schwann cells or the axon, IP(3)R2 and IP(3)R3 are expressed in distinct cellular domains, suggesting distinct signaling roles for the two receptors. IP(3)R3 is the most predominant isoform in Schwann cells, and is expressed in particularly dense patches in the paranodal region. In addition to IP(3)Rs, two other members of the metabotropic Ca(2+) signaling pathway, G(alpha)q, and P(2)Y1 type of purinoceptors were also found in Schwann cells. Their pattern of expression matches the expression of their signaling partners, the IP(3)Rs. One interesting finding to emerge from this study is the expression of connexin 32 (Cx32) in close proximity with IP(3)R3. Although IP(3)R3 and Cx32 are not colocalized, their expression in the same membrane areas raises the question whether Schwann cell Ca(2+) signals either control the function of the gap junctions, or whether the gap junctional channels serve as conduits for rapid radial spread of Ca(2+) signals initiated during action potential propagation.

SP-B and SP-C alter diffusion in bilayers of pulmonary surfactant.

The hydrophobic proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface by an unknown mechanism. We tested the hypothesis that these proteins accelerate adsorption by disrupting the structure of the lipid bilayer, either by a generalized increase in fluidity or by a focal induction of interfacial boundaries within the bilayer. We used fluorescence recovery after photobleaching to measure diffusion of nitrobenzoxadiazolyl-dimyristoyl-phosphatidylethanolamine between 11 and 54 degrees C in multilayers containing the complete set of lipids and proteins in calf lung surfactant extract (CLSE), or the complete set of neutral and phospholipids without the proteins. Above 35 degrees C, Arrhenius plots of diffusion were parallel for CLSE and neutral and phospholipids, but shifted to lower values for CLSE, suggesting that the proteins rigidify the lipid bilayer rather than producing the proposed increase in membrane fluidity. The slopes of the Arrhenius plots for CLSE were steeper below 35 degrees C, suggesting that the proteins induce phase separation at that temperature. The mobile fraction fell below 27 degrees C, consistent with a percolation threshold of coexisting gel and liquid-crystal phases. The induction of lateral phase separation in CLSE, however, does not correlate with apparent changes in adsorption kinetics at this temperature. Our results suggest that SP-B and SP-C accelerate adsorption through a mechanism other than the disruption of surfactant bilayers, possibly by stabilizing a high-energy, highly curved adsorption intermediate.

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface.

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.

Pulmonary surfactant: phase behavior and function.

Pulmonary surfactant functions by first flowing rapidly into the alveolar air/water interface, but then resisting collapse from the surface when the adsorbed interfacial film is compressed during exhalation. Widely accepted models emphasize the importance of phase behavior in both processes. Recent studies show, however, that fluidity is a relatively minor determinant of adsorption and that solid films, which resist collapse, can form by kinetic processes unrelated to equilibrium phase behavior.