Rabbit Anti-Human THY1 monoclonal antibody, clone KG21-10

Corticofugal projection neurons are key components in connecting the neocortex and the subcortical regions. In the barrel field, these neurons have various projection targets and play crucial roles in the rodent whisker sensorimotor system. However, the projection features of corticofugal projection neurons at the single-axon level are far from comprehensive elucidation. Based on a brain-wide positioning system with high-resolution imaging for Thy1-GFP M-line mice brains, we reconstructed and analyzed more than one hundred corticofugal projection neurons in both layer V and VI of barrel cortex. The dual-color imaging made it possible to locate the neurons' somata, trace their corresponding dendrites and axons and then distinguish the neurons as L5 type I/II or L6 type. The corticofugal projection pattern showed significant diversity across individual neurons. Usually, the L5 type I neurons have greater multi-region projection potential. The thalamus and the midbrain are the most frequent projection targets among the investigated multidirectional projection neurons, and the hypothalamus is particularly unique in that it only appears in multidirectional projection situations. Statistically, the average branch length of apical dendrites in multi-region projection groups is larger than that of single-region projection groups. This study demonstrated a single-axon-level analysis for barrel corticofugal projection neurons, which could provide a micro-anatomical basis for interpreting whisker sensorimotor circuit function.

The interplay between hemodynamic-based markers of cortical activity (e.g. fMRI and optical intrinsic signal imaging), which are an indirect and relatively slow report of neural activity, and underlying synaptic electrical and metabolic activity through neurovascular coupling is a topic of ongoing research and debate. As application of resting state functional connectivity measures is extended further into topics such as brain development, aging and disease, the importance of understanding the fundamental physiological basis for functional connectivity will grow. Here we extend functional connectivity analysis from hemodynamic-to calcium- based imaging. Transgenic mice (n = 7) expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketamine/xylazine) and awake states. Sequential LED illumination (lambda = 454, 523, 595, 640nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. Functional connectivity network maps were constructed for infraslow (0.009-0.08Hz), intermediate (0.08-0.4Hz), and high (0.4-4.0Hz) frequency bands. At infraslow and intermediate frequencies, commonly used in BOLD fMRI and fcOIS studies of functional connectivity and implicated in neurovascular coupling mechanisms, GCaMP6 and HbO(2) functional connectivity structures were in high agreement, both qualitatively and also quantitatively through a measure of spatial similarity. The spontaneous dynamics of both contrasts had the highest correlation when the GCaMP6 signal was delayed with a similar to 0.6-1.5s temporal offset. Within the higher-frequency delta band, sensitive to slow wave sleep oscillations in non-REM sleep and anesthesia, we evaluate the speed with which the connectivity analysis stabilized and found that the functional connectivity maps captured putative network structure within time window lengths as short as 30 seconds. Homotopic GCaMP6 functional connectivity maps at 0.4-4.0Hz in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. This structure is potentially explained in part by Dobserved propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas. During awake imaging, this spatio-temporal quality is altered, and a more complex and detailed functional connectivity structure is observed. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and neurovascular coupling and provide a framework for subsequent studies of neurological disease such as stroke.