in-vivo-three-dimensional-two-photon-microscopy-to-study-conducted

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

University of Copenhagen

Rigshospitalet

Brain's electrical activity correlates strongly to changes in cerebral blood flow (CBF) and the cerebral metabolic rate of oxygen (CMRO(2)). Subthreshold synaptic processes correlate better than the spike rates of principal neurons to CBF, CMRO(2) and positive BOLD signals. Stimulation-induced rises in CMRO(2) are controlled by the ATP turnover, which depends on the energy used to fuel the Na,K-ATPase to reestablish ionic gradients, while stimulation-induced CBF responses to a large extent are controlled by mechanisms that depend on Ca(2+) rises in neurons and astrocytes. This dichotomy of metabolic and vascular control explains the gap between the stimulation-induced rises in CMRO(2) and CBF, and in turn the BOLD signal. Activity-dependent rises in CBF and CMRO(2) vary within and between brain regions due to differences in ATP turnover and Ca(2+)-dependent mechanisms. Nerve cells produce and release vasodilators that evoke positive BOLD signals, while the mechanisms that control negative BOLD signals by activity-dependent vasoconstriction are less well understood. Activation of both excitatory and inhibitory neurons produces rises in CBF and positive BOLD signals, while negative BOLD signals under most conditions correlate to excitation of inhibitory interneurons, but there are important exceptions to that rule as described in this paper. Thus, variations in the balance between synaptic excitation and inhibition contribute dynamically to the control of metabolic and hemodynamic responses, and in turn the amplitude and polarity of the BOLD signal. Therefore, it is not possible based on a negative or positive BOLD signal alone to decide whether the underlying activity goes on in principal or inhibitory neurons.

Neuronal inhibition and excitation, and the dichotomic control of brain hemodynamic and oxygen responses.

Functional neuroimaging, such as fMRI, is based on coupling neuronal activity and accompanying changes in cerebral blood flow (CBF) and metabolism. However, the relationship between CBF and events at the level of the penetrating arterioles and capillaries is not well established. Recent findings suggest an active role of capillaries in CBF control, and pericytes on capillaries may be major regulators of CBF and initiators of functional imaging signals. Here, using two-photon microscopy of brains in living mice, we demonstrate that stimulation-evoked increases in synaptic activity in the mouse somatosensory cortex evokes capillary dilation starting mostly at the first- or second-order capillary, propagating upstream and downstream at 5-20 µm/s. Therefore, our data support an active role of pericytes in cerebrovascular control. The gliotransmitter ATP applied to first- and second-order capillaries by micropipette puffing induced dilation, followed by constriction, which also propagated at 5-20 µm/s. ATP-induced capillary constriction was blocked by purinergic P2 receptors. Thus, conducted vascular responses in capillaries may be a previously unidentified modulator of cerebrovascular function and functional neuroimaging signals.

Stimulation-induced increases in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses.

The endothelial cells that form the blood-brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules-sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran-from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

Article	Year
Neuronal inhibition and excitation, and the dichotomic control of brain hemodynamic and oxygen responses. NeuroImage\| PubMed ID: 22261372	2012
Stimulation-induced increases in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29866853	2018
Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood-brain barrier. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30217895	2018

Martin Lauritzen

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