Lecture 11: Neurovascular Coupling

Why Must Cerebral Blood Flow Be Tightly Regulated?

• Neurons cannot store energy efficiently (e.g. limited glycogen), unlike muscle.

• Active neurons have a high metabolic demand, requiring increased oxygen and nutrient delivery to sustain function

• Different brain regions are active at different times, so energy demand is region-specific.

• Active neurons require efficient removal of metabolic waste → prevents toxic metabolite accumulation

  • This is achieved by increasing blood flow to the brain

• Global increases in cerebral blood flow are dangerous, as they can cause brain oedema and damage.

• Therefore, changes in cerebral blood flow must be precisely and locally matched to neuronal activity.

How is Cerebral Blood Flow Tightly Regulated?

• Increased blood flow is achieved through neurovascular coupling

  • Neurovascular coupling: small local increases in blood flow to brain regions with increased activity → active areas

• Active brain regions signal blood vessels that more energy is required, and to deliver more blood and oxygen to these areas, supplying the necessary energy for the activity.

How Does a Functional fMRI Measure Brain Activity?

• An individual is placed in the MRI machine

• Neuronal activity is tracked to identify active areas in the brain

• Functional readout: differences in blood flow are measured (indicating active areas of the brain)

  • Blood flow acts as a surrogate measure for neuronal activity, as the more active an area in the brain, the more blood required

• Helps identify damaged areas of the brain and shows how different brain regions are connected.

How Do Neurons Regulate Blood Flow Along the Cerebral Vascular Tree in Neurovascular Coupling?

• Neurons interact with blood vessels throughout the entire vascular tree.

• Neurovascular coupling (NVC) allows neurons to increase local blood flow via the neurovascular unit:

  • Capillaries

  • Pericytes

  • Astrocyte end-feet

• Pial surface vessels (on the brain surface) are involved in autoregulation and are innervated by perivascular nerves that surround the arteriole wall and release vasoactive factors.

• As pial vessels penetrate brain tissue, perivascular neuronal activity and innervation are lost.

  • Regulation is taken over by astrocytes (glial cells).

• At the capillary level, blood flow regulation (NVC) is driven by astrocytes, capillaries, and pericyte signalling.

How Does the Neurovascular Unit Contribute to Cerebral Blood Flow Regulation

• At the capillary level, there is a loss of smooth muscle cells, which control blood through contraction and relaxation

  • Capillaries: single layer of endothelial cells (SMC absent)

• Pericytes surround the capillary, modulating blood flow through contractility

• Astrocytic end-feet cover most of the brain vasculature, maintaining homeostasis and regulating neuronal activity and blood flow.

  • astrocytes located between neurons andblood vessels

• NVU coordinates blood flow to match neuronal activity via pericyte and astrocyte signalling

How Can Blood Flow Be Rapidly Increased, According to Poiseuille’s Law?

• Changes in the diameter of arterioles allow rapid adjustments in blood flow to active brain areas.

• Hyperpolarisation of smooth muscle stimulates vasodilation, increasing blood flow.

How Do Voltage Gated Ca²⁺ Channels Regulate Vascular Reactivity?

• Voltage-Gated Ca²⁺ Channels Regulate Vascular Reactivity → contraction and relaxation

• Depolarisation increases Ca2+ influx → contraction of VSMC

• Voltage-Gated Ca²⁺ channel signals regulate the phosphorylation of myosin to allow cross bridge formation and shorterning to allow for contraction

How Do Depolarisation and Hyperpolarisatioin Regulate Vascular Reactivity

• Depolarisation mechanisms drive vasoconstriction

  • Mediated by the release of NA, NPY and 5-HT

    • 5-HT can cause vasodilation

  • Stimulation of contractile pathways depolarises smooth muscle cells, initiating vasoconstriction

• Hyperpolarisation mechanisms drive vasodilation

  • Endothelial cells generate Ca²⁺ signals, which activate IK/SK channels

  • Hyperpolarisation is transmitted via gap junctions to smooth muscle, initiating vasodilatation (relaxation).

  • At small arterioles/capillaries, ACh and NO increase Ca²⁺ in the endothelium, hyperpolarising smooth muscle → vasodilation of smaller arterioles.

What are the two main cells that control blood flow in the brain?

• Smooth muscle cells → mediate contraction in arterioles

• Pericytes → regulate capillary diameter.

• Vasodilation is driven by the hyperpolarisation of these cells, which leads to relaxation and increased vessel diameter, increasing blood flow.

• NVC ensures blood supply matches neuronal activity via these mechanisms

What Mediators Drive Neurovascular Coupling (NVC) and How Do They Affect Blood Flow ?

• Neurons: release neurotransmitters during AP firing:

  • Glutamate: sensed by endothelial cells, driving hyperpolarisation and vasodilation

  • Nitric oxide (NO): vasodilator

• Astrocytes release vasoactive signals and transmitters on blood vessels in response to neuronal activity:

  • EETs, ATP, adenosine, prostaglandins → vasodilation or vasoconstriction

• Byproducts of neuronal activity are sensed by endothelial cells, smooth muscle cells, and pericytes:

  • K⁺, reactive oxygen species (ROS) → initiate vasodilation

• Overall effect → increase local blood flow to active brain regions via hyperpolarisation and vessel relaxation

How does ATP act in neurovascular coupling and regulate endothelial cell activity

• ATP is an energy source, a cellular sensor, neurotransmitter

• It binds to purinergic receptors on endothelial cells, driving receptor subtype-dependent activity

• Astrocytic ATP can act on GqPCR on endothelial cells, depleting PIP2 and relieving the block on TRPV4 channels, stimulating their activity

What Are Key Features of TRPV4 Channels?

• Channels involved in endothelial cel activity in the brain

  • Non-selective cation channel

  • high Ca²⁺ permeability

  • Mechanosensitive (?)

  • Activated by EETs

How are TRPV4 Channels Regulated In Brain Capillary Endothelial Cells?

• TRPV4 channels are expressed on brain capillary endothelial cells.

• They are activated by neurotransmitters (e.g., prostaglandin), which drive GPCR signalling and PIP2 hydrolysis

  • Unlike TRPM4, PIP2 inhibits TRPV4, so hydrolysis removes the block and stimulates channel activation

• Experimental evidence → IV curve shows GPCR agonist (PGs) increases TRPV4 current and channel activity, which is blocked by RuR (TRPV4 inhibitor)

• Demonstrates that TRPV4 channels present on endothelial cells stimulate Ca²⁺ signalling and modulate local cerebral blood flow

What are K_ATP Channels?

• ATP-sensitive K⁺ channels, present on endothelial cells and pericytes

• Member of inwardly rectifying K⁺ channel (Kir) family → more effective for K⁺ movement inside the cell rather than outside the cell.

• A tetrameric receptor consisting of:

  • K_IR6.1 or K_IR6.2 subunits (KCNJ8 & KCNJ11 genes)

  • Sulfonylurea receptor auxiliary subunits (bind ATP/ADP)

• Involved in hyperpolarisation and driving vasodilation → couples vascular metabolism and adenosine signalling to increase blood flow to metabolically active areas.

• Inhibited by high intracellular ATP levels; activated when ADP increases, and ATP decreases

  • Channel activity is linked to the ATP/ADP ratio

• Channel targeted in diabetes

How Does Adenosine Modulate Cerebral Blood Flow?

• Adenosine is a signalling molecule produced from extracellular ATP,

  • Both ATP and adenosine can activate GPCR

• Adenosine released from neurons or astrocyte end-feet acts locally on capillary endothelial cells and pericytes

• Adenosine binds to adenosine receptors (Gs-GCPRs), which activate PKA, which can stimulate the K_ATP channel directly

• K_ATP channel opening leads to K⁺ efflux and hyperpolarisation, which is sensed by endothelial cells and pericytes, causing vasodilation

• The signal can be transmitted upstream via gap junctions, increasing blood flow to the active brain region

• Two-pronged effect → local hyperpolarisation and signal transmission upstream

How Do K_ATP Channels Contribute to NVC

• K_ATP channels are involved in signalling pathways that help regulate blood flow during NVC.

• Adding adenosine to cells (help at Vm) induces an inward current (downward deflection) by activating KATP channel activity through PKA activation.

  • K_ATP channel activity blocked by Beclamide, leading to a loss of current.

• Current density is 2-3x larger in pericytes than in endothelial cells, suggesting pericytes may play a larger role in detecting adenosine and regulating blood flow.

What are Kir Channels and How Do They Modulate Cerebral Blood Flow?

• Inwardly rectifying K+ channels → K⁺ sensitive K⁺ channels

  • Sensitive to extracellular [K⁺]; unusual kinetics (intracellular Mg²⁺/polyamine block of outward current)

• Encoded by KCNJ2 gene

• Increases in [K⁺]_o from 3-15nm cause vasodilation;

  • K+ channels detect increased extracellular K⁺ and promote further K⁺ release into the extracellular environment

  • This K⁺ efflux from the endothelial cell causes hyperpolarisation, which is transmitted to smooth muscle cells to mediate vasodilation

• Removal of [K⁺]_o causes vasoconstriction

What are the Key Features of the K_ir (Inwardly Rectifying) Channels?

• Sensitive to extracellular K⁺ concentration → sense changes in K⁺ to regulate efflux.

• Pore blocked by Ba²⁺ → selective inhibitor that abolishes Kir current.

• Channel activity increases with hyperpolarisation → greater conductance at more negative potentials.

• Kir IV curve:

  • Full IV curve generated at Vm range:-140 mV to +50 mV (experimental, not physiological)

  • Shows inward rectification – preferential K⁺ influx at negative potentials.

What happens to Kir2.1 channel activity at normal (physiological) extracellular K⁺ ([K⁺]o ≈ 3 mM) and depolarised membrane potential (Vm ≈ -35 mV)?

• Vessel is constricted; cell is depolarised.

• When depolarised, Kir2.1 channel is blocked by polyamines and Mg²⁺.

  • Polyamines: positively charged, block the pore.

  • Mg²⁺: binds the cytosolic region, blocking the pore.

• This results in little to no channel activity → no K⁺ current

• Reversal potential (Ek) ≈ -103 mV → drives K⁺ entry, but is blocked by polyamines/Mg²⁺.

What happens to Kir2.1 channel activity at extracellular K⁺ ([K⁺]o ≈ 3 mM) and hyperpolarised membrane potential (Vm ≈ -55 mV)?

• Hyperpolarisation stimulates channel activity, increasing the electrochemical driving force for K⁺ influx (due to a negative intracellular environment) → increases channel conductance

• K⁺ influx displaces polyamines and Mg²⁺, blocking the pore

• Removal of pore block allows Kir channels to open

• Channel closure occurs through depolarisation or [K⁺]_O removal (non-physiological)

What happens to Kir2.1 channel activity when extracellular [K⁺] increases?

• At physiological [K⁺]o (≈3 mM), Ek ≈ −103 mV, there is a strong drive for K⁺ entry, but little current is generated due to polyamine/Mg²⁺ block

• Neuronal activity increases [K⁺]o, in response to neuronal repolarisation, shifts the Ek towards −76 mV and decreases the membrane potential

• This shifts the I–V relationship rightwards, activating Kir channels

• Once open, Kir channels drive Vm toward Ek via sustained K⁺ efflux

  • The channel is consistently activated to drive down the reversal potential

• At −40 mV and 8 mM [K⁺]o → ~62-fold increase in channel activity until Ek is released

• → increased extracellular K+ increases channel conductance

How Can Kir Channels Be Activated by Neuronal and Astrocyte Action Potential Firing?

• Both neuronal and astrocyte firing can activate Kir channels.

• Astrocyte action potentials cause Ca²⁺ influx, followed by hyperpolarisation in response to the AP depolarisation to restore membrane potential.

• This results in the release of K⁺ near smooth muscle cells or the endothelium.

• The K⁺ released is sensed by the Kir2.1 channels, and the hyperpolarisation decreases Ca²⁺ influx via Voltage-Gated Calcium Channels

• If there is excessive neuronal activity, vasoconstriction can be stimulated.

  • Excess K⁺ in the extracellular space can also lead to vasoconstriction, often seen in certain diseases.

How Can Excess K+ In The Extracellular Environment Lead to Vasoconstriction?

• Excess K⁺ in the extracellular environment can alter the reversal potential and trigger vasoconstriction.

• Stimulation of neuronal activity leads to [K⁺]ₒ increasing to 15mM.

• This causes the reversal potential to shift to -60mV.

• The membrane potential of VSMCs lies within a range of -55mV and -35mV to allows both vasoconstriction and vasodilation.

  • Hyperpolarisation from increased K⁺ allows for vasodilation.

• This increase in [K⁺]ₒ to 15mM activates Kir channels and pushes the reversal potential (E_K) to -60mV, which promotes vasodilation.

• Further increase in [K⁺]ₒ to 40mM shifts the reversal potential to -35mV (Vm for full constriction).

  • K⁺ Channels have no significant effect on the vasculature, promoting vasoconstriction instead.

Why is It Important For Cells in the Brain Modulate Extracellular K^+?

• To control neuronal activity.

• Migraines are linked to an increase in extracellular K⁺, which can stimulate vasoconstriction in the brain.

How Do Neurons & Astrocytes Regulate Blood Flow via K⁺ Efflux?

• When neurons and astrocytes are depolarised, they must repolarise by effluxing K⁺.

• This K⁺ efflux occurs near capillary endothelial cells.

• The efflux of K⁺ hyperpolarises endothelial cells, which then causes hyperpolarisation in the pericytes (contractile cells around capillaries) which is then transmitted upstream to the arteriole.

• Hyperpolarisation reaches the arteriole smooth muscle, and initiates vasodilation, increasing blood flow to the area.

• Pressure myography can be used to monitor small arteries and simulate physiological events, e.g. K⁺ stimulation in the capillary bed can mimic the process of neuronal and astrocyte regulation of blood flow in experimental setups

How Do KIR2.1 Channels Regulate Vasodilation and Blood Flow?

• KIR2.1 channels are present at the capillary level and help sense ATP and adenosine to initiate vasodilation.

• Neuronal activity stimulates contractile cells (in either arterioles or capillaries) to increase vessel diameter, increasing blood flow to the area.

• KIR channels play a critical role in sensing K⁺ levels.

  • in a physiological setting, there's no immediate turn-off of KIR channel activity, allowing sustained regulation.

• Vasodilation leads to hyperpolarisation and a decrease in the reversal potential.

• This process is stopped via Peizo1, a mechanosensitive ion channel that helps regulate the vasodilation response.

What is Peizo1?

• A stretch-sensitive, mechanosensitive ion channel, found on endothelial cells, including capillary endothelial cells in the brain.

• It is a large protein that forms a trimer.

  • The channel bends the membrane within its structure, creating kinks due to its hydrophobic and hydrophilic components.

• When the membrane is stretched, it opens the channel, allowing cation entry.

  • It is a non-selective cation channel with high Ca²⁺ permeability.

How is Piezo1 Studies in the Cerebral Circulation

• Negative pressure is applied via a pipette to stretch the membrane underneath.

• Results with Varying Stretch:

  • Little to no stretch: No channel activity.

  • Increasing negative stretch: Causes single-channel openings at a given conductance.

How Was Piezo1 Shown to Regulate Neurovascular Coupling (NVC)?

• Whisker stimulation in a mouse model

• Whisker stimulation increases neuronal activity in the barrel cortex, which also increases blood flow to that area.

• This neuronal activity stimulates Kir2.1 channels via K⁺ release, causing hyperpolarisation and vasodilation.

• Increased vasodilation increases blood flow through capillaries, increasing shear stress.

• This shear stress (due to RBCs squeezing through capillaries, generating friction against endothelial cells) stimulates Piezo1 channel activity as the membrane stretches.

• Piezo1 activation leads to depolarisation due to cation influx, which inhibits Kir2.1 channel activity, reducing vasodilation.

• Application of Yoda1 (a Piezo1 activator) reduces neurovascular coupling (NVC), blunting the vasodilation response.

• Enhancing Piezo1 activity decreases NVC by depolarising endothelial cells, which decreases Kir channel activity.

How Does Piezo1 Act as a "Brake" in Blood Flow Regulation?

• Piezo1 functions like a brake in a car, regulating blood flow and endothelial cell activity.

• Stimulation of Kir2.1 = hyperpolarisation of endothelial cells, similar to pressing the accelerator to increase blood flow.

• Once the increased blood flow is sensed, Piezo1 acts as the brake, slowing things down.

• Changes in blood flow alter hemodynamic forces (like shear stress and blood volume), stretch the capillary membrane.

• This stretch activates Piezo1, causing an influx of Ca²⁺, Na⁺, and K⁺, which depolarises the membrane.

• This depolarisation switches off the Kir2.1 channels, stoping vasodilation and reducing blood flow.

How Do Vascular Ion Channels Sense Neuronal Activity?

• Via astrocyte signalling or neuronal signalling byproducts

• Capillary endothelial cells are well-positioned to sense neuronal activity.

  • They send hyperpolarising signals upstream to arterial smooth muscle to initiate vasodilation.

• Astrocytes can also signal directly to arteriole smooth muscle to influence blood flow.