Cortical Plasticity is associated with Blood-Brain-Barrier Modulation

Reviewer #1 (Public Review):

The goal of the current study was to evaluate the effect of neuronal activity on blood-brain barrier permeability in the healthy brain, and to determine whether changes in BBB dynamics play a role in cortical plasticity. The authors used a variety of well-validated approaches to first demonstrate that limb stimulation increases BBB permeability. Using in vivo-electrophysiology and pharmacological approaches, the authors demonstrate that albumin is sufficient to induce cortical potentiation and that BBB transporters are necessary for stimulus-induced potentiation. The authors include a transcriptional analysis and differential expression of genes associated with plasticity, TGF-beta signaling, and extracellular matrix were observed following stimulation. Overall, the results obtained in rodents are compelling and support the authors' conclusions that neuronal activity modulates the BBB in the healthy brain and that mechanisms downstream of BBB permeability changes play a role in stimulus-evoked plasticity. These findings were further supported with fMRI and BBB permeability measurements performed in healthy human subjects performing a simple sensorimotor task. While there are many strengths in this study, there is literature to suggest that there are sex differences in BBB dysfunction in pathophysiological conditions. The authors only used males in this study and do not discuss whether they would also expect to sex differences in stimulation-evoked BBB changes in the healthy brain. Another minor limitation is the authors did not address the potential impact of anesthesia which can impact neurovascular coupling in rodent studies. The authors could have also better integrated the RNAseq findings into mechanistic experiments, including testing whether the upregulation of OAT3 plays a role in cortical plasticity observed following stimulation. Overall, this study provides novel insights into how neurovascular coupling, BBB permeability, and plasticity interact in the healthy brain.

Reviewer #2 (Public Review):

Summary:
This study builds upon previous work that demonstrated that brain injury results in leakage of albumin across the blood-brain barrier, resulting in activation of TGF-beta in astrocytes. Consequently, this leads to decreased glutamate uptake, reduced buffering of extracellular potassium, and hyperexcitability. This study asks whether such a process can play a physiological role in cortical plasticity. They first show that stimulation of a forelimb for 30 minutes in a rat results in leakage of the blood-brain barrier and extravasation of albumin on the contralateral but not ipsilateral cortex. The authors propose that the leakage is dependent upon neuronal excitability and is associated with an enhancement of excitatory transmission. Inhibiting the transport of albumin or the activation of TGF-beta prevents the enhancement of excitatory transmission. In addition, gene expression associated with TGF-beta activation, synaptic plasticity, and extracellular matrix are enhanced on the "stimulated" hemisphere. That this may translate to humans is demonstrated by a breakdown in the blood-brain barrier following activation of brain areas through a motor task.

Strengths:
This study is novel and the results are potentially important as they demonstrate an unexpected breakdown of the blood-brain barrier with physiological activity and this may serve a physiological purpose, affecting synaptic plasticity.

The strengths of the study are:

1. The use of an in vivo model with multiple methods to investigate the blood-brain barrier response to a forelimb stimulation.
2. The determination of a potential functional role for the observed leakage of the blood-brain barrier from both a genetic and electrophysiological viewpoint.
3. The demonstration that inhibiting different points in the putative pathway from activation of the cortex to transport of albumin and activation of the TGF-beta pathway, the effect on synaptic enhancement could be prevented.
4. Preliminary experiments demonstrating a similar observation of activity-dependent breakdown of the blood-brain barrier in humans.

Weaknesses:
There are both conceptual and experimental weaknesses.

1. The stimulation is in an animal anesthetized with ketamine, which can affect critical receptors (ie NMDA receptors) in synaptic plasticity.

2. The stimulation protocol is prolonged and it would be helpful to know if briefer stimulations have the same effect or if longer stimulations have a greater effect ie does the leakage give a "readout" of the stimulation intensity/length.

3. For some of the experiments (see below), the numbers of animals are low and the statistical tests used may not be the most appropriate, making the results less clear cut.

4. The experimental paradigms are not entirely clear, especially the length of time of drug application and the authors seem to try to detect enhancement of a blocked SEP.

5. It is not clear how long the enhancement lasts. There is a remark that it lasts longer than 5 hours but there is no presentation of data to support this.

6. It is not clear if this enhancement of synaptic transmission has any physiological role.

7. The spatial and temporal specificity of this effect is unclear (other than hemispheric in rats) and even less clear in humans.

8. It is not clear to what extent the experimenters and those doing the analysis were blinded to group. If neither were blind to group, then considerable biases could be introduced.

9. The experimenters rightly use separate controls for most of the experiments but this is not always the case, also raising the possibility that the application of drugs was not done randomly or interleaved, but possibly performed in blocks of animals, which can also affect results.

10. Methyl-beta-cyclodextrin clears cholesterol so the effect on albumin transport is not specific, it could be mediating its effect through some other pathway.

11. Since the breakdown of the blood-brain barrier can be inhibited by a TGF-beta inhibitor, then this implies that TGF-beta is necessary for the breakdown of the blood-brain barrier. This does not sit well with the hypothesis that TGF-beta activation depends upon blood-brain barrier leakage.

Reviewer #3 (Public Review):

Summary:
This study used prolonged stimulation of a limb to examine possible plasticity in somatosensory evoked potentials induced by the stimulation. They also studied the extent that the blood-brain barrier (BBB) was opened by prolonged stimulation and whether that played a role in the plasticity. They found that there was potentiation of the amplitude and area under the curve of the evoked potential after prolonged stimulation and this was long-lasting (>5 hrs). They also implicated extravasation of serum albumin, caveolae-mediated transcytosis, and TGFb signalling, as well as neuronal activity and upregulation of PSD95. Transcriptomics was done and implicated plasticity-related genes in the changes after prolonged stimulation, but not proteins associated with the BBB or inflammation. Next, they address the application to humans using a squeeze ball task. They imaged the brain and suggested that the hand activity led to an increased permeability of the vessels, suggesting modulation of the BBB.

Strengths:
The strengths of the paper are the novelty of the idea that stimulation of the limb can induce cortical plasticity in a normal condition, and it involves the opening of the BBB with albumin entry. In addition, there are many datasets and both rat and human data.

Weaknesses:
The conclusions are not compelling however because of a lack of explanation of methods and quantification. It also is not clear whether the prolonged stimulation in the rat was normal conditions. To their credit, the authors recorded the neuronal activity during stimulation, but it seemed excessive excitation. Since seizures open the BBB this result calls into question one of the conclusions. that the results reflect a normal brain. The authors could either conduct studies with stimulation that is more physiological or discuss the caveats of using a supraphysiological stimulus to infer healthy brain function.