Assessment of neurovascular coupling and cortical spreading depression in mixed mouse models of atherosclerosis and Alzheimer’s disease

Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in mouse preparations that modelled Alzheimer’s disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9 and 12 m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, altered levels of oxyhaemoglobin and deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3 x increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimer’s-related amyloid-plaques.

Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

Background Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD. Methods CSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay. Results The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1β and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1β gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. Conclusion This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine.

Validating a computational framework for ionic electrodiffusion with cortical spreading depression as a case study

Cortical spreading depression (CSD) is a wave of pronounced depolarization of brain tissue accompanied by substantial shifts in ionic concentrations and cellular swelling. Here, we validate a computational framework for modelling electrical potentials, ionic movement, and cellular swelling in brain tissue during CSD. We consider different model variations representing wild type or knock-out/knock-down mice and systematically compare the numerical results with reports from a selection of experimental studies. We find that the data for several CSD hallmarks obtained computationally, including wave propagation speed, direct current shift duration, peak in extracellular K + concentration as well as a pronounced shrinkage of extracellular space, are well in line with what has previously been observed experimentally. Further, we assess how key model parameters including cellular diffusivity, structural ratios, membrane water and/or K + permeabilities affect the set of CSD characteristics.

TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1β gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1β mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14–22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.

Aura Mapping: Where Vision and Somatosensation Meet

While migraine auras are most frequently visual, somatosensory auras are also relatively common. Both are characterized by the spread of activation across a cortical region containing a spatial mapping of the sensory (retinal or skin) surface. When both aura types occur within a single migraine episode, they may offer an insight into the neural mechanism which underlies them. Could they both be initiated by a single neural event, or do the timing and laterality relationships between them demand multiple triggers? The observations reported here were carried out 25 years ago by a group of six individuals with migraine with aura. They timed, described and mapped their visual and somatosensory auras as they were in progress. Twenty-nine episode reports are summarized here. The temporal relationship between the onset of the two auras was quite variable within and across participants. Various forms of the cortical spreading depression hypothesis of migraine aura are evaluated in terms of whether they can account for the timing, pattern of symptom spread and laterality of the recorded auras.

Cortical Spreading Depression Can Be Triggered by Sensory Stimulation in Primed Wild Type Mouse Brain: a Mechanistic Insight to Migraine Aura Generation

Background: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight how migraine starts in an otherwise healthy brain, remains largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. Methods: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low dose of Na+/K+-ATPase blocker ouabain that does not induce repetitive CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake by astrocytes, with shRNA. Stimulation-induced CSDs and extracellular K+ changes were monitored in vivo electrophysiologically or with a K+-sensitive fluoroprobe (IPG-4). Results: After priming with ouabain, photic stimulation increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p<0.001). Whisker stimulation was less effective (14.9 vs. 2.4%, p=0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested but triggered stimulus-induced CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during stimulation after subthreshold ouabain or shRNA treatment unlike controls. In line with higher CSD susceptibility, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the incidence of stimulus-induced CSDs, we applied an A1-receptor (DPCPX) or GABA-A (bicuculine) antagonist over the occipital cortex, because adenosine formed during stimulation or inhibitory interneuron activity can reduce CSD susceptibility. DPCPX induced CSDs or CSD-like small-DC shifts during photic stimulation, whereas bicuculine was not effective. Conclusions: Our findings indicate that normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has the potential to trigger a CSD when co-existing conditions can bring extracellular K+ and glutamate concentrations over threshold via reduced uptake of K+ and glutamate (e.g. inefficient fueling of α2-Na+/K+-ATPase due to reduced glycogen breakdown) or facilitated glutamate release (e.g. reduced presynaptic adenosinergic inhibition).

Efficacy profile of noninvasive vagus nerve stimulation on cortical spreading depression susceptibility and the tissue response

Background Noninvasive vagus nerve stimulation (nVNS) has recently emerged as a promising therapy for migraine. We previously demonstrated that vagus nerve stimulation inhibits cortical spreading depression (CSD), the electrophysiological event underlying migraine aura and triggering headache; however, the optimal nVNS paradigm has not been defined. Methods Various intensities and doses of nVNS were tested to improve efficacy on KCl-evoked CSD frequency and electrical threshold of CSD in a validated rat model. Chronic efficacy was evaluated by daily nVNS delivery for four weeks. We also examined the effects of nVNS on neuroinflammation and trigeminovascular activation by western blot and immunohistochemistry. Results nVNS suppressed susceptibility to CSD in an intensity-dependent manner. Two 2-minute nVNS 5 minutes apart afforded the highest efficacy on electrical CSD threshold and frequency of KCl-evoked CSD. Daily nVNS for four weeks did not further enhance efficacy over a single nVNS 20 minutes prior to CSD. The optimal nVNS also attenuated CSD-induced upregulation of cortical cycloogenase-2, calcitonin gene-related peptide in trigeminal ganglia, and c-Fos expression in trigeminal nucleus caudalis. Conclusion Our study provides insight on optimal nVNS parameters to suppress CSD and suggests its benefit on CSD-induced neuroinflammation and trigeminovascular activation in migraine treatment.

Src Family Kinases Activity is Required for Transmitting Purinergic P2X7 Receptor Signaling in Cortical Spreading Depression and Neuroinflammation

Background Purinergic P2X7 receptor plays a key role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and CSD-induced neuroinflammation. Methods CSD was recorded using electrophysiology in rats, and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA expression were detected using qPCR. Glutamate release in mouse brain slices was detected using glutamate assay. Results The data showed that systematic deactivation of SFKs by PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1b and TNF-a gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor-SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, CSD-induced IL-1b gene expression and glutamate release were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA and disrupting Fyn-NMDA interaction using TAT-Fyn (39-57), but not disrupting Src-NMDA receptor using TAT-Src (40-49), reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. Conclusion This study reveals that SFKs activity mediates P2X7 receptor pore formation facilitating CSD propagation, CSD-induced neuroinflammation and glutamate release, of particular relevance to migraine.

Review and Hypothesis: A Potential Common Link Between Glial Cells, Calcium Changes, Modulation of Synaptic Transmission, Spreading Depression, Migraine, and Epilepsy—H+

There is significant evidence to support the notion that glial cells can modulate the strength of synaptic connections between nerve cells, and it has further been suggested that alterations in intracellular calcium are likely to play a key role in this process. However, the molecular mechanism(s) by which glial cells modulate neuronal signaling remains contentiously debated. Recent experiments have suggested that alterations in extracellular H+ efflux initiated by extracellular ATP may play a key role in the modulation of synaptic strength by radial glial cells in the retina and astrocytes throughout the brain. ATP-elicited alterations in H+ flux from radial glial cells were first detected from Müller cells enzymatically dissociated from the retina of tiger salamander using self-referencing H+-selective microelectrodes. The ATP-elicited alteration in H+ efflux was further found to be highly evolutionarily conserved, extending to Müller cells isolated from species as diverse as lamprey, skate, rat, mouse, monkey and human. More recently, self-referencing H+-selective electrodes have been used to detect ATP-elicited alterations in H+ efflux around individual mammalian astrocytes from the cortex and hippocampus. Tied to increases in intracellular calcium, these ATP-induced extracellular acidifications are well-positioned to be key mediators of synaptic modulation. In this article, we examine the evidence supporting H+ as a key modulator of neurotransmission, review data showing that extracellular ATP elicits an increase in H+ efflux from glial cells, and describe the potential signal transduction pathways involved in glial cell—mediated H+ efflux. We then examine the potential role that extracellular H+ released by glia might play in regulating synaptic transmission within the vertebrate retina, and then expand the focus to discuss potential roles in spreading depression, migraine, epilepsy, and alterations in brain rhythms, and suggest that alterations in extracellular H+ may be a unifying feature linking these disparate phenomena.