Whisker-evoked neurovascular coupling is preserved during hypoglycemia in mouse cortical arterioles and capillaries.

Hypoglycemia is a serious complication of insulin treatment of diabetes that can lead to coma and death. Neurovascular coupling, which mediates increased local blood flow in response to neuronal activity, increases glucose availability to active neurons. This mechanism could be essential for neuronal health during hypoglycemia, when total glucose supplies are low. Previous studies suggest, however, that neurovascular coupling (a transient blood flow increase in response to an increase in neuronal activity) may be reduced during hypoglycemia. Such a reduction in blood flow increase would exacerbate the effects of hypoglycemia, depriving active neurons of glucose. We have reexamined the effects of hypoglycemia on neurovascular coupling by simultaneously monitoring neuronal and vascular responses to whisker stimulation in the awake mouse somatosensory cortex. We find that neurovascular coupling at both penetrating arterioles and at 2nd order capillaries did not change significantly during insulin-induced hypoglycemia compared to euglycemia. In addition, we show that the basal diameter of both arterioles and capillaries increases during hypoglycemia (10.3 and 9.7% increases, respectively). Our results demonstrate that both neurovascular coupling and basal increases in vessel diameter are active mechanisms which help to maintain an adequate supply of glucose to the brain during hypoglycemia.

Cellular mechanisms mediating activity-dependent extracellular space shrinkage in the retina.

Volume transmission plays an essential role in CNS function, with neurotransmitters released from synapses diffusing through the extracellular space (ECS) to distant sites. Changes in the ECS volume fraction (α) will influence the diffusion and the concentration of transmitters within the ECS. We have recently shown that neuronal activity evoked by physiological photic stimuli results in rapid decreases in ECS α as large as 10% in the retina. We now characterize the cellular mechanisms responsible for this ECS shrinkage. We find that block of inwardly rectifying K+ channels with Ba2+ , inhibition of the Na+ /K+ /2Cl- cotransporter with bumetanide, or block of AQP4 water channels with TGN-020 do not diminish the light-evoked ECS decrease. Inhibition of the Na+ /HCO3 - cotransporter by removing HCO3 - from the superfusate, in contrast, reduces the light-evoked ECS decrease by 95.6%. Inhibition of the monocarboxylate transporter with alpha-cyano-4-hydroxycinnamate (4-CIN) also reduces the ECS shrinkage, but only by 32.5%. We tested whether the swelling of Müller cells, the principal glial cells of the retina, is responsible for the light-evoked ECS shrinkage. Light stimulation evoked a 6.3% increase in the volume of the fine processes of Müller cells. This volume increase was reduced by 97.1% when HCO3 - was removed from the superfusate. We conclude that a large fraction of the activity-dependent decrease in ECS α is generated by the activation of the Na+ /HCO3 - cotransporter in Müller cells. The monocarboxylate transporter may also contribute to the response.

Astrocyte regulation of cerebral blood flow during hypoglycemia.

Hypoglycemia triggers increases in cerebral blood flow (CBF), augmenting glucose supply to the brain. We have tested whether astrocytes, which can regulate vessel tone, contribute to this CBF increase. We hypothesized that hypoglycemia-induced adenosine signaling acts to increase astrocyte Ca2+ activity, which then causes the release of prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs), leading to the dilation of brain arterioles and blood flow increases. We used an awake mouse model to investigate the effects of insulin-induced hypoglycemia on arterioles and astrocytes in the somatosensory cortex. During insulin-induced hypoglycemia, penetrating arterioles dilated and astrocyte Ca2+ signaling increased when blood glucose dropped below a threshold of ∼50 mg/dL. Application of the A2A adenosine receptor antagonist ZM-241385 eliminated hypoglycemia-evoked astrocyte Ca2+ increases and reduced arteriole dilations by 44% (p < 0.05). SC-560 and miconazole, which block the production of the astrocyte vasodilators PGs and EETs respectively, reduced arteriole dilations in response to hypoglycemia by 89% (p < 0.001) and 76% (p < 0.001). Hypoglycemia-induced arteriole dilations were decreased by 65% (p < 0.001) in IP3R2 knockout mice, which have reduced astrocyte Ca2+ signaling compared to wild-type. These results support the hypothesis that astrocytes contribute to hypoglycemia-induced increases in CBF by releasing vasodilators in a Ca2+-dependent manner.

Dilation of cortical capillaries is not related to astrocyte calcium signaling.

The brain requires an adequate supply of oxygen and nutrients to maintain proper function as neuronal activity varies. This is achieved, in part, through neurovascular coupling mechanisms that mediate local increases in blood flow through the dilation of arterioles and capillaries. The role of astrocytes in mediating this functional hyperemia response is controversial. Specifically, the function of astrocyte Ca2+ signaling is unclear. Cortical arterioles dilate in the absence of astrocyte Ca2+ signaling, but previous work suggests that Ca2+ increases are necessary for capillary dilation. This question has not been fully addressed in vivo, however, and we have reexamined the role of astrocyte Ca2+ signaling in vessel dilation in the barrel cortex of awake, behaving mice. We recorded evoked vessel dilations and astrocyte Ca2+ signaling in response to whisker stimulation. Experiments were carried out on WT and IP3R2 KO mice, a transgenic model where astrocyte Ca2+ signaling is substantially reduced. Compared to WT mice at rest, Ca2+ signaling in astrocyte endfeet contacting capillaries increased by 240% when whisker stimulation evoked running. In contrast, Ca2+ signaling was reduced to 9% of WT values in IP3R2 KO mice. In all three conditions, however, the amplitude of capillary dilation was largely unchanged. In addition, the latency to the onset of astrocyte Ca2+ signaling lagged behind dilation onset in most trials, although a subset of rapid onset Ca2+ events with latencies as short as 0.15 s occurred. In summary, we found that whisker stimulation-evoked capillary dilations occurred independent of astrocyte Ca2+ increases in the cerebral cortex.

Spatial Organization and Dynamics of the Extracellular Space in the Mouse Retina.

The extracellular space (ECS) plays an important role in the physiology of neural circuits. Despite our detailed understanding of the cellular architecture of the mammalian retina, little is known about the organization and dynamics of the retinal ECS. We developed an optical technique based on two-photon imaging of fluorescently labeled extracellular fluid to measure the ECS volume fraction (α) in the ex vivo retina of male and female mice. This method has high spatial resolution and can detect rapid changes in α evoked by osmotic challenge and neuronal activity. The measured ECS α varied dramatically in different layers of the adult mouse retina, with α equaling ∼0.050 in the ganglion cell layer, ∼0.122 in the inner plexiform layer (IPL), ∼0.025 in the inner nuclear layer (INL), ∼0.087 in the outer plexiform layer, and ∼0.026 in the outer nuclear layer (ONL). ECS α was significantly larger early in retinal development; α was 67% larger in the IPL and 100% larger in the INL in neonatal mice compared with adults. In adult retinas, light stimulation evoked rapid decreases in ECS α. Light-driven reductions in ECS α were largest in the IPL, where visual stimuli decreased α values ∼10%. These light-evoked decreases demonstrate that a physiological stimulus can lead to rapid changes in ECS α and indicate that activity-dependent regulation of extracellular space may contribute to visual processing in the retina.SIGNIFICANCE STATEMENT The volume fraction of the extracellular space (ECS α), that portion of CNS tissue occupied by interstitial space, influences the diffusion of neurotransmitters from the synaptic cleft and the volume transmission of transmitters. However, ECS α has never been measured in live retina, and little is known about how ECS α varies following physiological stimulation. Here we show that ECS α values vary dramatically between different retinal layers and decrease by 10% following light stimulation. ECS α differences within the retina will influence volume transmission and light-evoked α variations may modulate synaptic transmission and visual processing in the retina. Activity-dependent ECS α variations may represent a mechanism of synaptic modulation throughout the CNS.

Cortical spreading depolarizations induced by surgical field blood in a mouse model of neurosurgery.

OBJECTIVE: Cortical spreading depolarization (CSD) has been linked to poor clinical outcomes in the setting of traumatic brain injury, malignant stroke, and subarachnoid hemorrhage. There is evidence that electrocautery during neurosurgical procedures can also evoke CSD waves in the brain. It is unknown whether blood contacting the cortical surface during surgical bleeding affects the frequency of spontaneous or surgery-induced CSDs. Using a mouse neurosurgical model, the authors tested the hypothesis that electrocautery can induce CSD waves and that surgical field blood (SFB) is associated with more CSDs. The authors also investigated whether CSD can be reliably observed by monitoring the fluorescence of GCaMP6f expressed in neurons. METHODS: CSD waves were monitored by using confocal microscopy to detect fluorescence increases at the cortical surface in mice expressing GCaMP6f in CamKII-positive neurons. The cortical surface was electrocauterized through an adjacent burr hole. SFB was simulated by applying a drop of tail vein blood to the brain through the same burr hole. RESULTS: CSD waves were readily detected in GCaMP6f-expressing mice. Monitoring GCaMP6f fluorescence provided far better sensitivity and spatial resolution than detecting CSD events by observing changes in the intrinsic optical signal (IOS). Forty-nine percent of the CSD waves identified by GCaMP6f had no corresponding IOS signal. Electrocautery evoked CSD waves. On average, 0.67 ± 0.08 CSD events were generated per electrocautery episode, and multiple CSD waves could be induced in the same mouse by repeated cauterization (average, 7.9 ± 1.3 events; maximum number in 1 animal, 13 events). In the presence of SFB, significantly more spontaneous CSDs were generated (1.35 ± 0.37 vs 0.13 ± 0.16 events per hour, p = 0.002). Ketamine effectively decreased the frequency of spontaneous CSD waves (1.35 ± 0.37 to 0.36 ± 0.15 CSD waves per hour, p = 0.016) and electrocautery-stimulated CSD waves (0.80 ± 0.05 to 0.18 ± 0.08 CSD waves per electrocautery, p = 0.00002). CONCLUSIONS: CSD waves are detected with far greater sensitivity and fidelity by monitoring GCaMP6f signals in neurons than by monitoring IOSs. Electrocautery reliably evokes CSD waves, and the frequency of spontaneous CSD waves is increased when blood is applied to the cortical surface. These experimental conditions recapitulate common scenarios in the neurosurgical operating room. Ketamine, a clinically available pharmaceutical agent, can block stimulated and spontaneous CSDs. More research is required to understand the clinical importance of intraoperative CSD.

Role of PEPT2 in glycylsarcosine transport in astrocyte and glioma cultures.

The aims of the current study were (1) to quantify the role of PEPT2 in the uptake of glycylsarcosine (GlySar) in cultured neonatal astrocytes and (2) to examine GlySar transport and PEPT2 expression in two glioma cell lines. The uptake of [(14)C]GlySar was measured in astrocytes cultured from neonatal mouse (PEPT2(+/+) and PEPT2(-/-)) and rat, as well as rat C6 and F98 glioma cells. PEPT2 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). Neonatal astrocytes from PEPT2(-/-) mice had a 94% reduction in [(14)C]GlySar uptake compared to wild type mice and there was no saturable transport. In PEPT2(+/+) mice, [(14)C]GlySar uptake was saturable (V(max) 58 +/- 12 pmol/mg/min, K(m) 107 +/- 46 microM, K(d) 0.043 +/- 0.004 microl/mg/min). In neonatal rat astrocytes, kinetic analysis also suggested that [(14)C]GlySar uptake was via a single transporter. The inhibitor profile and pH dependence of that transport process was consistent with PEPT2. In C6 and F98 glioma cells, [(14)C]GlySar uptake was markedly reduced ( approximately 96-98%) compared to that in neonatal astrocytes and this was reflected by an absence of PEPT2 mRNA expression. These results indicate that PEPT2 is the sole transporter involved in the uptake of GlySar into neonatal cultured astrocytes. However, PEPT2 mRNA appears to be absent from two glioma cell lines.