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Tonotopic organization of the auditory cortex has been extensively studied in many mammalian species using various methodologies and physiological preparations. Tonotopy mapping in primates, however, is more limited due to constraints such as cortical folding, use of anesthetized subjects, and mapping methodology. Here we applied a combination of through-skull and through-window intrinsic optical signal imaging, wide-field calcium imaging, and neural probe recording techniques in awake marmosets (Callithrix jacchus), a New World monkey with most of its auditory cortex located on a flat brain surface. Coarse tonotopic gradients, including a recently described rostral-temporal (RT) to parabelt gradient, were revealed by the through-skull imaging of intrinsic optical signals and were subsequently validated by single-unit recording. Furthermore, these tonotopic gradients were observed with more detail through chronically implanted cranial windows with additional verifications on the experimental design. Moreover, the tonotopy mapped by the intrinsic-signal imaging methods was verified by wide-field calcium imaging in an AAV-GCaMP labeled subject. After these validations and with further effort to expand the field of view more rostrally in both windowed and through-skull subjects, an additional putative tonotopic gradient was observed more rostrally to the area RT, which has not been previously described by the standard model of tonotopic organization of the primate auditory cortex. Together, these results provide the most comprehensive data of tonotopy mapping in an awake primate species with unprecedented coverage and details in the rostral proportion and support a caudal-rostrally arranged mesoscale organization of at least three repeats of functional gradients in the primate auditory cortex, similar to the ventral stream of primate visual cortex.

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The early detection and pathological classification of brain edema are very important for symptomatic treatment. The dual-optical imaging system (DOIS) consists of intrinsic optical signal imaging (IOSI) and laser speckle contrast imaging (LSCI), which can acquire cerebral hemodynamic parameters of mice in real-time, including changes of oxygenated hemoglobin concentration ( Δ C HbO 2 ), deoxyhemoglobin concentration (ΔCHbR) and relative cerebral blood flow (rCBF) within the field of view. The slope sum of Δ C HbO 2 , ΔCHbR and rCBF was proposed to classify vasogenic edema (VE) and cytotoxic edema (CE). The slope sum values in the VE and CE group remain statistically different and the classification results provide higher accuracy of more than 93% for early brain edema detection. In conclusion, the differences of hemodynamic parameters between VE and CE in the early stage were revealed and the method helps in the classification of early brain edema.

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Functional neuroimaging, which measures hemodynamic responses to brain activity, has great potential for monitoring recovery in stroke patients and guiding rehabilitation during recovery. However, hemodynamic responses after stroke are almost always altered relative to responses in healthy subjects and it is still unclear if these alterations reflect the underlying brain physiology or if the alterations are purely due to vascular injury. In other words, we do not know the effect of stroke on neurovascular coupling and are therefore limited in our ability to use functional neuroimaging to accurately interpret stroke pathophysiology. To address this challenge, we simultaneously captured neural activity, through fluorescence calcium imaging, and hemodynamics, through intrinsic optical signal imaging, during longitudinal stroke recovery. Our data suggest that neurovascular coupling was preserved in the chronic phase of recovery (2 weeks and 4 weeks post-stoke) and resembled pre-stroke neurovascular coupling. This indicates that functional neuroimaging faithfully represents the underlying neural activity in chronic stroke. Further, neurovascular coupling in the sub-acute phase of stroke recovery was predictive of long-term behavioral outcomes. Stroke also resulted in increases in global brain oscillations, which showed distinct patterns between neural activity and hemodynamics. Increased neural excitability in the contralesional hemisphere was associated with increased contralesional intrahemispheric connectivity. Additionally, sub-acute increases in hemodynamic oscillations were associated with improved sensorimotor outcomes. Collectively, these results support the use of hemodynamic measures of brain activity post-stroke for predicting functional and behavioral outcomes.

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Spreading depolarizations (SDs) have been linked to infarct volume expansion following ischemic stroke. Therapeutic hypothermia provides a neuroprotective effect after ischemic stroke. This study aimed to evaluate the effect of hypothermia on the propagation of SDs and infarct volume in an ischemic swine model. Through left orbital exenteration, middle cerebral arteries were surgically occluded (MCAo) in 16 swine. Extensive craniotomy and durotomy were performed. Six hypothermic and five normothermic animals were included in the analysis. An intracranial temperature probe was placed right frontal subdural. One hour after ischemic onset, mild hypothermia was induced and eighteen hours of electrocorticographic (ECoG) and intrinsic optical signal (IOS) recordings were acquired. Postmortem, 4 mm-thick slices were stained with 2,3,5-triphenyltetrazolium chloride to estimate the infarct volume. Compared to normothermia (36.4 ± 0.4°C), hypothermia (32.3 ± 0.2°C) significantly reduced the frequency and expansion of SDs (ECoG: 3.5 ± 2.1, 73.2 ± 5.2% vs. 1.0 ± 0.7, 41.9 ± 21.8%; IOS 3.9 ± 0.4, 87.6 ± 12.0% vs. 1.4 ± 0.7, 67.7 ± 8.3%, respectively). Further, infarct volume among hypothermic animals (23.2 ± 1.8% vs. 32.4 ± 2.5%) was significantly reduced. Therapeutic hypothermia reduces infarct volume and the frequency and expansion of SDs following cerebral ischemia.

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[This corrects the article DOI: 10.3389/fncel.2020.00027.].

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Intrinsic optical signal (IOS) imaging of the retina, also termed as optoretinogram or optoretinography (ORG), promises a non-invasive method for the objective assessment of retinal function. By providing the unparalleled capability to differentiate individual retinal layers, functional optical coherence tomography (OCT) has been actively investigated for intrinsic signal ORG measurements. However, clinical deployment of functional OCT for quantitative ORG is still challenging due to the lack of a standardized imaging protocol and the complication of IOS sources and mechanisms. This article aims to summarize recent developments of functional OCT for ORG measurement, OCT intensity- and phase-based IOS processing. Technical challenges and perspectives of quantitative IOS analysis and ORG interpretations are discussed.

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OBJECTIVE: Spreading depolarization (SD) has been regarded as one cause of neuronal injury in subarachnoid hemorrhage (SAH). However, SD in the hyperacute phase of SAH is still unclear. The objective of this study was to detect real-time spatial-temporal patterns of SD, assess the effect of SD on cerebral blood flow, and test the relationship between SD and brain injury in the acute phase of SAH. METHODS: Twenty-eight mice were separated into two groups: 16 animals in the SAH group and 12 animals in the sham group. Experimental SAH was done with an endovascular filament perforation model. Changes in optical reflection were registered with intrinsic optical signal imaging (IOSI) after SAH. Spatial-temporal patterns of SDs were analyzed and brain injury including brain edema and infarction was tested. RESULTS: Totally, 117 SDs occurred after SAH. According to the hemodynamic response and duration, SDs could be classified into Type I (short SD), Type II (intermediate SD), and Type III (persistent SD). Most of SDs originated from the somatosensory and visual cortex. SDs demonstrated distinct spreading patterns. Moreover, the number and duration of SDs associated with brain water content (p < 0.05, p < 0.01). SDs, especially, persistent SDs associated with infarct volume in the hyperacute phase of SAH (p < 0.001, p < 0.001). CONCLUSION: Our results suggest that SD occurs with a high incidence during the acute stage of SAH in mice. And the lissencephalic mouse brain is capable of different SD propagation patterns. Additionally, SD may aggravate brain edema and induce brain infarction, contributing to early brain injury after SAH.

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SIGNIFICANCE: Cerebral blood flow (CBF) regulation at neurovascular coupling (NVC) plays an important role in normal brain functioning to support oxygen delivery to activating neurons. Therefore, studying the mechanisms of CBF adjustment is crucial for the improved understanding of brain activity. AIM: We investigated the temporal profile of hemodynamic signal change in mouse cortex caused by neural activation and its variation over cortical depth. APPROACH: Following the cranial window surgery, intrinsic optical signal imaging (IOSI) was used to spatially locate the activated region in mouse cortex during whisker stimulation. Optical microangiography (OMAG), the functional extension of optical coherence tomography, was applied to image the activated and control regions identified by IOSI. Temporal profiles of hemodynamic response signals obtained by IOSI and OMAG were compared, and OMAG signal was analyzed over cortical layers. RESULTS: Our results showed that the hemodynamic response to neural activity revealed by blood flow change signal signal through IOSI is slower than that observed by OMAG signal. OMAG also indicated the laminar variation of the response over cortical depth, showing the largest response in cortical layer IV. CONCLUSIONS: Overall, we demonstrated the development and application of dual-modality imaging system composed of IOSI and OMAG, which may have potential to enable the future investigations of depth-resolved CBF and to provide the insights of hemodynamic events associated with the NVC.

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Pericytes are perivascular mural cells that enwrap brain capillaries and maintain blood-brain barrier (BBB) integrity. Most studies suggest that pericytes regulate cerebral blood flow (CBF) and oxygen delivery to activated brain structures, known as neurovascular coupling. While we have previously shown that congenital loss of pericytes leads over time to aberrant hemodynamic responses, the effects of acute global pericyte loss on neurovascular coupling have not been studied. To address this, we used our recently reported inducible pericyte-specific Cre mouse line crossed to iDTR mice carrying Cre-dependent human diphtheria toxin (DT) receptor, which upon DT treatment leads to acute pericyte ablation. As expected, DT led to rapid progressive loss of pericyte coverage of cortical capillaries up to 50% at 3 days post-DT, which correlated with approximately 50% reductions in stimulus-induced CBF responses measured with laser doppler flowmetry (LDF) and/or intrinsic optical signal (IOS) imaging. Endothelial response to acetylcholine, microvascular density, and neuronal evoked membrane potential responses remained, however, unchanged, as well as arteriolar smooth muscle cell (SMC) coverage and functional responses to adenosine, as we previously reported. Together, these data suggest that neurovascular uncoupling in this model is driven by pericyte loss, but not other vascular deficits or neuronal dysfunction. These results further support the role of pericytes in CBF regulation and may have implications for neurological conditions associated with rapid pericyte loss such as hypoperfusion and stroke, as well as conditions where the exact time course of global regional pericyte loss is less clear, such as Alzheimer's disease (AD) and other neurogenerative disorders.

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BACKGROUND: The main objective of this study was to generate a hemodynamically stable swine model to detect spreading depolarizations (SDs) using electrocorticography (ECoG) and intrinsic optical signal (IOS) imaging and laser speckle flowmetry (LSF) after a 30-h middle cerebral artery (MCA) occlusion (MCAo) in German Landrace Swine. METHODS: A total of 21 swine were used. The study comprised a training group (group 1, n = 7), a group that underwent bilateral craniectomy and MCAo (group 2, n = 10) and a group used for 2,3,5-triphenyltetrazolium (TTC) staining (group 3, n = 5). RESULTS: In group 2, nine animals that underwent MCAo survived for 30 h, and one animal survived for 12 h. We detected MCA variants with 2 to 4 vessels. In all cases, all of the MCAs were occluded. The intensity changes exhibited by IOS and LSF after clipping were closely correlated and indicated a lower blood volume and reduced blood flow in the middle cerebral artery territory. Using IOS, we detected a mean of 2.37 ± (STD) 2.35 SDs/h. Using ECoG, we detected a mean of 0.29 ± (STD) 0.53 SDs/h. Infarctions were diagnosed using histological analysis. TTC staining in group 3 confirmed that the MCA territory was compromised and that the anterior and posterior cerebral arteries were preserved. CONCLUSIONS: We confirm the reliability of performing live monitoring of cerebral infarctions using our MCAo protocol to detect SDs.

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Spreading depolarization (SD) has been suggested as a pathomechanism for delayed cerebral ischemia after subarachnoid hemorrhage (SAH). However, the role of SD during the acute phase of SAH is still unclear. The objective of this study was to investigate (a) the occurrence of SD with intrinsic optical signal (IOS) imaging, (b) the effect of ketamine on SD, and (c) the resulting brain edema (brain water content (BWC)) during the acute stage of experimental SAH in mice. SAH was elicited by the endovascular filament perforation method. After SAH or sham operation, ketamine or saline, 30 mg/kg, was given every half hour. Changes in tissue light reflectance were recorded with IOS. BWC was measured during the acute stage. Overall, 199 SDs occurred in SAH groups and 33 SDs appeared in sham groups. These SDs displayed distinct originating and spreading patterns. Compared with saline, ketamine decreased SD spread and influenced the amplitude, duration, and speed of SD. However, the occurrence of SD was not prevented by ketamine. Moreover, ketamine did not reduce BWC after SAH. These results demonstrate that SD occurs with a high incidence during the acute stage of SAH. SDs are heterogeneous in incidence, origination, and propagation. It remains unclear whether ketamine effects on SD may be viewed as therapeutically beneficial after SAH.

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The olfactory environment is first represented by glomerular activity patterns in the olfactory bulb. It remains unclear how these representations intersect with sampling behavior to account for the time required to discriminate odors. Using different chemical classes, we investigate glomerular representations and sniffing behavior during olfactory decision-making. Mice rapidly discriminate odorants and learn to increase sniffing frequency at a fixed latency after trial initiation, independent of odor identity. Relative to the increase in sniffing frequency, monomolecular odorants are discriminated within 10-40 ms, while binary mixtures require an additional 60-70 ms. Intrinsic imaging of glomerular activity in anesthetized and awake mice reveals that Euclidean distance between activity patterns and the time needed for discriminations are anti-correlated. Therefore, the similarity of glomerular patterns and their activation strengths, rather than sampling behavior, define the extent of neuronal processing required for odor discrimination, establishing a neural metric to predict olfactory discrimination time.

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The latent period, a seizure-free phase, is the duration between brain injury and the onset of spontaneous recurrent seizures (SRSs) during epileptogenesis. The latent period is thought to involve several progressive pathophysiological events that lead to the evolution of the chronic epilepsy phase. Hence, it is vital to investigate the changes in the latent period during epileptogenesis in order to better understand temporal lobe epilepsy (TLE), and to achieve early diagnosis and appropriate management of the condition. Accordingly, recent studies with patients with TLE using resting-state functional magnetic resonance imaging (rs-fMRI) have reported that alterations of resting-state functional connectivity (rsFC) during the chronic period are associated with some clinical manifestations, including learning and memory impairments, emotional instability, and social behavior deficits, in addition to repetitive seizure episodes. In contrast, the changes in the intrinsic rsFC during epileptogenesis, particularly during the latent period, remain unclear. In this study, we investigated the alterations in intrinsic rsFC during the latent and chronic periods in a pilocarpine-induced TLE mouse model using intrinsic optical signal imaging (IOSI). This technique can monitor the changes in the local hemoglobin concentration according to neuronal activity and can help investigate large-scale brain intrinsic networks. After seeding on the anatomical regions of interest (ROIs) and calculating the correlation coefficients between each ROI, we established and compared functional correlation matrices and functional connectivity maps during the latent and chronic periods of epilepsy. We found a decrease in the interhemispheric rsFC at the frontal and temporal regions during both the latent and chronic periods. Furthermore, a significant decrease in the interhemispheric rsFC was observed in the somatosensory area during the chronic period. Changes in network configurations during epileptogenesis were examined by graph theoretical network analysis. Interestingly, increase in the power of low frequency oscillations was observed during the latent period. These results suggest that, even if there are no apparent ictal seizure events during the latent period, there are ongoing changes in the rsFC in the epileptic brain. Furthermore, these results suggest that the pathophysiology of epilepsy may be related to widespread altered intrinsic functional connectivity. These findings can help enhance our understanding of epileptogenesis, and accordingly, changes in intrinsic functional connectivity can serve as an early diagnosis.

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In the recently published article, "Heterogeneous incidence and propagation of spreading depolarizations," it is shown, in vivo and in vitro, how KCl-induced spreading depolarizations in mouse and rat brains can be highly variable, and that they are not limited, as once thought, to a concentric, isotropic, or homogenous depolarization wave in space or in time. The reported results serve as a link between the different species, and this paper contributes to changing the way in which SD expansion is viewed in the lissencephalic brain. Here, we discuss their results with our previous observations made in the gyrencephalic swine brain, in computer simulations, and in the human brain.

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Spreading depolarization (SD) generates significant alterations in cerebral haemodynamics, which can have detrimental consequences on brain function and integrity. Ketamine has shown an important capacity to modulate SD; however, its impact on SD haemodynamic response is incompletely understood. We investigated the effect of two therapeutic ketamine dosages, a low-dose of 2 mg/kg/h and a high-dose of 4 mg/kg/h, on the haemodynamic response to SD in the gyrencephalic swine brain. Cerebral blood volume, pial arterial diameter and cerebral blood flow were assessed through intrinsic optical signal imaging and laser-Doppler flowmetry. Our findings indicate that frequent SDs caused a persistent increase in the baseline pial arterial diameter, which can lead to a diminished capacity to further dilate. Ketamine infused at a low-dose reduced the hyperemic/vasodilative response to SD; however, it did not alter the subsequent oligemic/vasoconstrictive response. This low-dose did not prevent the baseline diameter increase and the diminished dilative capacity. Only infusion of ketamine at a high-dose suppressed SD and the coupled haemodynamic response. Therefore, the haemodynamic response to SD can be modulated by continuous infusion of ketamine. However, its use in pathological models needs to be explored to corroborate its possible clinical benefit.

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Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

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The aim was to characterize the effects of magnesium sulfate, using i.v. bolus and local administration, using intrinsic signal imaging, and on electrocorticographic activity during the induction and propagation of spreading depolarizations in the gyrencephalic porcine brain. Local application of magnesium sulfate led to a complete inhibition of spreading depolarizations. One hour after washing out the topical magnesium sulfate, re-incidence of the spreading depolarizations was observed in 50% of the hemispheres. Those spreading depolarizations showed attenuation in hemodynamic characteristics and speed in intrinsic optical signal imaging. The electrical amplitude decreased through electrocorticographic activity. Intravenous magnesium therapy showed no significant effects on spreading depolarization incidence and characteristics.

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BACKGROUND: PET allows the measurement of CBF, CBV and CMRO2 in human and plays an important role in the diagnosis of pathologic conditions and clinical research. On the other hand, in animal studies, there is no optical imaging system for evaluating changes in CBF and CBV, and oxygen metabolism, from the same brain area under awake condition. NEW METHOD: In the present study, we developed a simultaneous measurement system of LSI and IOSI, which was verified by LDF. Moreover, to evaluate oxygen metabolism, FAI was performed from the same brain area as LSI and IOSI measurements. RESULTS: The change in CBF according to LSI was correlated with that by LDF. Similarly, the change in CBV obtained by IOSI was also correlated with RBC concentration change measured by LDF. The change in oxygen metabolism by FAI was associated with that in CBF obtained by LSI, although the change in CBF was greater than that in oxygen metabolism. COMPARISON WITH EXISTING METHOD(S): We revealed that the relationship between oxygen metabolism and CBF as measured by our system was in good agreement with the relationship between CMRO2 and CBF in human PET studies. CONCLUSIONS: Our measurement system of CBF, CBV and oxygen metabolism is not only useful for studying neurovascular coupling, but also easily corroborates human PET studies.

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