Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Cranial window for longitudinal and multimodal imaging of the whole mouse cortex.

Significance: All functional brain imaging methods have technical drawbacks and specific spatial and temporal resolution limitations. Unraveling brain function requires bridging the data acquired with cellular and mesoscopic functional imaging. This imposes the access to animal preparations, allowing longitudinal and multiscale investigations of brain function in anesthetized and awake animals. Such preparations are optimal to study normal and pathological brain functions while reducing the number of animals used. Aim: To fulfill these needs, we developed a chronic and stable preparation for a broad set of imaging modalities and experimental design. Approach: We describe the detailed protocol for a chronic cranial window, transparent to light and ultrasound, devoid of BOLD functional magnetic resonance imaging (fMRI) artifact and allowing stable and longitudinal multimodal imaging of the entire mouse cortex. Results: The inexpensive, transparent, and curved polymethylpentene cranial window preparation gives access to the entire mouse cortex. It is compatible with standard microscopic and mesoscopic neuroimaging methods. We present examples of data on the neurovascular unit and its activation using two-photon, functional ultrasound imaging, and BOLD fMRI. Conclusion: This preparation is ideal for multimodal imaging in the same animal.

In vivo detection of carnosine and its derivatives using chemical exchange saturation transfer.

PURPOSE: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T. METHODS: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats. RESULTS: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 µ$$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results. CONCLUSION: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules.

Diversity of neurovascular coupling dynamics along vascular arbors in layer II/III somatosensory cortex.

The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial. Here we utilized a single whisker stimulation model to map out functional hyperemia along vascular arbours from layer II/III to the surface of primary somatosensory cortex, in anesthetized and awake Thy1-GCaMP6 mice. We demonstrate that sensory stimulation triggers an increase in blood velocity within the mid-capillary bed and a dilation of upstream large capillaries, and the penetrating and pial arterioles. We report that under physiological stimulation, response onset times are highly variable across compartments of different vascular arbours. Furthermore, generating transfer functions (TFs) between neuronal Ca2+ and vascular dynamics across different brain states demonstrates that anesthesia decelerates neurovascular coupling (NVC). This spatial-temporal pattern of vascular events demonstrates functional diversity not only between different brain regions but also at the level of different vascular arbours within supragranular layers of the cerebral cortex.

Iliski, a software for robust calculation of transfer functions.

Understanding the relationships between biological processes is paramount to unravel pathophysiological mechanisms. These relationships can be modeled with Transfer Functions (TFs), with no need of a priori hypotheses as to the shape of the transfer function. Here we present Iliski, a software dedicated to TFs computation between two signals. It includes different pre-treatment routines and TF computation processes: deconvolution, deterministic and non-deterministic optimization algorithms that are adapted to disparate datasets. We apply Iliski to data on neurovascular coupling, an ensemble of cellular mechanisms that link neuronal activity to local changes of blood flow, highlighting the software benefits and caveats in the computation and evaluation of TFs. We also propose a workflow that will help users to choose the best computation according to the dataset. Iliski is available under the open-source license CC BY 4.0 on GitHub (https://github.com/alike-aydin/Iliski) and can be used on the most common operating systems, either within the MATLAB environment, or as a standalone application.

Transfer functions linking neural calcium to single voxel functional ultrasound signal.

Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.

Perivascular spaces in the brain: anatomy, physiology and pathology.

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance.

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation.

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2-3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.

Mesoscopic and microscopic imaging of sensory responses in the same animal.

Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales. Under these conditions, functional hyperaemia occurred at the threshold of neuronal activation and fUS-CBV signals could be detected at the level of single voxels with activation maps varying according to blood velocity. Both neuronal and vascular responses increase non-linearly as a function of odour concentration, whereas both microscopic and mesoscopic vascular responses are linearly correlated to local neuronal calcium. These data establish strengths and limits of mesoscopic imaging techniques to report neural activity.

Light controls cerebral blood flow in naive animals.

Optogenetics is increasingly used to map brain activation using techniques that rely on functional hyperaemia, such as opto-fMRI. Here we test whether light stimulation protocols similar to those commonly used in opto-fMRI or to study neurovascular coupling modulate blood flow in mice that do not express light sensitive proteins. Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging, we report that in the naive mouse brain, light per se causes a calcium decrease in arteriolar smooth muscle cells, leading to pronounced vasodilation, without excitation of neurons and astrocytes. This photodilation is reversible, reproducible and energy-dependent, appearing at about 0.5 mJ. These results impose careful consideration on the use of photo-activation in studies involving blood flow regulation, as well as in studies requiring prolonged and repetitive stimulations to correct cellular defects in pathological models. They also suggest that light could be used to locally increase blood flow in a controlled fashion.

High-frequency oscillations and seizure-like discharges in the entorhinal cortex of the in vitro isolated guinea pig brain.

We analyzed the patterns of seizure-like activity and associated high-frequency oscillations (HFOs) induced by the K+ channel blocker 4-aminopyridine (4AP, 50µM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 50µM) in the in vitro isolated guinea pig brain preparation. Extracellular field recordings were obtained from the medial entorhinal cortex (EC) using glass pipettes or silicon probes; 4AP or BMI were applied through the basilar artery. Ripples (80-200Hz) or fast ripples (250-500Hz) occurred at higher rates shortly before ictal events induced by 4AP or BMI, respectively. In addition, during the ictal period, ripples were mostly associated with 4AP-induced ictal events whereas fast ripples predominated during ictal discharges induced by BMI. Finally, ripples occurred at higher rates during the clonic phase of 4AP-induced ictal events compared to the tonic phase, while higher rates of fast ripples characterized the clonic phase in both 4AP- and BMI-induced ictal discharges. These differences in HFO occurrence presumably reflect the diverse action of these two convulsants on GABAA receptor signaling.

Simultaneous enhancement of excitation and postburst inhibition at the end of focal seizures.

OBJECTIVE: Comprehension of the events that lead to seizure termination contributes to the development of strategies to confine propagation of ictal discharges. It is commonly assumed that the inhibitory control fails during seizures and recovers after the end of the ictal event. We examine the possibility that a progressive increase of inhibition that counters an increase in the strength of excitation contributes to terminating a focal seizure. METHODS: We analyzed seizures acutely induced by pharmacological manipulations (bicuculline and 4-aminopyridine) in the entorhinal cortex and in the hippocampus of the in vitro isolated guinea pig brain. RESULTS: As seizures ended, extracellular and intracellular recordings showed periodic bursting that progressively decreased in frequency. During the late bursting phase, the duration, number, and rate of occurrence of spikes within single bursts remained constant, whereas cumulative spike amplitude (index of excitation during a burst) and interburst interval (index of inhibition between bursts) progressively increased. The increment of average/cumulative burst excitation and interburst interval toward seizure end was confirmed in human focal seizures recorded with intracerebral electrodes in patients with drug-resistant partial epilepsies. A postburst refractory period of circa 2 seconds that increases with time toward the end of the seizure was confirmed in the experimental model by probing interburst epochs in the CA1 region with local dentate gyrus stimulation just suprathreshold for burst generation. INTERPRETATION: Our findings support the concept that focal seizures are terminated by the simultaneous and opposing enhancement of excitation (burst activity) in addition to postburst inhibition. We hypothesize that a seizure stops when postburst inhibition becomes large enough to prevent reactivation of excitation.

Stimulus-evoked potentials contribute to map the epileptogenic zone during stereo-EEG presurgical monitoring.

Presurgical monitoring with intracerebral electrodes in patients with drug-resistant focal epilepsy represents a standard invasive procedure to localize the sites of seizures origin, defined as the epileptogenic zone (EZ). During presurgical evaluation, intracerebral single-pulse electrical stimulation (SPES) is performed to define the boundaries of eloquent areas and to evoke seizure-associated symptoms. Extensive intracranial exploration and stimulation generate a large dataset on brain connectivity that can be used to improve EZ detection and to understand the organization of the human epileptic brain. We developed a protocol to analyse field responses evoked by intracranial stimulation. Intracerebral recordings were performed with 105-162 recording sites positioned in fronto-temporal regions in 12 patients with pharmacoresistant focal epilepsy. Recording sites were used for bipolar SPES at 1 Hz. Reproducible early and late phases (<60 ms and 60-500 ms from stimulus artefact, respectively) were identified on averaged evoked responses. Phase 1 and 2 responses recorded at all and each recording sites were plotted on a 3D brain reconstructions. Based on connectivity properties, electrode contacts were primarily identified as receivers, mainly activators or bidirectional. We used connectivity patterns to construct networks and applied cluster partitioning to study the proprieties between potentials evoked/stimulated in different regions. We demonstrate that bidirectional connectivity during phase 1 is a prevalent feature that characterize contacts included in the EZ. This study shows that the application of an analytical protocol on intracerebral stimulus-evoked recordings provides useful information that may contribute to EZ detection and to the management of surgical-remediable epilepsies.

Network dynamics during the progression of seizure-like events in the hippocampal-parahippocampal regions.

Seizure patterns in temporal lobe epilepsies have been described both in humans and in animal models. The involvement of specific hippocampal-parahippocampal subregions in the initiation and progression of temporal lobe seizures is not defined yet. We analyzed limbic network dynamics during seizures induced by 3-min arterial perfusion of 50 µM bicuculline in the in vitro isolated guinea pig brain preparation. As for human and animal temporal lobe epilepsies, 2 seizure types characterized at onset by either fast activity (FA) or hypersynchronous activity (HSA) were observed in our acute model. Simultaneous extracellular recordings were performed from ventral hippocampal-parahippocampal subregions with multichannel electrodes, and laminar analysis and propagation directions were computed to define reciprocal interactions during seizures. FA seizures started with fast oscillations generated in CA1-subiculum and entorhinal cortex, followed by irregular spikes and progressively regular bursts well defined in all subfields, with the exception of pre- and parasubiculum that do not participate in seizure activity. Dentate gyrus was not involved at FA seizure onset and became prominent during the transition to bursting in both FA and HSA patterns. HSA seizures were similar to FA events, but lacked initial FA. During seizures, reliable and steady propagation within the intra-hippocampal re-entrant loop was observed.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability.

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na(+) channels, thereby decreasing the neuronal Na(+) current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, re-establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

Synaptic and extrasynaptic origin of the excitation/inhibition imbalance in the hippocampus of synapsin I/II/III knockout mice.

Synapsins (Syn I, Syn II, and Syn III) are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes have been identified as major epilepsy susceptibility genes in humans and synapsin I/II/III triple knockout (TKO) mice are epileptic. However, excitatory and inhibitory synaptic transmission and short-term plasticity have never been analyzed in intact neuronal circuits of TKO mice. To clarify the generation and expression of the epileptic phenotype, we performed patch-clamp recordings in the CA1 region of acute hippocampal slices from 1-month-old presymptomatic and 6-month-old epileptic TKO mice and age-matched controls. We found a strong imbalance between basal glutamatergic and γ-aminobutyric acid (GABA)ergic transmission with increased evoked excitatory postsynaptic current and impaired evoked inhibitory postsynaptic current amplitude. This imbalance was accompanied by a parallel derangement of short-term plasticity paradigms, with enhanced facilitation of glutamatergic transmission in the presymptomatic phase and milder depression of inhibitory synapses in the symptomatic phase. Interestingly, a lower tonic GABA(A) current due to the impaired GABA release is responsible for the more depolarized resting potential found in TKO CA1 neurons, which makes them more susceptible to fire. All these changes preceded the appearance of epilepsy, indicating that the distinct changes in excitatory and inhibitory transmission due to the absence of Syns initiate the epileptogenic process.

Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel.

Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as DeltaPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca(2+) channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca(2+) channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca(2+) channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca(2+) channels and strong effect as potentiators on the DeltaPhe508, G551D, and G1349D CFTR mutants.