©3D brainbow astrocytes

Developed by researchers from École Polytechnique, Sorbonne Université, Inserm and the CNRS, ChroMS is a new microscopy technique bringing together color, 3D and high-resolution imaging, and is nothing short of a revolution in vertebrate brain imaging. The ChroMS technique is described in detail in a recently published article in Nature Communications.

Until now, researchers have had to choose between resolution and volume when performing vertebrate brain imaging. They could either obtain very high resolution over small volumes using three-dimensional electron microscopy or an image of the whole brain at resolutions that are far too low to understand the details.

The main advantage of the ChroMS (Chromatic Multiphoton Serial imaging) technique is that it provides a truly high-resolution virtual view (at the cellular level) of certain parts of the brain that are essential for understanding the development of neuronal circuits. Although the visit is virtual, the data are real. They are obtained from the brains of transgenic mice whose neurons produce fluorescent markers originating from jellyfish or coral. When stimulated by an infrared laser, these markers show up as colors.

“This instrument is ideal for making extremely precise, 3D reconstructions of regions of the brain with a volume of a few cubic millimeters, which is a breakthrough with this image quality, and this is the appropriate scale for what we want to observe”, explains Emmanuel Beaurepaire, researcher from the Laboratory for Optics and Biosciences (LOB – a joint research lab between École Polytechnique, the CNRS and Inserm). “Using the current version of our instrument, we can also reconstitute a complete mouse brain, albeit at a lower level of precision”.

“We are particularly interested in cell lineage,” states Jean Livet, researcher from the Institut de la Vision (Sorbonne Université, Inserm, CNRS). “In other words, the manner in which the brain develops from neural stem cells: what are the daughter cells from a given stem cell, how can a stem cell mutation influence their development, and how are groups of cells generated by different stem cells organized in relation to one another? The high-volume, color-coded images produced by ChroMS reveal the developmental history of an individual region of the brain”.

ChroMS should enable us to answer questions that neuroscientists have been asking for a long time, such as whether neurons arising from the same stem cell connect to each other preferentially to fulfil a given function, and whether pathologies such as epilepsy could be linked to localized problems affecting certain neural stem cells.

Although the ChroMS technique is ideally suited to the study of highly complex organs such as the brain, it can be used on all organs and should also prove a very effective tool for embryogenesis studies.

(A) Principle of ChroMS microscopy, combining color two-photon excitation by frequency mixing and automated serial slicing of brain tissue. (B) Image acquired with the “whole brain tomography” mode showing the cortex and hippocampus of a Brainbow mouse. (C) 3D reconstruction and view at different scales of a 4.8 mm3 volume of mouse cortex in which astrocytes are marked with fluorescent proteins of different colors. (D) 3D view of color-marked neurons in the mouse cortex. Adapted from: Abdeladim et al, Nat Commun 2019.

©Photo by Cris Saur on Unsplash

How can the quality of life of patients with narcolepsy, the severest sleep disorder in humans, be improved? An international scientific team led by Yves Dauvilliers, a researcher at Inserm and Université de Montpellier, is working on Solriamfetol – a promising new drug that stimulates alertness and improves resistance to sleepiness. The results of the Phase 3 clinical trial published in Annals of Neurology show that when compared with existing treatments, Solriamfetol is not just more effective and long-lasting, but also has fewer side effects.

Narcolepsy is a chronic rare neurological disorder caused by a loss of neurons that synthesize the protein hypocretin. It is characterized by excessive daytime sleepiness and difficulty staying awake. Being the severest sleep disorder in humans, it is an excellent model for studying other pathologies of the same type.

Existing treatments to improve the symptoms of narcolepsy are thin on the ground, inconsistent in their efficacy and sometimes linked to side effects. And they only treat the symptoms, not the root cause. Since research is as yet unable to produce synthetic hypocretin, these treatments make do with compensating the lack of hypocretin: they stimulate alertness by acting primarily on dopamine transporters.

It is on the development of a more effective drug in improving narcolepsy symptoms that is working Yves Dauvilliers, researcher at Inserm and Université de Montpellier in the “Neuropsychiatry: Epidemiological and Clinical Research” laboratory (Inserm/Université de Montpellier), in collaboration with international teams. The research he is leading is centered around Solriamfetol[1], a drug that not only inhibits the transporters of dopamine but also those of norepinephrine – another neurotransmitter involved in the regulation of waking.

In this Phase 3 clinical trial, 240 narcoleptic patients were followed for 12 weeks in order to evaluate the efficacy and safety of Solriamfetol in humans. The tests were performed under double-blind conditions on groups of 60 patients receiving different doses of Solriamfetol or placebo. In addition to patient feedback on the day-to-day changes in their sleepiness, the trial also involved tests in which the patients were required to try to stay awake in an atmosphere conducive to sleep.

The research team observed that those patients receiving a daily dose of 150 mg or 300 mg Solriamfetol managed to fight sleepiness for around 20 minutes versus 10 without treatment, i.e. for twice as long. The currently-prescribed treatments extend this alertness by only 2 to 3 minutes. This efficacy was maintained throughout the 12 weeks of treatment, with few side effects and no need to increase dosage.

“By enabling them to better resist sleepiness, Solriamfetol therefore proves to be highly promising when it comes to improving the quality of life of people with narcolepsy and also in the other disorders linked to sleepiness, such as sleep apnea syndrome – in which it offers the same efficacy”, states Dauvilliers. In order to evaluate its efficacy and safety over time, the researchers have launched a new clinical trial lasting one year.

[1]The study protocol was developed, in collaboration with the authors, by Jazz Pharmaceuticals, which is funding the trial and holds a license to develop and commercialize Solriamfetol

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Physical inactivity is a common factor in lifestyle diseases – and one that is often linked to the excessive consumption of fatty and/or sugary foods. The opposite scenario of excessive physical activity at the expense of caloric intake can also be harmful, as cases of anorexia nervosa illustrate. These data therefore point to the crucial need to research the neurobiological processes that control the respective motivations for exercise and food intake. A study by Inserm and CNRS researchers published on March 7, 2019 in JCI Insight reveals that the cannabinoid type 1 (CB1) receptors play an essential role in the choice between running and eating chocolatey food.

The authors of this paper had previously reported that the cannabinoid type-1 (CB1) receptors, present on several types of neurons, play a key role in performance during physical activity in mice. A conclusion based on the performances achieved by animals with free access to an exercise wheel – a model in which it was not possible to distinguish the mechanism involved (motivation, pleasure…). Given that the motivation for a reward can only be estimated by measuring the efforts that the individual – whether human or animal – is prepared to make to get that reward, the researchers devised a model in which each access to the wheel was conditional on a prior effort. This involved the animal repeatedly introducing its snout into a recipient, an essential prerequisite for unlocking the wheel. After a training period during which the level of effort required to unlock the wheel remained the same, the mice were confronted with a test in which the effort required was gradually increased. When exposed to this test, the mice lacking CB1 receptors showed an 80 % deficit in the maximum effort they were prepared to make to unlock the wheel, and without a decrease in performance during their access to it. This finding indicates that the CB1 receptors play a major role in controlling motivation for exercise. The use of other genetically-modified mice also enabled the researchers to demonstrate that these CB1 receptors controlling motivation for exercise are located on GABAergic neurons.

The researchers then examined whether the CB1 receptors in the GABAergic neurons control the motivation for another reward: chocolatey food (like humans, mice love it even when they are otherwise well-fed). While the CB1 receptors also play a role in motivation for food – albeit to a lesser extent than in motivation for exercise – the CB1 receptors located on the GABAergic neurons are not implicated in the motivation for eating chocolatey food.

In our daily life, we are faced with an ongoing choice between various rewards. A fact which has encouraged the researchers to develop a model in which following a learning period the mice had the choice – in return for the efforts described above – between exercise and chocolatey food. The motivation for exercise was greater than that for chocolatey food, with the exception of the mice lacking CB1 – whether generally or just on GABAergic neurons – whose preference was for the food.

In addition to these findings indicating that the cannabinoid receptor is essential for the motivation for exercise, this study opens up avenues for researching the neurobiological mechanisms behind pathological increases in this motivation. One illustration is provided by anorexia nervosa which often combines the decreased motivation to eat with an increased motivation to exercise.

Left to right : Pr Elisabeth Tournier-Lasserve, Pr Hugues Chabriat, Pr Marie-Germaine Bousser, Dr Anne Joutel

Awarded by the Danish Lundbeck Foundation, the “Brain Prize” is a major international award for scientific importance of their research in neuroscience. It has a total of one million euros.

He puts this year in honor of the work begun there nearly forty years by four French scientists on CADASIL, a hereditary cerebrovascular disease, which causes migraine attacks, stroke and cognitive decline. Today is the genetic disease of small cerebral vessels most frequently diagnosed.

The four winners of the French “Brain Prize 2019” are:

> Professor Elisabeth Tournier-Lasserve , head of the hospital neurovascular genetics department Lariboisière AP-HP, medical genetics professor at Paris Diderot University and head of the research team “cerebrovascular diseases, genomics , imaging and personalized medicine “within the unit Paris Diderot – Inserm 1141” NeuroDiderot “.

> Professor Hugh Chabriat , head of the neurology department of the hospital Lariboisière AP-HP, coordinator of the reference center for rare vascular diseases of the brain and the eye (CERVCO), professor of neurology at the University Paris Diderot and researcher at the research team “cerebrovascular diseases, genomics, imaging and personalized medicine” in the unit of Paris-Diderot – Inserm 1141 “NeuroDiderot”. Prof. Chabriat also co-coordinates the University Hospital Department (DHU) neurovasc Sorbonne Paris Cité.

> Professor Marie-Germaine Bousser , former head of the neurology department at the hospital Lariboisière AP-HP and emeritus professor of neurology at the University of Paris Diderot.

> Dr. Anne Joutel, Research Director Inserm, director of the team “pathophysiological mechanisms of diseases of the small cerebral vessels” at the Institute of Psychiatry and Neurosciences Paris Descartes – UMR1266 Inserm and professor in the pharmacology department University of Vermont (USA).

The four scientists showed that CADASIL (for “Cerebral Autosomal Dominant arteriopathy with sub-cortical Infarcts and Leukoencephalopathy”) is an inherited cerebrovascular disease caused by mutations in the NOTCH3 gene on chromosome 19. This condition is responsible for migraine attacks and stroke, can lead to severe motor and cognitive disorders. Patients may also suffer from depression, difficulty concentrating, a slowdown and balance disorders.

The discovery of CADASIL has allowed the development of diagnostic tests and the development of mouse models of the disease, essential to understand the mechanisms of brain damage and the development of therapeutic. The identification and clinical and preclinical study of this disease are also a major step to identify and better understand other diseases of cerebral vessels.

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Hospital teams Pitié-Salpêtrière Hospital AP-HP, Inserm and the Institute of the brain and spinal cord – AP-HP / CNRS / Inserm / University-Sorbonne showed appreciation caregivers (nurses and nursing auxiliaries) for the state of consciousness of patients represented a real added value to medical and electrophysiological exams and conventional brain imaging diagnostics. This work, published in the journal The British Medical Journal Open, apply the principle of “collective intelligence” (or “wisdom of the crowds”).

The waning of severe brain injury, initially a coma patient can progress to altered state of consciousness such as vegetative or minimally conscious state. The determination of the level of consciousness is important both to better assess the patient’s condition, to explain to his family, and also because of the prognostic value of that information.

However it is sometimes difficult to establish and then requires an approach called “multimodal” combining clinical expertise and neuroimaging. The recent international recommendations stress in particular the need to repeat clinical evaluations using a specific scale (the “Coma Recovery Scale – Revised”), and the utility of complement by specialized brain imaging examinations (EEG, evoked potentials cognitive, PET scans and functional MRI).

It is in this context that researchers from Inserm, a medical team of the neurological intensive care hospital Pitié-Salpêtrière AP-HP, led by Dr. Sophie Demeret and a team of the neurophysiology department clinic Pitié-Salpêtrière hospital AP-HP and laboratory “PICNIB lab” at the Institute of the brain and spinal cord led by Prof. Lionel Naccache, professor of physiology at Sorbonne University, wanted to add to this multimodal approach an additional source of information on the principle of “collective intelligence” (or “wisdom of the crowds”): the expertise of the medical staff is constantly in contact with patients throughout their hospital stay.

The tool, called “DoC-feeling” (DoC for Disorders of Consciousness) using a visual analog scale (as used for pain assessment) to collect the subjective perception vis-à-vis the status of carers awareness of the patient quickly and easily. The synthesis of all the measurements taken over a week and provides a “collective” score between 0 and 100.

Forty-nine patients hospitalized for expert evaluation of the level of consciousness in the neurological intensive care unit of the hospital of the Pitié-Salpêtrière AP-HP were included in a year and a half. Nearly 700 evaluations by more than 80 carers were collected. The study, supervised by two referent nurses and supported by health managers, has shown that the median value of individual assessments by caregivers was closely correlated with depth specialized clinical assessments. Another advantage is that this approach allowed to significantly increase the number of patient observations, the state of consciousness may fluctuate over time.

The teams conclude that this approach, in addition to the medical clinic assessment and examinations electrophysiological and brain imaging, should improve the diagnostic accuracy of patients’ state of consciousness. This work puts more and before the interest of the collective intelligence and a collaborative approach to a complex known clinical question.

“This work initiated by two nurses in neurological intensive care hospital Pitié-Salpêtrière AP-HP: Gwen Goudard and Karine Courcoux, which involved more than 80 carers over a period of more than a year, demonstrates extraordinary motivation and the enormous potential of research in allied unity, “ says Dr. Benjamin Rohaut who oversaw the study. He added : “The support of service managers, Louise Richard Gili and Julie Bourmaleau and the help of Dr. Bertrand Hermann, Inserm researcher at the PICNIC-lab for data analysis and writing of the article, were key assets for conducting the study to completion. “

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A painkiller nanomedicine has just been developed by Patrick Couvreur’s team at Institut Galien Paris-Sud (Université Paris-Sud/CNRS)[i]. This new drug specifically targets the area of painful inflammation without causing the addiction phenomena of current medication.

In pain management, morphine and synthetic opioids are currently effective drugs. Unfortunately, their side effects are serious, in particular respiratory depression, but especially addiction. Opioid addiction is indeed a global healthcare scourge. The United States is particularly affected with 11 million dependent patients and around 175 daily deaths as a result of overdoses.

The use of certain neuropeptides, natural peptides used by neurons to communicate with each other, is undoubtedly an interesting alternative to these drugs. In fact, acting mainly on the neurotransmitter receptors that specifically modulate the pain response function, these natural molecules do not cause these side effects. Unfortunately, after administration these molecules degrade in a few minutes without having time to act.

An effective mode of action without side effects

The “Innovative Nanomedicines for the Treatment of Serious Illnesses” team, led by Patrick Couvreur at the Faculty of Pharmacy at Université Paris-Sud, had the idea of synthesising samples of nanoparticles made up of Leu-enkephalin and coupling them to squalene, a naturally occurring lipid in the body, whose specificity is to be able to provide a protective membrane for the whole compound.

Together with scientists from the Paris Institute of Psychiatry and Neuroscience (Inserm/Université Paris Descartes) and the Neuropharmacology Laboratory (Université Paris-Sud/Inserm), the team demonstrated that these nanomedicines induced a significant and prolonged pain-relief effect in rats, with a higher efficacy than morphine.

The researchers particularly noted that, unlike morphine, the Leu-enkephalin-squalene nanoparticles spared brain tissue and acted solely on peripheral receptors. Furthermore, imaging showed that the nanoparticles were capable of delivering the neuropeptide specifically to the area of painful inflammation, thus avoiding the central effects responsible for addiction phenomena.

Biochemical and histological investigations conducted on the treated animals, have moreover demonstrated that this new painkiller medicine did not cause any toxicity or side effects.

This study, published in the journal Science Advances, on 13 February 2019, represents a breakthrough in pain treatment due to the nanomedicine. Further research is still necessary before being able to move to clinical trials. A start-up could be established in the coming months to raise the funds needed for their financing.

[i]  Together with scientists from the Paris Institute of Psychiatry and Neurosciences (Inserm/Université Paris Descartes) and the Neuropharmacology Laboratory (Université Paris-Sud/Inserm).

Images en cryo-TEM des nanoparticules de Leu-Enképhaline-squalène d’une taille comprise entre 70 et 100 nm.© Institut Galien Paris-Sud, Faculté de Pharmacie  

Effet antidouleur des nanoparticules (courbe bleue) aboli après traitement par l’antagoniste naloxone méthiodide (courbe verte) et l’antagoniste naloxone (courbe rouge). Le traitement par la leu-enképhaline libre ou le vecteur seul est sans effet (courbe noire et grise) © Institut Galien Paris-Sud, Faculté de Pharmacie  

Nanoparticules de Leu-enképhaline-squalène libérant le peptide spécifiquement au niveau du site de la douleur inflammatoire (zone rouge). © Institut Galien Paris-Sud, Faculté de Pharmacie   

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What happens inside our brains when we sleep? To answer this question, French researchers have, for the first time, filmed the entire brain in sleeping rats, thanks to innovative ultrasound imaging technology. They were thus able to closely observe brain function in rodents, particularly during the REM sleep phase. These results were obtained in shared Inserm, ESPCI Paris, CNRS, and Sorbonne University laboratories. Published in Nature Communications, these findings allow this period to be redefined as a brain hypersynchronization phase, characterized by massive peaks in blood flow, particularly in the hippocampus. These new data, which question the role currently ascribed to REM sleep, have yet to be confirmed in humans.

REM sleep is a specific sleeping state in which brain activity is similar to the waking state, while being associated with inhibition of muscle activity. It is notably characterized by rapid eye movements, and has long been considered to coincide only with dreaming and emotional processes. However, recent studies showed that it also played a major role in neuronal plasticity in the hippocampus, i.e., the ability of neurons to reconfigure their connections.

To have a better understanding of brain function during REM sleep, researchers from Inserm unit 979 “Wave Physics for Medicine” managed by Mickaël Tanter at Institut Langevin (ESPCI Paris/CNRS), and recently accredited as a “Biomedical ultrasound” technology research accelerator in partnership with the Paris-Seine Neuroscience Laboratory (Sorbonne University/CNRS/Inserm), studied brain activity in sleeping rats. To do so, they combined electroencephalography (EEG), which records neuronal electrical activity, with an ultrarapid ultrasound imaging technique known as fUS (functional ultrasound). This innovative technique, developed by the team led by Mickaël Tanter, allows highly precise visualization of variations in blood flow related to neuronal activity throughout the brain in alert, moving rats.

The research team observed that REM sleep in rats is associated with substantially increased blood flow in the brain, in the form of waves which first reach the subcortical regions and then move along the hippocampus, followed by the cortex. In comparison, non-REM sleep and waking phases in inactive rats show relatively low cerebral blood volumes.

This vascular hyperactivity during REM sleep is characterized by two phases: one, similar to the findings observed when recording active rats, and the other, unknown until now, consisting of sudden elevations in blood flow which the researchers describe as “vascular surges”. Although lasting 5 to 30 seconds on average, these can persist for 1 minute in the cortical regions, and are particularly strong in the hippocampus.

The researchers successfully identified an electrical signal in the hippocampus (a crucial zone for memory), characteristic of these elevated blood flow peaks. This signal – high-frequency gamma oscillations – is ordinarily observed in alert rats. Their intensity during REM sleep is directly correlated with vascular surge intensity, which suggests that these local oscillations could control vascular flow throughout the brain. “This information is crucial,” points out Antoine Bergel, co-author of the study, “as it allows us to target very precise regions of the brain potentially involved in the genesis of these intense vascular events.”

The scientists also noted that, during REM sleep, a vascular synchronization phenomenon exists between distant areas of the brain (cortex, hippocampus, and thalamus), which is much more extensive compared to any other sleeping or waking states.

These studies reveal the very first films of the entire brain during REM sleep and confirm the benefit of neurofunctional ultrasound to fundamental neuroscience research. At present, the fUS technique is still difficult to apply in adult humans. However, these results can now be confirmed in neonates, although caution should be exercised in terms of extrapolation to human physiology. These results nonetheless represent major progress in understanding the connection between electrical and vascular activity (a phenomenon involved in numerous human disorders, such as stroke or epilepsy), and challenge our understanding of REM sleep, the function of which has yet to be elucidated.

Images obtained using the fUS technique and EEG signals generated in a rat brain during the waking, deep sleep and REM sleep phases. In contrast to the highly similar electrical signals between waking and REM sleep, vascular brain activity is much more intense and more “synchronized” than during the waking state. The brain structures are identified by superimposing a brain atlas onto the vascular network images.