Tribute to Roger Guillemin, a pioneer in neuroendocrinology (1924-2024), Nobel Prize in Physiology or Medicine.

Roger Guillemin discovered and characterized the hypothalamic factors that control anterior pituitary functions. He consequently demonstrated that these brain peptides regulate a large number of major body activities through neuroendocrine mechanisms. This especially include growth, fertility and reproduction, endocrine gland functions and stress. These seminal works paved the way to major applications in many fields of physiology and medicine for diagnosis, pharmacology and therapy, far beyond the initial discovery and properties of these molecules, including in cancerology, immunology, inflammation, drug addiction and behavior.

Roger Guillemin a mis en évidence et caractérisé les facteurs hypothalamiques qui contrôlent les fonctions de l'adénohypophyse. Cette découverte majeure a permis de démontrer que ces peptides du cerveau régulent par voie neuroendocrine un grand nombre de fonctions importantes de l'organisme. C'est le cas de la croissance, de la fertilité et de la reproduction, des fonctions des glandes endocriniennes et du stress. Ces travaux pionniers ont ouvert la voie à des applications innovantes dans de nombreux domaines de la physiologie et de la médecine pour le diagnostic, la pharmacologie et la thérapie, bien au-delà de la découverte initiale et des propriétés de ces molécules. Ces domaines comprennent en particulier la cancérologie, l'immunologie, les problèmes inflammatoires, les addictions et le comportement.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Regulation of perineuronal nets in the adult cortex by the activity of the cortical network.

Perineuronal net (PNN) accumulation around parvalbumin-expressing (PV) inhibitory interneurons marks the closure of critical periods of high plasticity, whereas PNN removal reinstates juvenile plasticity in the adult cortex. Using targeted chemogenetic in vivo approaches in the adult mouse visual cortex, we found that transient inhibition of PV interneurons, through metabotropic or ionotropic chemogenetic tools, induced PNN regression. Electroencephalographic recordings indicated that inhibition of PV interneurons did not elicit unbalanced network excitation. Likewise, inhibition of local excitatory neurons also induced PNN regression, whereas chemogenetic excitation of either PV or excitatory neurons did not reduce the PNN. We also observed that chemogenetically inhibited PV interneurons exhibited reduced PNN compared to their untransduced neighbors, and confirmed that single PV interneurons express multiple genes enabling individual regulation of their own PNN density. Our results indicate that PNN density is regulated in the adult cortex by local changes of network activity that can be triggered by modulation of PV interneurons. PNN regulation may provide adult cortical circuits with an activity-dependent mechanism to control their local remodeling.SIGNIFICANCE STATEMENTThe perineuronal net is an extracellular matrix, which accumulates around individual parvalbumin-expressing inhibitory neurons during postnatal development, and is seen as a barrier that prevents plasticity of neuronal circuits in the adult cerebral cortex. We found that transiently inhibiting parvalbumin-expressing or excitatory cortical neurons triggers a local decrease of perineuronal net density. Our results indicate that perineuronal nets are regulated in the adult cortex depending on the activity of local microcircuits. These findings uncover an activity-dependent mechanism by which adult cortical circuits may locally control their plasticity.

New light on cortical neuropeptides and synaptic network plasticity.

Neuropeptides, members of a large and evolutionarily ancient family of proteinaceous cell-cell signaling molecules, are widely recognized as extremely potent regulators of brain function and behavior. At the cellular level, neuropeptides are known to act mainly via modulation of ion channel and synapse function, but functional impacts emerging at the level of complex cortical synaptic networks have resisted mechanistic analysis. New findings from single-cell RNA-seq transcriptomics now illuminate intricate patterns of cortical neuropeptide signaling gene expression and new tools now offer powerful molecular access to cortical neuropeptide signaling. Here we highlight some of these new findings and tools, focusing especially on prospects for experimental and theoretical exploration of peptidergic and synaptic networks interactions underlying cortical function and plasticity.

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks.

Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.

Neuronal Pentraxin 2 Binds PNNs and Enhances PNN Formation.

The perineuronal net (PNN) is a mesh-like proteoglycan structure on the neuronal surface which is involved in regulating plasticity. The PNN regulates plasticity via multiple pathways, one of which is direct regulation of synapses through the control of AMPA receptor mobility. Since neuronal pentraxin 2 (Nptx2) is a known regulator of AMPA receptor mobility and Nptx2 can be removed from the neuronal surface by PNN removal, we investigated whether Nptx2 has a function in the PNN. We found that Nptx2 binds to the glycosaminoglycans hyaluronan and chondroitin sulphate E in the PNN. Furthermore, in primary cortical neuron cultures, the addition of NPTX2 to the culture medium enhances PNN formation during PNN development. These findings suggest Nptx2 as a novel PNN binding protein with a role in the mechanism of PNN formation.

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

Comment on "Principles of connectivity among morphologically defined cell types in adult neocortex".

Jiang et al (Research Article, 27 November 2015, aac9462) describe detailed experiments that substantially add to the knowledge of cortical microcircuitry and are unique in the number of connections reported and the quality of interneuron reconstruction. The work appeals to experts and laypersons because of the notion that it unveils new principles and provides a complete description of cortical circuits. We provide a counterbalance to the authors' claims to give those less familiar with the minutiae of cortical circuits a better sense of the contributions and the limitations of this study.

Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1(-/y) mice.

Hypersensitivity in response to sensory stimuli and neocortical hyperexcitability are prominent features of Fragile X Syndrome (FXS) and autism spectrum disorders, but little is known about the dendritic mechanisms underlying these phenomena. We found that the primary somatosensory neocortex (S1) was hyperexcited in response to tactile sensory stimulation in Fmr1(-/y) mice. This correlated with neuronal and dendritic hyperexcitability of S1 pyramidal neurons, which affect all major aspects of neuronal computation, from the integration of synaptic input to the generation of action potential output. Using dendritic electrophysiological recordings, calcium imaging, pharmacology, biochemistry and a computer model, we found that this defect was, at least in part, attributable to the reduction and dysfunction of dendritic h- and BKCa channels. We pharmacologically rescued several core hyperexcitability phenomena by targeting BKCa channels. Our results provide strong evidence pointing to the utility of BKCa channel openers for the treatment of the sensory hypersensitivity aspects of FXS.

Chronic assessment of cerebral hemodynamics during rat forepaw electrical stimulation using functional ultrasound imaging.

Functional ultrasound imaging is a method recently developed to assess brain activity via hemodynamics in rodents. Doppler ultrasound signals allow the measurement of cerebral blood volume (CBV) and red blood cells' (RBCs') velocity in small vessels. However, this technique originally requires performing a large craniotomy that limits its use to acute experiments only. Moreover, a detailed description of the hemodynamic changes that underlie functional ultrasound imaging has not been described but is essential for a better interpretation of neuroimaging data. To overcome the limitation of the craniotomy, we developed a dedicated thinned skull surgery for chronic imaging. This procedure did not induce brain inflammation nor neuronal death as confirmed by immunostaining. We successfully acquired both high-resolution images of the microvasculature and functional movies of the brain hemodynamics on the same animal at 0, 2, and 7 days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1 mA) ranging from 1 (200 µs) to 25 pulses (5s). Our results indicate that CBV HRF parameters such as the peak amplitude, the time to peak, the full width at half-maximum and the spatial extent of the activated area increase with stimulus duration. Functional ultrasound imaging was sensitive enough to detect hemodynamic responses evoked by only a single pulse stimulus. We also observed that the RBC velocity during activation could be separated in two distinct speed ranges with the fastest velocities located in the upper part of the cortex and slower velocities in deeper layers. For the first time, functional ultrasound imaging demonstrates its potential to image brain activity chronically in small animals and offers new insights into the spatiotemporal evolution of cerebral hemodynamics.

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.

New insights into the classification and nomenclature of cortical GABAergic interneurons.

A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts' assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus.

Diversity of GABAergic interneurons in layer VIa and VIb of mouse barrel cortex.

Neocortical layer VI modulates the thalamocortical transfer of information and has a significant impact on sensory processing. This function implicates local γ-aminobutyric acidergic (GABAergic) interneurons that have only been partly described at the present time. Here, we characterized 85 layer VI GABAergic interneurons in acute slices of mouse somatosensory barrel cortex, using whole-cell current-clamp recordings, single-cell reverse transcription-polymerase chain reaction, and biocytin labeling followed by Neurolucida reconstructions. Unsupervised clustering based on electrophysiological molecular and morphological properties disclosed 4 types of interneurons. The 2 major classes were fast-spiking cells transcribing parvalbumin (PV) (51%) and adapting interneurons transcribing somatostatin (SOM) (26%). The third population (18%) transcribed neuropeptide Y (NPY) and appeared very similar to neurogliaform cells. The last class (5%) was constituted by well-segregated GABAergic interneurons transcribing vasoactive intestinal peptide (VIP). Using transgenic mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we investigated the densities of GABAergic cells immunolabeled against PV, SOM, VIP, and NPY through the depth of layer VI. This analysis revealed that PV and NPY translating interneurons concentrate in the upper and lower parts of layer VI, respectively. This study provides an extensive characterization of the properties of layer VI interneurons.

Activation of cortical 5-HT(3) receptor-expressing interneurons induces NO mediated vasodilatations and NPY mediated vasoconstrictions.

GABAergic interneurons are local integrators of cortical activity that have been reported to be involved in the control of cerebral blood flow (CBF) through their ability to produce vasoactive molecules and their rich innervation of neighboring blood vessels. They form a highly diverse population among which the serotonin 5-hydroxytryptamine 3A receptor (5-HT(3A))-expressing interneurons share a common developmental origin, in addition to the responsiveness to serotonergic ascending pathway. We have recently shown that these neurons regroup two distinct subpopulations within the somatosensory cortex: Neuropeptide Y (NPY)-expressing interneurons, displaying morphological properties similar to those of neurogliaform cells and Vasoactive Intestinal Peptide (VIP)-expressing bipolar/bitufted interneurons. The aim of the present study was to determine the role of these neuronal populations in the control of vascular tone by monitoring blood vessels diameter changes, using infrared videomicroscopy in mouse neocortical slices. Bath applications of 1-(3-Chlorophenyl)biguanide hydrochloride (mCPBG), a 5-HT(3)R agonist, induced both constrictions (30%) and dilations (70%) of penetrating arterioles within supragranular layers. All vasoconstrictions were abolished in the presence of the NPY receptor antagonist (BIBP 3226), suggesting that they were elicited by NPY release. Vasodilations persisted in the presence of the VIP receptor antagonist VPAC1 (PG-97-269), whereas they were blocked in the presence of the neuronal Nitric Oxide (NO) Synthase (nNOS) inhibitor, L-NNA. Altogether, these results strongly suggest that activation of neocortical 5-HT(3A)-expressing interneurons by serotoninergic input could induces NO mediated vasodilatations and NPY mediated vasoconstrictions.

Characterization of Type I and Type II nNOS-Expressing Interneurons in the Barrel Cortex of Mouse.

IN THE NEOCORTEX, NEURONAL NITRIC OXIDE (NO) SYNTHASE (NNOS) IS ESSENTIALLY EXPRESSED IN TWO CLASSES OF GABAERGIC NEURONS: type I neurons displaying high levels of expression and type II neurons displaying weaker expression. Using immunocytochemistry in mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we studied the distribution of type I and type II neurons in the barrel cortex and their expression of parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal peptide (VIP). We found that type I neurons were predominantly located in deeper layers and expressed SOM (91.5%) while type II neurons were concentrated in layer II/III and VI and expressed PV (17.7%), SOM (18.7%), and VIP (10.2%). We then characterized neurons expressing nNOS mRNA (n = 42 cells) ex vivo, using whole-cell recordings coupled to single-cell reverse transcription-PCR and biocytin labeling. Unsupervised cluster analysis of this sample disclosed four classes. One cluster (n = 7) corresponded to large, deep layer neurons, displaying a high expression of SOM (85.7%) and was thus very likely to correspond to type I neurons. The three other clusters were identified as putative type II cells and corresponded to neurogliaform-like interneurons (n = 19), deep layer neurons expressing PV or SOM (n = 9), and neurons expressing VIP (n = 7). Finally, we performed nNOS immunohistochemistry on mouse lines in which GFP labeling revealed the expression of two specific developmental genes (Lhx6 and 5-HT(3A)). We found that type I neurons expressed Lhx6 but never 5-HT(3A), indicating that they originate in the medial ganglionic eminence (MGE). Type II neurons expressed Lhx6 (63%) and 5-HT(3A) (34.4%) supporting their derivation either from the MGE or from the caudal ganglionic eminence (CGE) and the entopeduncular and dorsal preoptic areas. Together, our results in the barrel cortex of mouse support the view that type I neurons form a specific class of SOM-expressing neurons while type II neurons comprise at least three classes.

Impaired neurovascular coupling in the APPxPS1 mouse model of Alzheimer's disease.

The tight coupling between neuronal activity and the local increase of blood flow termed neurovascular coupling is essential for normal brain function. This mechanism of regulation is compromised in Alzheimer's Disease (AD). In order to determine whether a purely vascular dysfunction or a neuronal alteration of blood vessels diameter control could be responsible for the impaired neurovascular coupling observed in AD, blood vessels reactivity in response to different pharmacological stimulations was examined in double transgenic APPxPS1 mice model of AD. Blood vessels movements were monitored using infrared videomicroscopy ex vivo, in cortical slices of 8 month-old APPxPS1 and wild type (WT) mice. We quantified vasomotor responses induced either by direct blood vessel stimulation with a thromboxane A2 analogue, the U46619 (9,11-dideoxy-11a,9a-epoxymethanoprostaglandin F2α) or via the stimulation of interneurons with the nicotinic acetylcholine receptor (nAChRs) agonist DMPP (1,1-Dimethyl-4- phenylpiperazinium iodide). Using both types of stimulation, no significant differences were detected for the amplitude of blood vessel diameter changes between the transgenic APPxPS1 mice model of AD and WT mice, although the kinetics of recovery were slower in APPxPS1 mice. We find that activation of neocortical interneurons with DMPP induced both vasodilation via Nitric Oxide (NO) release and constriction via Neuropeptide Y (NPY) release. However, we observed a smaller proportion of reactive blood vessels following a neuronal activation in transgenic mice compared with WT mice. Altogether, these results suggest that in this mouse model of AD, deficiency in the cortical neurovascular coupling essentially results from a neuronal rather than a vascular dysfunction.

Deciphering the Neuronal Circuitry Controlling Local Blood Flow in the Cerebral Cortex with Optogenetics in PV::Cre Transgenic Mice.

Although it is know since more than a century that neuronal activity is coupled to blood supply regulation, the underlying pathways remains to be identified. In the brain, neuronal activation triggers a local increase of cerebral blood flow (CBF) that is controlled by the neurogliovascular unit composed of terminals of neurons, astrocytes, and blood vessel muscles. It is generally accepted that the regulation of the neurogliovascular unit is adjusted to local metabolic demand by local circuits. Today experimental data led us to realize that the regulatory mechanisms are more complex and that a neuronal system within the brain is devoted to the control of local brain-blood flow. Recent optogenetic experiments combined with functional magnetic resonance imaging have revealed that light stimulation of neurons expressing the calcium binding protein parvalbumin (PV) is associated with positive blood oxygen level-dependent (BOLD) signal in the corresponding barrel field but also with negative BOLD in the surrounding deeper area. Here, we demonstrate that in acute brain slices, channelrhodopsin-2 (ChR2) based photostimulation of PV containing neurons gives rise to an effective contraction of penetrating arterioles. These results support the neurogenic hypothesis of a complex distributed nervous system controlling the CBF.

Neurotransmitter release at the thalamocortical synapse instructs barrel formation but not axon patterning in the somatosensory cortex.

To assess the impact of synaptic neurotransmitter release on neural circuit development, we analyzed barrel cortex formation after thalamic or cortical ablation of RIM1 and RIM2 proteins, which control synaptic vesicle fusion. Thalamus-specific deletion of RIMs reduced neurotransmission efficacy by 67%. A barrelless phenotype was found with a dissociation of effects on the presynaptic and postsynaptic cellular elements of the barrel. Presynaptically, thalamocortical axons formed a normal whisker map, whereas postsynaptically the cytoarchitecture of layer IV neurons was altered as spiny stellate neurons were evenly distributed and their dendritic trees were symmetric. Strikingly, cortex-specific deletion of the RIM genes did not modify barrel development. Adult mice with thalamic-specific RIM deletion showed a lack of activity-triggered immediate early gene expression and altered sensory-related behaviors. Thus, efficient synaptic release is required at thalamocortical but not at corticocortical synapses for building the whisker to barrel map and for efficient sensory function.

On-bead tryptic proteolysis: an attractive procedure for LC-MS/MS analysis of the Drosophila caspase 8 protein complex during immune response against bacteria.

This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.

Genetic targeting of specific neuronal cell types in the cerebral cortex.

Understanding the structure and function of cortical circuits requires the identification of and control over specific cell types in the cortex. To address these obstacles, recent optogenetic approaches have been developed. The capacity to activate, silence, or monitor specific cell types by combining genetics, virology, and optics will decipher the role of specific groups of neurons within circuits with a spatiotemporal resolution that overcomes standard approaches. In this review, the various strategies for selective genetic targeting of a defined neuronal population are discussed as well as the pros and cons of the use of transgenic animals and recombinant viral vectors for the expression of transgenes in a specific set of neurons.