Plasticity of thalamocortical axons is regulated by serotonin levels modulated by preterm birth.

Sensory inputs are conveyed to distinct primary areas of the neocortex through specific thalamocortical axons (TCA). While TCA have the ability to reorient postnatally to rescue embryonic mistargeting and target proper modality-specific areas, how this remarkable adaptive process is regulated remains largely unknown. Here, using a mutant mouse model with a shifted TCA trajectory during embryogenesis, we demonstrated that TCA rewiring occurs during a short postnatal time window, preceded by a prenatal apoptosis of thalamic neurons-two processes that together lead to the formation of properly innervated albeit reduced primary sensory areas. We furthermore showed that preterm birth, through serotonin modulation, impairs early postnatal TCA plasticity, as well as the subsequent delineation of cortical area boundary. Our study defines a birth and serotonin-sensitive period that enables concerted adaptations of TCA to primary cortical areas with major implications for our understanding of brain wiring in physiological and preterm conditions.

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.

Imaging the brain in action: a motorized optical rotary joint for wide field fibroscopy in freely moving animals.

Significance: The study of neuronal processes governing behavior in awake behaving mice is constantly boosted by the development of technological strategies, such as miniaturized microscopes and closed-loop virtual reality systems. However, the former limits the quality of recorded signals due to constrains in size and weight and the latter suffers from the restriction of the movement repertoire of the animal, therefore, hardly reproducing the complexity of natural multisensory scenes. Aim: Another strategy that takes advantage of both approaches consists of the use of a fiber-bundle interface to carry optical signals from a moving animal to a conventional imaging system. However, as the bundle is usually fixed below the optics, its torsion resulting from rotations of the animal inevitably constrains the behavior over long recordings. Our aim was to overcome this major limitation of fibroscopic imaging. Approach: We developed a motorized optical rotary joint controlled by an inertial measurement unit at the animal's head. Results: We show its principle of operation, demonstrate its efficacy in a locomotion task, and propose several modes of operation for a wide range of experimental designs. Conclusions: Combined with an optical rotary joint, fibroscopic approaches represent an outstanding tool to link neuronal activity with behavior in mice at the millisecond timescale.

Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice.

Tactile information is actively acquired and processed in the brain through concerted interactions between movement and sensation. Somatosensory input is often the result of self-generated movement during the active touch of objects, and conversely, sensory information is used to refine motor control. There must therefore be important interactions between sensory and motor pathways, which we chose to investigate in the mouse whisker sensorimotor system. Voltage-sensitive dye was applied to the neocortex of mice to directly image the membrane potential dynamics of sensorimotor cortex with subcolumnar spatial resolution and millisecond temporal precision. Single brief whisker deflections evoked highly distributed depolarizing cortical sensory responses, which began in the primary somatosensory barrel cortex and subsequently excited the whisker motor cortex. The spread of sensory information to motor cortex was dynamically regulated by behavior and correlated with the generation of sensory-evoked whisker movement. Sensory processing in motor cortex may therefore contribute significantly to active tactile sensory perception.

Serotonin 3A receptor subtype as an early and protracted marker of cortical interneuron subpopulations.

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT(3A) promoter (5-HT(3A):GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT(3A):GFP mice, we identified 2 populations of 5-HT(3A)-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT(3A) mRNA detection showed that cortical 5-HT(3A) interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT(3A) receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT(3A) interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT(3A) subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

Visualizing the cortical representation of whisker touch: voltage-sensitive dye imaging in freely moving mice.

Voltage-sensitive dye imaging resolves the spatiotemporal dynamics of supragranular subthreshold cortical activity with millisecond temporal resolution and subcolumnar spatial resolution. We used a flexible fiber optic image bundle to visualize voltage-sensitive dye dynamics in the barrel cortex of freely moving mice while simultaneously filming whisker-related behavior to generate two movies matched frame-by-frame with a temporal resolution of up to 2 ms. Sensory responses evoked by passive whisker stimulation lasted longer and spread further across the barrel cortex in awake mice compared to anesthetized mice. Passively evoked sensory responses were large during behaviorally quiet periods and small during active whisking. However, as an exploring mouse approached an object while whisking, large-amplitude, propagating cortical sensory activity was evoked by active whisker-touch. These experiments demonstrate that fiber optics can be used to image cortical sensory activity with high resolution in freely moving animals. The results demonstrate differential processing of sensory input depending upon behavior.

Degenerative abnormalities in transgenic neocortical neuropeptide Y interneurons expressing tau-green fluorescent protein.

The introduction of a reporter gene into bacterial artificial chromosome (BAC) constructs allows a rapid identification of the cell type expressing the gene of interest. Here we used BAC transgenic mice expressing a tau-sapphire green fluorescent protein (GFP) under the transcriptional control of the neuropeptide Y (NPY) genomic sequence to characterize morphological and electrophysiological properties of NPY-GFP interneurons of the mouse juvenile primary somatosensory cortex. Electrophysiological whole-cell recordings and biocytin injections were performed to allow the morphological reconstruction of the recorded neurons in three dimensions. Ninety-six recorded NPY-GFP interneurons were compared with 39 wild-type (WT) NPY interneurons, from which 23 and 19 were reconstructed, respectively. We observed that 91% of the reconstructed NPY-GFP interneurons had developed an atypical axonal swelling from which emerge numerous ramifications. These abnormalities were very heterogeneous in shape and size. They were immunoreactive for the microtubule-associated protein tau and the lysosomal-associated membrane protein 1 (LAMP1). Moreover, an electron microscopic analysis revealed the accumulation of numerous autophagic and lysosomal vacuoles in swollen axons. Morphological analyses of NPY-GFP interneurons also indicated that their somata were smaller, their entire dendritic tree was thickened and presented a restricted spatial distribution in comparison with WT NPY interneurons. Finally, the morphological defects observed in NPY-GFP interneurons appeared to be associated with alterations of their electrophysiological intrinsic properties. Altogether, these results demonstrate that NPY-GFP interneurons developed dystrophic axonal swellings and severe morphological and electrophysiological defects that could be due to the overexpression of tau-coupled reporter constructs.

Spatiotemporal properties of whisker-evoked tactile responses in the mouse secondary somatosensory cortex.

The representation of rodents' mystacial vibrissae within the primary somatosensory (S1) cortex has become a major model for studying the cortical processing of tactile sensory information. However, upon vibrissal stimulation, tactile information first reaches S1 but also, almost simultaneously, the secondary somatosensory cortex (S2). To further understand the role of S2 in the processing of whisker inputs, it is essential to characterize the spatio-temporal properties of whisker-evoked response dynamics in this area. Here we describe the topography of the whiskers representation in the mouse S2 with voltage sensitive dye imaging. Analysis of the spatial properties of the early S2 responses induced by stimulating individually 22 to 24 whiskers revealed that they are spatially ordered in a mirror symmetric map with respect to S1 responses. Evoked signals in S2 and S1 are of similar amplitude and closely correlated at the single trial level. They confirm a short delay (~3 ms) between S1 and S2 early activation. In both S1 and S2 caudo-dorsal whiskers induce stronger responses than rostro-ventral ones. Finally, analysis of early C2-evoked responses indicates a faster activation of neighboring whisker representations in S2 relative to S1, probably due to the reduced size of the whisker map in S2.

Biphasic Impact of Prenatal Inflammation and Macrophage Depletion on the Wiring of Neocortical Inhibitory Circuits.

The etiology of neurodevelopmental disorders is linked to defects in parvalbumin (PV)-expressing cortical interneurons and to prenatal immune challenges. Mouse models of maternal immune activation (MIA) and microglia deficits increase the postnatal density of PV interneurons, raising the question of their functional integration. Here, we show that MIA and embryonic depletion of macrophages including microglia have a two-step impact on PV interneurons wiring onto their excitatory target neurons in the barrel cortex. In adults, both challenges reduced the inhibitory drive from PV interneurons, as reported in neurodevelopmental disorders. In juveniles, however, we found an increased density of PV neurons, an enhanced strength of unitary connections onto excitatory cells, and an aberrant horizontal inhibition with a reduced lateral propagation of sensory inputs in vivo. Our results provide a comprehensive framework for understanding the impact of prenatal immune challenges onto the developmental trajectory of inhibitory circuits that leads to pathological brain wiring.

Representation of tactile scenes in the rodent barrel cortex.

After half a century of research, the sensory features coded by neurons of the rodent barrel cortex remain poorly understood. Still, views of the sensory representation of whisker information are increasingly shifting from a labeled line representation of single-whisker deflections to a selectivity for specific elements of the complex statistics of the multi-whisker deflection patterns that take place during spontaneous rodent behavior - so called natural tactile scenes. Here we review the current knowledge regarding the coding of patterns of whisker stimuli by barrel cortex neurons, from responses to single-whisker deflections to the representation of complex tactile scenes. A number of multi-whisker tunings have already been identified, including center-surround feature extraction, angular tuning during edge-like multi-whisker deflections, and even tuning to specific statistical properties of the tactile scene such as the level of correlation across whiskers. However, a more general model of the representation of multi-whisker information in the barrel cortex is still missing. This is in part because of the lack of a human intuition regarding the perception emerging from a whisker system, but also because in contrast to other primary sensory cortices such as the visual cortex, the spatial feature selectivity of barrel cortex neurons rests on highly nonlinear interactions that remained hidden to classical receptive field approaches.

Supra-barrel Distribution of Directional Tuning for Global Motion in the Mouse Somatosensory Cortex.

Rodents explore their environment with an array of whiskers, inducing complex patterns of whisker deflections. Cortical neuronal networks can extract global properties of tactile scenes. In the primary somatosensory cortex, the information relative to the global direction of a spatiotemporal sequence of whisker deflections can be extracted at the single neuron level. To further understand how the cortical network integrates multi-whisker inputs, we imaged and recorded the mouse barrel cortex activity evoked by sequences of multi-whisker deflections generating global motions in different directions. A majority of barrel-related cortical columns show a direction preference for global motions with an overall preference for caudo-ventral directions. Responses to global motions being highly sublinear, the identity of the first deflected whiskers is highly salient but does not seem to determine the global direction preference. Our results further demonstrate that the global direction preference is spatially organized throughout the barrel cortex at a supra-columnar scale.

Review: How do spontaneous and sensory-evoked activities interact?

Twenty years ago, the seminal work of Grinvald et al. revolutionized the view cast on spontaneous cortical activity by showing how, instead of being a mere measure of noise, it profoundly impacts cortical responses to a sensory input and therefore could play a role in sensory processing. This paved the way for a number of studies on the interactions between spontaneous and sensory-evoked activities. Spontaneous activity has subsequently been found to be highly structured and to participate in high cognitive functions, such as influencing conscious perception in humans. However, its functional role remains poorly understood, and only a few speculations exist, from the maintenance of the cortical network to the internal representation of an a priori knowledge of the environment. Furthermore, elucidation of this functional role could stem from studying the opposite relationship between spontaneous and sensory-evoked activities, namely, how a sensory input influences subsequent internal activities. Indeed, this question has remained largely unexplored, but a recent study by the Grinvald laboratory shows that a brief sensory input largely dampens spontaneous rhythms, suggesting a more sophisticated view where some spontaneous rhythms might relate to sensory processing and some others not.

Spatially Structured Sparse Morphological Component Separation for voltage-sensitive dye optical imaging.

BACKGROUND: Voltage-sensitive dye optical imaging is a promising technique for studying in vivo neural assemblies dynamics where functional clustering can be visualized in the imaging plane. Its practical potential is however limited by many artifacts. NEW METHOD: We present a novel method, that we call "SMCS" (Spatially Structured Sparse Morphological Component Separation), to separate the relevant biological signal from noise and artifacts. It extends Generalized Linear Models (GLM) by using a set of convex non-smooth regularization priors adapted to the morphology of the sources and artifacts to capture. RESULTS: We make use of first order proximal splitting algorithms to solve the corresponding large scale optimization problem. We also propose an automatic parameters selection procedure based on statistical risk estimation methods. COMPARISON WITH EXISTING METHODS: We compare this method with blank subtraction and GLM methods on both synthetic and real data. It shows encouraging perspectives for the observation of complex cortical dynamics. CONCLUSIONS: This work shows how recent advances in source separation can be integrated into a biophysical model of VSDOI. Going beyond GLM methods is important to capture transient cortical events such as propagating waves.

Orofacial Neuropathic Pain Leads to a Hyporesponsive Barrel Cortex with Enhanced Structural Synaptic Plasticity.

Chronic pain is a long-lasting debilitating condition that is particularly difficult to treat due to the lack of identified underlying mechanisms. Although several key contributing processes have been described at the level of the spinal cord, very few studies have investigated the supraspinal mechanisms underlying chronic pain. Using a combination of approaches (cortical intrinsic imaging, immunohistochemical and behavioural analysis), our study aimed to decipher the nature of functional and structural changes in a mouse model of orofacial neuropathic pain, focusing on cortical areas involved in various pain components. Our results show that chronic neuropathic orofacial pain is associated with decreased haemodynamic responsiveness to whisker stimulation in the barrel field cortex. This reduced functional activation is likely due to the increased basal neuronal activity (measured indirectly using cFos and phospho-ERK immunoreactivity) observed in several cortical areas, including the contralateral barrel field, motor and cingulate cortices. In the same animals, immunohistochemical analysis of markers for active pre- or postsynaptic elements (Piccolo and phospho-Cofilin, respectively) revealed an increased immunofluorescence in deep cortical layers of the contralateral barrel field, motor and cingulate cortices. These results suggest that long-lasting orofacial neuropathic pain is associated with exacerbated neuronal activity and synaptic plasticity at the cortical level.

An automated workflow for the anatomo-functional mapping of the barrel cortex.

BACKGROUND: The rodent barrel cortex is a widely used model to study the cortical processing of tactile sensory information. It is notable by the cytoarchitecture of its layer IV, which contains distinguishable structural units called barrels that can be considered as anatomical landmarks of the functional columnar organization of the cerebral cortex. To study sensory integration in the barrel cortex it is therefore essential to map recorded functional data onto the underlying barrel topography, which can be reconstructed from the post hoc alignment of tangential brain slices stained for cytochrome oxidase. NEW METHOD: This article presents an automated workflow to perform the registration of histological slices of the barrel cortex followed by the 2-D reconstruction of the barrel map from the registered slices. The registration of two successive slices is obtained by computing a rigid transformation to align sets of detected blood vessel cross-sections. This is achieved by using a robust variant of the classical iterative closest point method. A single fused image of the barrel field is then generated by computing a nonlinear merging of the gradients from the registered images. COMPARISON WITH EXISTING METHODS: This novel anatomo-functional mapping tool leads to a substantial gain in time and precision compared to conventional manual methods. It provides a flexible interface for the user with only a few parameters to tune. CONCLUSIONS: We demonstrate here the usefulness of the method for voltage sensitive dye imaging of the mouse barrel cortex. The method could also benefit other experimental approaches and model species.

5-HT3 receptors mediate serotonergic fast synaptic excitation of neocortical vasoactive intestinal peptide/cholecystokinin interneurons.

Neocortical neurons expressing the serotonin 5-HT3 receptor (5-HT3R) were characterized in rat acute slices by using patch-clamp recordings combined with single-cell RT-PCR and histochemical labeling. The 5-HT3A receptor subunit was expressed selectively in a subset of GABAergic interneurons coexpressing cholecystokinin (CCK) and vasoactive intestinal peptide (VIP). The 5-HT3B subunit was never detected, indicating that 5-HT3Rs expressed by neocortical interneurons did not contain this subunit. In 5-HT3A-expressing VIP/CCK interneurons, serotonin induced fast membrane potential depolarizations by activating an inward current that was blocked by the selective 5-HT3R antagonist tropisetron. Furthermore, we observed close appositions between serotonergic fibers and the dendrites and somata of 5-HT3R-expressing neurons, suggestive of possible synaptic contacts. Indeed, in interneurons exhibiting rapid excitation by serotonin, local electrical stimulations evoked fast EPSCs of large amplitude that were blocked by tropisetron. Finally, 5-HT3R-expressing neurons were also excited by a nicotinic agonist, indicating that serotonergic and cholinergic fast synaptic transmission could converge onto VIP/CCK interneurons. Our results establish a clear correlation between the presence of the 5-HT3A receptor subunit in neocortical VIP/CCK GABAergic interneurons, its functional expression, and its synaptic activation by serotonergic afferent fibers from the brainstem raphe nuclei.

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.

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.

Straightforward synthesis of the near-infrared fluorescent voltage-sensitive dye RH1691 and analogues thereof.

A highly straightforward synthesis of the near-infrared voltage-sensitive dye RH1691 is reported featuring two sequential anionic additions of C-nucleophilic heterocycles on a cyanine. This convergent approach led to the synthesis of four new probes, which also exhibit fluorescence in the near-infrared region.