Capturing Cytoskeleton-Based Agitation of The Mouse Oocyte Nucleus Across Spatial Scales.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

Dynamic local mRNA localization and translation occurs during the postnatal molecular maturation of perivascular astrocytic processes.

Astrocytes are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe the brain's blood vessels. PvAPs are equipped with an enriched molecular repertoire that sustains astrocytic regulatory functions at the vascular interface. In the mouse, PvAP development starts after birth and is essentially complete by postnatal day (P) 15. Progressive molecular maturation also occurs over this period, with the acquisition of proteins enriched in PvAPs. The mechanisms controlling the development and molecular maturation of PvAPs have not been extensively characterized. We reported previously that mRNAs are distributed unequally in mature PvAPs and are locally translated. Since dynamic mRNA localization and local translation influence the cell's polarity, we hypothesized that they might sustain the postnatal maturation of PvAPs. Here, we used a combination of molecular biology and imaging approaches to demonstrate that the development of PvAPs is accompanied by the transport of mRNA and polysomal mRNA into PvAPs, the development of a rough endoplasmic reticulum (RER) network and Golgi cisternae, and local translation. By focusing on genes and proteins that are selectively or specifically expressed in astrocytes, we characterized the developmental profile of mRNAs, polysomal mRNAs and proteins in PvAPs from P5 to P60. We found that some polysomal mRNAs polarized progressively towards the PvAPs. Lastly, we found that expression and localization of mRNAs in developing PvAPs is perturbed in a mouse model of megalencephalic leukoencephalopathy with subcortical cysts. Our results indicate that dynamic mRNA localization and local translation influence the postnatal maturation of PvAPs.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Physiopathological changes of ferritin mRNA density and distribution in hippocampal astrocytes in the mouse brain.

Astrocytes are thought to play a crucial role in brain iron homeostasis. How they accomplish this regulation in vivo is unclear. In a recent transcriptomic analysis, we showed that polysomal Ftl1 and Fth1 mRNAs, encoding the ferritin light (Ftl) and heavy (Fth) chains that assemble into ferritin, a critical complex for iron storage and reduction, are enriched in perisynaptic astrocytic processes as compared to astrocytic soma. These data suggested that ferritin translation plays a specific role at the perisynaptic astrocytic interface and is tighly regulated by local translation. Here, we used our recently described AstroDot 3D in situ methodology to study the density and localization of ferritin mRNAs in astrocytes in the hippocampus in three different contexts in which local or systemic iron overload has been documented: aging, the hepcidin knock-out mouse model of hemochromatosis and the APP/PS1dE9 mouse model of Alzheimer's disease (AD). Our results showed that in wild type mice, Fth1 mRNA density was higher than Ftl1 and that both mRNAs were mostly distributed in astrocyte fine processes. Aging and absence of hepcidin caused an increased Fth1/Ftl1 ratio in astrocytes and in the case of aging, led to a redistribution of Fth1 mRNAs in astrocytic fine processes. In contrast, in AD mice, we observed a lower Fth1/Ftl1 ratio. Fth1 mRNAs became more somatic and Ftl1 mRNAs redistributed in large processes of astrocytes proximal to Amyloid beta (Aß) deposits. Hence, we propose that regulation of ferritin mRNA density and distribution in astrocytes contribute to iron homeostasis in physiology and pathophysiology.

In mice and humans, brain microvascular contractility matures postnatally.

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Angiopoietin-like 4-Induced 3D Capillary Morphogenesis Correlates to Stabilization of Endothelial Adherens Junctions and Restriction of VEGF-Induced Sprouting.

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell-cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.

Perturbed DNA methylation by Gadd45b induces chromatin disorganization, DNA strand breaks and dopaminergic neuron death.

Age is a major risk factor for neurodegenerative diseases like Parkinson's disease, but few studies have explored the contribution of key hallmarks of aging, namely DNA methylation changes and heterochromatin destructuration, in the neurodegenerative process. Here, we investigated the consequences of viral overexpression of Gadd45b, a multifactorial protein involved in DNA demethylation, in the mouse midbrain. Gadd45b overexpression induced global and stable changes in DNA methylation, particularly in introns of genes related to neuronal functions, as well as on LINE-1 transposable elements. This was paralleled by disorganized heterochromatin, increased DNA damage, and vulnerability to oxidative stress. LINE-1 de-repression, a potential source of DNA damage, preceded Gadd45b-induced neurodegeneration, whereas prolonged Gadd45b expression deregulated expression of genes related to heterochromatin maintenance, DNA methylation, or Parkinson's disease. Our data indicates that aging-related alterations contribute to dopaminergic neuron degeneration with potential implications for Parkinson's disease.

Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning.

Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. We study hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain the machinery for translation. Using a refined immunoprecipitation technique, we characterize the entire pool of ribosome-bound mRNAs in PAPs and compare it with the one expressed in the whole astrocyte. We find that a specific pool of mRNAs is highly polarized at the synaptic interface. These transcripts encode an unexpected molecular repertoire, composed of proteins involved in iron homeostasis, translation, cell cycle, and cytoskeleton. Remarkably, we observe alterations in global RNA distribution and ribosome-bound status of some PAP-enriched transcripts after fear conditioning, indicating the role of astrocytic local translation in memory and learning.

AstroDot - a new method for studying the spatial distribution of mRNA in astrocytes.

Astrocytes are morphologically complex and use local translation to regulate distal functions. To study the distribution of mRNA in astrocytes, we combined mRNA detection via in situ hybridization with immunostaining of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). mRNAs at the level of GFAP-immunolabelled astrocyte somata, and large and fine processes were analysed using AstroDot, an ImageJ plug-in and the R package AstroStat. Taking the characterization of mRNAs encoding GFAP-α and GFAP-δ isoforms as a proof of concept, we showed that they mainly localized on GFAP processes. In the APPswe/PS1dE9 mouse model of Alzheimer's disease, the density and distribution of both α and δ forms of Gfap mRNA changed as a function of the region of the hippocampus and the astrocyte's proximity to amyloid plaques. To validate our method, we confirmed that the ubiquitous Rpl4 (large subunit ribosomal protein 4) mRNA was present in astrocyte processes as well as in microglia processes immunolabelled for ionized calcium binding adaptor molecule 1 (Iba1; also known as IAF1). In summary, this novel set of tools allows the characterization of mRNA distribution in astrocytes and microglia in physiological or pathological settings.

Glucagon-like Peptide 1 Receptor Agonists, Diabetic Retinopathy and Angiogenesis: The AngioSafe Type 2 Diabetes Study.

AIMS: Recent trials provide conflicting results on the association between glucagon-like peptide 1 receptor agonists (GLP-1RA) and diabetic retinopathy (DR). The aim of the AngioSafe type 2 diabetes (T2D) study was to determine the role of GLP-1RA in angiogenesis using clinical and preclinical models. METHODS: We performed two studies in humans. In study 1, we investigated the effect of GLP-1RA exposure from T2D diagnosis on the severity of DR, as diagnosed with retinal imaging (fundus photography). In study 2, a randomized 4-week trial, we assessed the effect of liraglutide on circulating hematopoietic progenitor cells (HPCs), and angio-miRNAs.We then studied the experimental effect of Exendin-4, on key steps of angiogenesis: in vitro on human endothelial cell proliferation, survival and three-dimensional vascular morphogenesis; and in vivo on ischemia-induced neovascularization of the retina in mice. RESULTS: In the cohort of 3154 T2D patients, 10% displayed severe DR. In multivariate analysis, sex, disease duration, glycated hemoglobin (HbA1c), micro- and macroangiopathy, insulin therapy and hypertension remained strongly associated with severe DR, while no association was found with GLP-1RA exposure (o 1.139 [0.800-1.622], P = .47). We further showed no effect of liraglutide on HPCs, and angio-miRNAs. In vitro, we demonstrated that exendin-4 had no effect on proliferation and survival of human endothelial cells, no effect on total length and number of capillaries. Finally, in vivo, we showed that exendin-4 did not exert any negative effect on retinal neovascularization. CONCLUSIONS: The AngioSafe T2D studies provide experimental and clinical data confirming no effect of GLP-1RA on angiogenesis and no association between GLP-1 exposure and severe DR.

Active Fluctuations of the Nuclear Envelope Shape the Transcriptional Dynamics in Oocytes.

Nucleus position in cells can act as a developmental cue. Mammalian oocytes position their nucleus centrally using an F-actin-mediated pressure gradient. The biological significance of nucleus centering in mammalian oocytes being unknown, we sought to assess the F-actin pressure gradient effect on the nucleus. We addressed this using a dedicated computational 3D imaging approach, biophysical analyses, and a nucleus repositioning assay in mouse oocytes mutant for cytoplasmic F-actin. We found that the cytoplasmic activity, in charge of nucleus centering, shaped the nucleus while promoting nuclear envelope fluctuations and chromatin motion. Off-centered nuclei in F-actin mutant oocytes were misshaped with immobile chromatin and modulated gene expression. Restoration of F-actin in mutant oocytes rescued nucleus architecture fully and gene expression partially. Thus, the F-actin-mediated pressure gradient also modulates nucleus dynamics in oocytes. Moreover, this study supports a mechano-transduction model whereby cytoplasmic microfilaments could modulate oocyte transcriptome, essential for subsequent embryo development.

Motor axon navigation relies on Fidgetin-like 1-driven microtubule plus end dynamics.

During neural circuit assembly, extrinsic signals are integrated into changes in growth cone (GC) cytoskeleton underlying axon guidance decisions. Microtubules (MTs) were shown to play an instructive role in GC steering. However, the numerous actors required for MT remodeling during axon navigation and their precise mode of action are far from being deciphered. Using loss- and gain-of-function analyses during zebrafish development, we identify in this study the meiotic clade adenosine triphosphatase Fidgetin-like 1 (Fignl1) as a key GC-enriched MT-interacting protein in motor circuit wiring and larval locomotion. We show that Fignl1 controls GC morphology and behavior at intermediate targets by regulating MT plus end dynamics and growth directionality. We further reveal that alternative translation of Fignl1 transcript is a sophisticated mechanism modulating MT dynamics: a full-length isoform regulates MT plus end-tracking protein binding at plus ends, whereas shorter isoforms promote their depolymerization beneath the cell cortex. Our study thus pinpoints Fignl1 as a multifaceted key player in MT remodeling underlying motor circuit connectivity.

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment.

Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.

Organization of the Anterior Limb of the Internal Capsule in the Rat.

Dysfunction of the orbitofrontal (OFC) and anterior cingulate (ACC) cortices has been linked with several psychiatric disorders, including obsessive-compulsive disorder, major depressive disorder, posttraumatic stress disorder, and addiction. These conditions are also associated with abnormalities in the anterior limb of the internal capsule, the white matter (WM) bundle carrying ascending and descending fibers from the OFC and ACC. Furthermore, deep-brain stimulation (DBS) for psychiatric disorders targets these fibers. Experiments in rats provide essential information on the mechanisms of normal and abnormal brain anatomy, including WM composition and perturbations. However, whereas descending prefrontal cortex (PFC) fibers in primates form a well defined and topographic anterior limb of the internal capsule, the specific locations and organization of these fibers in rats is unknown. We address this gap by analyzing descending fibers from injections of an anterograde tracer in the rat ACC and OFC. Our results show that the descending PFC fibers in the rat form WM fascicles embedded within the striatum. These bundles are arranged topographically and contain projections, not only to the striatum, but also to the thalamus and brainstem. They can therefore be viewed as the rat homolog of the primate anterior limb of the internal capsule. Furthermore, mapping these projections allows us to identify the fibers likely to be affected by experimental manipulations of the striatum and the anterior limb of the internal capsule. These results are therefore essential for translating abnormalities of human WM and effects of DBS to rodent models.SIGNIFICANCE STATEMENT Psychiatric diseases are linked to abnormalities in specific white matter (WM) pathways, and the efficacy of deep-brain stimulation relies upon activation of WM. Experiments in rodents are necessary for studying the mechanisms of brain function. However, the translation of results between primates and rodents is hindered by the fact that the organization of descending WM in rodents is poorly understood. This is especially relevant for the prefrontal cortex, abnormal connectivity of which is central to psychiatric disorders. We address this gap by studying the organization of descending rodent prefrontal pathways. These fibers course through a subcortical structure, the striatum, and share important organization principles with primate WM. These results allow us to model primate WM effectively in the rodent.

Active diffusion positions the nucleus in mouse oocytes.

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Astrocytic and vascular remodeling in the injured adult rat spinal cord after chondroitinase ABC treatment.

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPG) is a primary cause for the failure of axons to regenerate after spinal cord injury (SCI), and the beneficial effect of their degradation by chondroitinase ABC (ChABC) is widely documented. Little is known, however, about the effect of ChABC treatment on astrogliosis and revascularization, two important factors influencing axon regrowth. This was investigated in the present study. Immediately after a spinal cord hemisection at thoracic level 8-9, we injected ChABC intrathecally at the sacral level, repeated three times until 10 days post-injury. Our results show an effective cleavage of CSPG glycosaminoglycan chains and stimulation of axonal remodeling within the injury site, accompanied by an extended period of astrocyte remodeling (up to 4 weeks). Interestingly, ChABC treatment favored an orientation of astrocytic processes directed toward the injury, in close association with axons at the lesion entry zone, suggesting a correlation between axon and astrocyte remodeling. Further, during the first weeks post-injury, ChABC treatment affected the morphology of laminin-positive blood vessel basement membranes and vessel-independent laminin deposits: hypertrophied blood vessels with detached or duplicated basement membrane were more numerous than in lesioned untreated animals. In contrast, at later time points, laminin expression increased and became more directly associated with newly formed blood vessels, the size of which tended to be closer to that found in intact tissue. Our data reinforce the idea that ChABC injection in combination with other synergistic treatments is a promising therapeutic strategy for SCI repair.

Spatial distribution of D1R- and D2R-expressing medium-sized spiny neurons differs along the rostro-caudal axis of the mouse dorsal striatum.

The striatum projection neurons are striatonigral and striatopallidal medium-sized spiny neurons (MSNs) that preferentially express D1 (D1R) and D2 (D2R) dopamine receptors, respectively. It is generally assumed that these neurons are physically intermingled, without cytoarchitectural organization although this has not been tested. To address this question we used BAC transgenic mice expressing enhanced green fluorescence (EGFP) under the control of Drd1a or Drd2 promoter and spatial point pattern statistics. We demonstrate that D1R- and D2R-expressing MSNs are randomly distributed in most of the dorsal striatum, whereas a specific region in the caudal striatum, adjacent to the GPe, lacks neurons expressing markers for indirect pathway neurons. This area comprises almost exclusively D1R-expressing MSNs. These neurons receive excitatory inputs from the primary auditory cortex and the medial geniculate thalamic nucleus and a rich dopamine innervation. This area contains cholinergic and GABAergic interneurons but apparently no D2R/A2aR modulation because no fluorescence was detected in the neuropil of Drd2-EGFP or Drd2-Cre, and Adora-Cre BAC transgenic mice crossed with reporter mice. This striatal area that expresses calbindin D28k, VGluT1 and 2, is poor in µ opiate receptors and preproenkephalin. Altogether, the differences observed in D1R-MSNs, D2R-MSNs, and interneurons densities, as well as the anatomical segregation of D1R- and D2R/A2aR-expressing MSNs suggest that there are regional differences in the organization of the striatum.

The rat prefrontostriatal system analyzed in 3D: evidence for multiple interacting functional units.

Previous studies in monkeys disclosed a specific arrangement of corticostriatal projections. Prefrontal and premotor areas form dense projection fields surrounded by diffuse terminal areas extending outside the densely innervated region and overlapping with projections from other areas. In this study, the mode of prefrontostriatal innervation was analyzed in rats using a 3D approach. Following injections of tracers in defined cortical areas, 3D maps from individual cases were elaborated and combined into a global 3D map allowing us to define putative overlaps between projection territories. In addition to providing a detailed 3D mapping of the topographic representation of prefrontal cortical areas in the rat striatum, the results stress important similarities between the rodent and primate prefrontostriatal projections. They share the dual pattern of focal and diffuse corticostriatal projections. Moreover, besides segregated projections consistent with parallel processing, the interweaving of projection territories establishes specific patterns of overlaps spatially organized along the dorsoventral, mediolateral, and anteroposterior striatal axis. In particular, the extensive striatal projection fields from the prelimbic and anterior cingulate areas, which partly overlap the terminal fields from medial, orbital, and lateral prefrontal cortical areas, provide putative domains of convergence for integration between reward, cognitive, and motor processes.

Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice.

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.