Characterization of a new mouse line triggering transient oligodendrocyte progenitor depletion.

Oligodendrocyte progenitor cells (OPC) are the main proliferative cells in the healthy adult brain. They produce new myelinating oligodendrocytes to ensure physiological myelin remodeling and regeneration after various pathological insults. Growing evidence suggests that OPC have other functions. Here, we aimed to develop an experimental model that allows the specific ablation of OPC at the adult stage to unravel possible new functions. We generated a transgenic mouse expressing a floxed human diphtheria toxin receptor under the control of the PDGFRa promoter, crossed with an Olig2Cre mouse to limit the recombination to the oligodendrocyte lineage in the central nervous system. We determined a diphtheria toxin dose to substantially decrease OPC density in the cortex and the corpus callosum without triggering side toxicity after a few daily injections. OPC density was normalized 7 days post-treatment, showing high repopulation capacity from few surviving OPC. We took advantage of this strong but transient depletion to show that OPC loss was associated with behavioral impairment, which was restored by OPC recovery, as well as disruption of the excitation/inhibition balance in the sensorimotor cortex, reinforcing the hypothesis of a neuromodulatory role of OPC in the adult brain.

Validating the sensitivity of inhomogeneous magnetization transfer (ihMT) MRI to myelin with fluorescence microscopy.

Inhomogeneous Magnetization Transfer (ihMT) is a development from the MT MRI technique. IhMT can be considered as a dipolar order relaxation time (T1D) weighted imaging modality whose signal has shown an enhanced selectivity for myelin-rich structures. However, a formal validation of the ihMT sensitivity relative to a gold standard myelin density measurement has not yet been reported. To address this need, we compared ihMT MRI with green fluorescence protein (GFP) microscopy, in a study performed on genetically-modified plp-GFP mice, considered as a reference technique for myelin-content assessment. Various ihMT protocols consisting of variable T1D-filtering and radiofrequency power temporal distributions, were used for comparison with fluorescence microscopy. Strong and significant linear relationships (r2 (0.87-0.96), p < 0.0001) were found between GFP and ihMT ratio signals across brain regions for all tested protocol variants. Conventional MT ratios showed weaker correlations (r2 (0.24-0.78), p ≤ 0.02) and a much larger signal fraction unrelated to myelin, hence corresponding to a much lower specificity for myelin. T1D-filtering reduced the ihMT signal fraction not attributed to myelin by almost twofold relative to zero filtering suggesting that at least half of the unrelated signal has a substantially shorter T1D than myelin. Overall, these results strongly support the sensitivity of ihMT to myelin content.

Olesoxime favors oligodendrocyte differentiation through a functional interplay between mitochondria and microtubules.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by episodes of immune attacks and oligodendrocyte death leading to demyelination and progressive functional deficits. New therapeutic strategies are needed to stimulate the spontaneous regenerative process observed in some patients. Spontaneous myelin repair relies on the mobilization and differentiation of endogenous oligodendrocyte progenitors at the lesion site. Olesoxime, a cholesterol-like compound, has been shown to favor oligodendrocyte maturation in culture and promote myelin regeneration in rodents. Here, we study the mode of action of this compound and show that it binds to oligodendrocyte mitochondria, leading to their hyperfilamentation. This is accompanied by a reduction of basal superoxide levels, and accumulation of End Binding Protein 1 (EB1) at growing ends of microtubules. In parallel, we demonstrate that Reactive Oxygen Species (ROS) scavengers also promote oligodendrocyte differentiation, together with increasing mitochondrial filamentation and EB1-dependent microtubule polymerization. Altogether, our data uncover the mechanisms by which olesoxime promotes oligodendrocyte maturation. They also reveal that a bidirectional relationship between mitochondria hyperfilamentation and ROS level modulation controls oligodendrocyte maturation. This study identifies new cellular mechanisms to target for the development of regenerative treatments for MS.

A role for nitric oxide in sensory-induced neurogenesis in an adult insect brain.

In the adult cricket, neurogenesis occurs in the mushroom bodies, the main integrative structures of the insect brain. Mushroom body neuroblast proliferation is modulated in response to environmental stimuli. However, the mechanisms underlying these effects remain unspecified. In the present study, we demonstrate that electrical stimulation of the antennal nerve mimics the effects of olfactory activation and increases mushroom body neurogenesis. The putative role of nitric oxide (NO) in this activity-regulated neurogenesis was then explored. In vivo and in vitro experiments demonstrate that NO synthase inhibition decreases, and NO donor application stimulates neuroblast proliferation. NADPH-d activity, anti-L-citrulline immunoreactivity, as well as in situ hybridization with a probe specific for Acheta NO synthase were used to localize NO-producing cells. Combining these three approaches we clearly establish that mushroom body interneurons synthesize NO. Furthermore, we demonstrate that experimental interventions known to upregulate neuroblast proliferation modulate NO production: rearing crickets in an enriched sensory environment induces an upregulation of Acheta NO synthase mRNA, and unilateral electrical stimulation of the antennal nerve results in increased L-citrulline immunoreactivity in the corresponding mushroom body. The present study demonstrates that neural activity modulates progenitor cell proliferation and regulates NO production in brain structures where neurogenesis occurs in the adult insect. Our results also demonstrate the stimulatory effect of NO on mushroom body neuroblast proliferation. Altogether, these data strongly suggest a key role for NO in environmentally induced neurogenesis.

Pregnenolone sulfate enhances neurogenesis and PSA-NCAM in young and aged hippocampus.

Age-dependent cognitive impairments have been correlated with functional and structural modifications in the hippocampal formation. In particular, the brain endogenous steroid pregnenolone-sulfate (Preg-S) is a cognitive enhancer whose hippocampal levels have been linked physiologically to cognitive performance in senescent animals. However, the mechanism of its actions remains unknown. Because neurogenesis is sensitive to hormonal influences, we examined the effect of Preg-S on neurogenesis, a novel form of plasticity, in young and old rats. We demonstrate that in vivo infusion of Preg-S stimulates neurogenesis and the expression of the polysialylated forms of NCAM, PSA-NCAM, in the dentate gyrus of 3- and 20-month-old rats. These influences on hippocampal plasticity are mediated by the modulation of the gamma-aminobutyric acid receptor complex A (GABA(A)) receptors present on hippocampal neuroblasts. In vitro, Preg-S stimulates the division of adult-derived spheres suggesting a direct influence on progenitors. These data provide evidence that neurosteroids represent one of the local secreted signals controlling hippocampal neurogenesis. Thus, therapies which stimulate neurosteroidogenesis could preserve hippocampal plasticity and prevent the appearance of age-related cognitive disturbances.

Biochemical and histological changes in the brain of the cricket Nemobius sylvestris infected by the manipulative parasite Paragordius tricuspidatus (Nematomorpha).

Hairworms (nematomorpha) alter the behaviour of their insect hosts, making them commit 'suicide' by jumping into an aquatic environment required by the adult parasite for the continuation of its life cycle. To explore the physiological and neuronal basis of this behavioural manipulation, we first performed a biochemical study to quantify different neurotransmitters or neuromodulators (monoamines and amino acids) in the brain of crickets (Nemobius sylvestris) uninfected and infected by the hairworm Paragordius tricuspidatus. We also analysed several polyamines and amino-acids having no known neuromodulatory function. The presence/absence of the parasite explained the largest part of the variation in compound concentrations, with infected individuals displaying on average lower concentrations than uninfected individuals. However, for three amino acids (taurine, valine and tyrosine), a significant part of the variation was also correlated with the manipulative process. In order to compare neurogenesis between infected and uninfected crickets, we also performed a histological study on mushroom bodies in the cricket's brain. The mitotic index exhibited a two-fold increase in infected crickets as compared with uninfected crickets. This is the first study to document changes in the brain of insects infected by nematomorphs.

Short- and long-chain natural polyamines play specific roles in adult cricket neuroblast proliferation and neuron differentiation in vitro.

In the house cricket (Acheta domesticus) mushroom bodies, neurogenesis still occurs during adulthood. Using in vitro approaches, the respective roles of natural polyamines in neurogenesis were examined. Mushroom body neuroblast proliferation was assayed in organotypic culture using 5-bromo, 2'-deoxyuridine labeling. The number of labeled cells was significantly increased when putrescine was added to culture medium, whereas spermidine and spermine supplementation did not alter cell proliferation. Conversely, in vitro morphometric studies on mushroom body neurons cultured in a defined medium showed that putrescine addition failed to alter any morphological character of these interneurons, whereas addition of the long-chain polyamines, spermidine and spermine, stimulated neuron differentiation. These two polyamines significantly increased total neurite length; moreover, spermidine-treated cells exhibited more branches than the controls. The present data demonstrate that putrescine has a mitogenic effect on mushroom body neuronal precursors, and that spermidine and spermine, which failed to induce neuroblast proliferation, act on neuronal differentiation, inducing neurite outgrowth. Our results indicate that short- and long-chain polyamines play specific roles during neurogenesis, and provide a basis for further studies on neuronal precursor proliferation and differentiation.

Influence of environmental stimulation on neurogenesis in the adult insect brain.

Mushroom bodies are the main integrative structures of insect brain. They receive sensory information from the eyes, the palps, and the antennae. In the house cricket, Acheta domesticus, a cluster of mushroom body neuroblasts keeps producing new interneurons during an insect's life span. The aim of the present work is to study the impact of environmental stimuli on mushroom body neurogenesis during adulthood. Crickets were reared either in an enriched environment, where they received complex environmental and congeneric stimulations or isolated in small cages and deprived of most visual, auditory, and olfactory stimuli. They then were injected with a S-phase marker, 5-bromo, 2'-deoxyuridine (BrdU) and sacrificed at different periods of their life. Neurogenesis and cell survival were estimated by counting the number of BrdU-labeled cells in the mushroom bodies. Environmentally enriched crickets were found to have an increased number of newborn cells in their mushroom bodies compared with crickets housed in cages with an impoverished environment. This effect of external factors on neurogenesis seems to be limited to the beginning of imaginal life. Furthermore, no cell loss could be detected among the newborn neurons in either environmental situation, suggesting that cell survival was not affected by the quality of the environment. Considering vertebrate studies which showed that enriched environment increases hippocampal cell survival and improves animal performances in spatial learning tests, we suggest that the increased number of interneurons produced in an integrative brain structure after exposure to enriched environment could contribute to adaptive behavioral performances in adult insects.

Dual effect of ecdysone on adult cricket mushroom bodies.

Mushroom bodies, which are the main integrative centre for insect sensorial information, play a critical role in associative olfactory learning and memory. This paired brain structure contains interneurons grouped in a cortex, sending their axons into organized neuropiles. In the house cricket (Acheta domesticus) brain, persistent neuroblasts proliferate throughout adult life. Juvenile hormone (JH) has been shown to stimulate this proliferation [Cayre, M., Strambi, C. & Strambi, A. (1994) Nature, 368, 57-59]. In the present study, the effect of morphogenetic hormones on mushroom body cells maintained in primary culture was examined. Whereas JH did not significantly affect neurite growth, ecdysone significantly stimulated neurite elongation. Moreover, ecdysone also acted on neuroblast proliferation, as demonstrated by the reduced number of cells labelled with 5-bromodeoxyuridine following ecdysone application. Heterospecific antibodies raised against ecdysone receptor protein and ultraspiracle protein, the two heterodimers of ecdysteroid receptors, showed positive immunoreactivity in nervous tissue extracts and in nuclei of mushroom body cells, indicating the occurrence of putative ecdysteroid receptors in cricket mushroom body cells. These data indicate a dual role for ecdysone in adult cricket mushroom bodies: this hormone inhibits neuroblast proliferation and stimulates interneuron differentiation. These results suggest that a constant remodelling of mushroom body structure could result from physiological changes in hormone titres during adult life.

Fate of neuroblast progeny during postembryonic development of mushroom bodies in the house cricket, Acheta domesticus.

Mushroom bodies represent the main sensory integrative center of the insect brain and probably play a major role in the adaptation of behavioral responses to the environment. Taking into account the continuous neurogenesis of cricket mushroom bodies, we investigated ontogenesis of this brain structure. Using BrdU labeling, we examined the fate of neuroblast progeny during the postembryonic development. Preimaginal Kenyon cells survived throughout larval and imaginal moults and persisted during adulthood. Our results indicate that the location of labelled Kenyon cells in the cortex of the adult cricket mainly depends upon the period when they were produced during development. The present data demonstrate that cricket mushroom bodies grow from the inside out and that, at any developmental stage, the center of the cortex contains the youngest Kenyon cells. This study also allowed us to observe the occurrence of quiescent neuroblasts. Kenyon cell death during postembryonic and adult life seems to be reduced. Although preimaginal Kenyon cells largely contribute to adult mushroom body structure, a permanent remodeling of the mushroom body occurs throughout the whole insect life due to the persistence of neurogenesis in the house cricket. Further studies are needed to understand the functional significance of these findings.

Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine. The ACh response was insensitive to atropine (50 microM) but was reduced by mecamylamine (50 microM) and alpha-bungarotoxin (alpha-bgt, 10 microM). ACh-induced inward ion currents (n = 28, EACh approximately 0 mV) were also blocked by 1 microM mecamylamine and by 1 microM alpha-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional alpha-bgt-sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. gamma-Aminobutyric acid (GABA, 100 microM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 microM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30-100 microM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to -glutamate (100 microM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 microM) -glutamate with rapidly desensitizing, inward currents that reversed at approximately -30 mV. Dopamine (100 microM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 microM) in approximately 54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).

Adult insect mushroom body neurons in primary culture: cell morphology and characterization of potassium channels.

The dissociation and maintenance in culture of cells derived from the mushroom bodies of adult crickets (Acheta domesticus) are described. This primary culture was developed in order to investigate maturation and differentiation of mushroom-body cells including Kenyon cells, the major intrinsic interneurons of mushroom bodies, which have been shown to be involved in learning and memory in insects. Three distinct cell types were observed, all identified as neural cells on the basis of their size, morphology and immunocytochemical staining with horseradish peroxidase. These cells appear to correspond to the three cell types observed in vivo: Kenyon cells, ganglion mother cells and neuroblasts. Some cells showed neurite growth, usually with long unipolar processes, occasionally with either bipolar or, more rarely, multipolar processes. Neuronal cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell, patch-clamp electrophysiological recordings. Depolarization of the cell under voltage-clamp resulted in at least two types of outwardly directed potassium currents: a delayed rectifier-type of current that was sensitive to tetraethylammonium, and a cadmium-sensitive current with rapid inactivation. Neither type of current was affected by quinidine, a blocker of potassium currents recorded from pupal honeybee Kenyon cells. Other ionic currents, which have yet to be characterized, were also observed.

Immunocytochemical mapping of an RDL-like GABA receptor subunit and of GABA in brain structures related to learning and memory in the cricket Acheta domesticus.

The distribution of putative RDL-like GABA receptors and of gamma-aminobutyric acid (GABA) in the brain of the adult house cricket Acheta domesticus was studied using specific antisera. Special attention was given to brain structures known to be related to learning and memory. The main immunostaining for the RDL-like GABA receptor was observed in mushroom bodies, in particular the upper part of mushroom body peduncle and the two arms of the posterior calyx. Weaker immunostaining was detected in the distal part of the peduncle and in the alpha and beta lobes. The dorso- and ventrolateral protocerebrum neuropils appeared rich in RDL-like GABA receptors. Staining was also detected in the glomeruli of the antennal lobe, as well as in the ellipsoid body of the central complex. Many neurons clustered in groups exhibit GABA-like immunoreactivity. Tracts that were strongly immunostained innervated both the calyces and the lobes of mushroom bodies. The glomeruli of the antennal lobe, the ellipsoid body, as well as neuropils of the dorso- and ventrolateral protocerebrum were also rich in GABA-like immunoreactivity. The data demonstrated a good correlation between the distribution of the GABA-like and of the RDL-like GABA receptor immunoreactivity. The prominent distribution of RDL-like GABA receptor subunits, in particular areas of mushroom bodies and antennal lobes, underlines the importance of inhibitory signals in information processing in these major integrative centers of the insect brain.

Specific requirement of putrescine for the mitogenic action of juvenile hormone on adult insect neuroblasts.

Persistent neurogenesis in an adult insect brain was recently shown to be stimulated by juvenile hormone (JH). This morphogenetic hormone was also shown to act on polyamine biosynthesis. To analyze the possible involvement of polyamines in the neurogenic action of JH, two series of experiments were carried out with adult female crickets, Acheta domesticus: (i) inhibition of the first key enzyme in polyamine biosynthesis, ornithine decarboxylase, with alpha-difluoromethylornithine (alpha-DFMO), and examination of the effects of this treatment on the neuroblast proliferation response to JH; and (ii) examination of the effects of putrescine supplementation on the mitotic index of JH-deprived and alpha-DFMO-treated females. In control females, alpha-DFMO treatment, as well as JH deprivation, greatly reduced neuroblast proliferation. Putrescine supplementation in alpha-DFMO-treated insects overcame the effects of alpha-DFMO, and allowed for detection of putrescine in the neural tissue and stimulation of brain neurogenesis. In JH-deprived females, alpha-DFMO treatment completely prevented the stimulatory action of JH on neuroblast proliferation and on brain putrescine levels. By contrast, putrescine feeding of JH-deprived animals was able to mimic the stimulatory effect of JH: brain putrescine levels increased and neuroblast proliferation was restored. To our knowledge, this report demonstrates for the first time that in vivo administration of putrescine can mimic the effects of a morphogenetic hormone on adult neuroblast proliferation, and shows the importance of polyamines, especially putrescine, in the transduction of JH message in neural tissue.

Inhibition of polyamine biosynthesis alters oviposition behavior in female crickets.

The role of polyamines in the expression of cricket oviposition, a juvenile hormone-dependent behavior, was investigated using a specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (alpha-DFMO). The fat body of treated female house crickets (Acheta domesticus) did not show any putrescine and presented reduced levels of spermidine, whereas spermine titres were significantly enhanced. In nervous tissue, alpha-DFMO did not affect spermine titres but induced a severe drop in spermidine levels. In polyamine depleted females, the expression of egg-laying behavior was delayed and was expressed less frequently compared with controls. As drug treatment did not seem to affect juvenile hormone titres, the data suggest that juvenile hormone might act on behavior by way of polyamine metabolism. These results support the view that, in insects, as in vertebrates, the ornithine decarboxylase-polyamine system is involved in the maturation of complex behaviors.

Neurogenesis in adult insect mushroom bodies.

The occurrence of neurogenesis in mushroom bodies of adult insects belonging to several orthopteroid and coleopteran families is described. Using injections of 5-bromo, T2'-deoxyuridine, we showed that neuroblasts, which are progenitors of Kenyon cells during preimaginal instars, continue to divide in adult Acheta domesticus. Their progeny constitute a central column in mushroom body cortices of 3-week-old females. Other Gryllidae, Gryllus bimaculatus and Gryllomorpha dalmatina, show the same pattern of neuroblast activity and migration of their progeny. Immunocytochemical staining of glial cells failed to reveal any immunoreactivity, either in proliferating regions or in the resulting cells. In another orthopteran, Locusta migratoria, discrete clusters of cells, located dorsolateral to the Kenyon cells, incorporated 5-bromo, 2'-deoxyuridine, but we could not detect any neuronal progeny migrating to the mushroom body cortices. These cells were strongly labeled with an antiglial antibody, indicating that the replicating cells are glioblasts rather than neuroblasts. In Periplaneta americana (Dictyoptera), cells replicating their DNA were similarly shown to immunoreact with glial antibodies. In contrast, three coleopterans (Tenebrio molitor, Zophobas species, Harmonia axyridis) have two large neuroblasts located in the middle of the mushroom body cortices. These produce cells which migrate within the group of Kenyon cells, their nuclei having the same shape and size as those of surrounding Kenyon cells. In adult insects, neurogenesis in mushroom bodies occurs in Gryllidae and several coleopteran families, but could not be demonstrated in Dictyoptera and Acrididae. Its occurrence and distribution raise the issue of unexpected plasticity in the adult insect brain.