Transient demyelination causes long-term cognitive impairment, myelin alteration and network synchrony defects.

In the adult brain, activity-dependent myelin plasticity is required for proper learning and memory consolidation. Myelin loss, alteration, or even subtle structural modifications can therefore compromise the network activity, leading to functional impairment. In multiple sclerosis, spontaneous myelin repair process is possible, but it is heterogeneous among patients, sometimes leading to functional recovery, often more visible at the motor level than at the cognitive level. In cuprizone-treated mouse model, massive brain demyelination is followed by spontaneous and robust remyelination. However, reformed myelin, although functional, may not exhibit the same morphological characteristics as developmental myelin, which can have an impact on the activity of neural networks. In this context, we used the cuprizone-treated mouse model to analyze the structural, functional, and cognitive long-term effects of transient demyelination. Our results show that an episode of demyelination induces despite remyelination long-term cognitive impairment, such as deficits in spatial working memory, social memory, cognitive flexibility, and hyperactivity. These deficits were associated with a reduction in myelin content in the medial prefrontal cortex (mPFC) and hippocampus (HPC), as well as structural myelin modifications, suggesting that the remyelination process may be imperfect in these structures. In vivo electrophysiological recordings showed that the demyelination episode altered the synchronization of HPC-mPFC activity, which is crucial for memory processes. Altogether, our data indicate that the myelin repair process following transient demyelination does not allow the complete recovery of the initial myelin properties in cortical structures. These subtle modifications alter network features, leading to prolonged cognitive deficits in mice.

Inhomogeneous Magnetization Transfer (ihMT) imaging in the acute cuprizone mouse model of demyelination/remyelination.

BACKGROUND: To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology, and to assess the extent of tissue damage and repair from MRI data. METHODS: Acute demyelination by feeding 0.2% cuprizone for five weeks, followed by a four-week remyelination period was applied on genetically modified plp-GFP mice. Animals were scanned at different time points of the demyelination and remyelination phases of the cuprizone model using a multimodal MRI protocol, including ihMT T1D-filters, MPF (Macromolecular Proton Fraction) and R1 (longitudinal relaxation rate). For histology, plp-GFP (proteolipid protein - Green Fluorescent Protein) microscopy and LFB (Luxol Fast Blue) staining were employed as references for the myelin content. Comparison of MRI with histology was performed in the medial corpus callosum (mCC) and cerebral cortex (CTX) at two brain levels whereas ROI-wise and voxel-based analyses of the MRI metrics allowed investigating in vivo the spatial extent of myelin alterations. RESULTS: IhMT high-pass T1D-filters, targeted toward long T1D components, showed significant temporal variations in the mCC consistent with the effects induced by the cuprizone toxin. In addition, the corresponding signals correlated strongly and significantly with the myelin content assessed by GFP fluorescence and LFB staining over the demyelination and the remyelination phases. The signal of the band-pass T1D-filter, which isolates short T1D components, showed changes over time that were poorly correlated with histology, hence suggesting a sensitivity to pathological processes possibly not related to myelin. Although MPF was also highly correlated to histology, ihMT high-pass T1D-filters showed better capability to characterize the spatial-temporal patterns during the demyelination and remyelination phases of the acute cuprizone model (e.g., rostro-caudal gradient of demyelination in the mCC previously described in the literature). CONCLUSIONS: IhMT sequences selective for long T1D components are specific and sensitive in vivo markers of demyelination and remyelination and have successfully captured the spatially heterogeneous pattern of the demyelination and remyelination mechanisms in the cuprizone model. Interestingly, differences in signal variations between the ihMT high-pass and band-pass T1D-filter, suggest a sensitivity of the ihMT sequences targeted to short T1Ds to alterations other than those of myelin. Future studies will need to further address these differences by examining more closely the origin of the short T1D components and the variation of each T1D component in pathology.

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain.

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.

T1D -weighted ihMT imaging - Part II. Investigating the long- and short-T1D components correlation with myelin content. Comparison with R1 and the macromolecular proton fraction.

PURPOSE: To investigate the long- and short-T1D components correlation with myelin content using inhomogeneous magnetization transfer (ihMT) high-pass and band-pass T1D -filters and to compare ihMT, R1 , and the macromolecular proton fraction (MPF) for myelin specific imaging. METHODS: The 3D ihMT rapid gradient echo (ihMTRAGE) sequences with increasing switching times (Δt) were used to derive ihMT high-pass T1D -filters with increasing T1D cutoff values and an ihMT band-pass T1D -filter for components in the 100 µs to 1 ms range. 3D spoiled gradient echo quantitative MT (SPGR-qMT) protocols were used to derive R1 and MPF maps. The specificity of R1 , MPF, and ihMT T1D -filters was evaluated by comparison with two histological reference techniques for myelin imaging. RESULTS: The higher contribution of long-T1D s as compared to the short components as Δt got longer led to an increase in the specificity to myelination. In contrast, focusing on the signal originating from a narrow range of short-T1D s (< 1 ms) as isolated by the band-pass T1D -filter led to lower specificity. In addition, the significantly lower r2 correlation coefficient of the band-pass T1D -filter suggests that the origin of short-T1D components is mostly associated with non-myelin protons. Also, the important contribution of short-T1D s to the estimated MPF, explains its low specificity to myelination as compared to the ihMT high-pass T1D -filters. CONCLUSION: Long-T1D components imaging by means of ihMT high-pass T1D -filters is proposed as an MRI biomarker for myelin content. Future studies should enable the investigation of the sensitivity of ihMT T1D -filters for demyelinating processes.

T1D -weighted ihMT imaging - Part I. Isolation of long- and short-T1D components by T1D -filtering.

PURPOSE: To identify T1D -filtering methods, which can specifically isolate various ranges of T1D components as they may be sensitive to different microstructural properties. METHODS: Modified Bloch-Provotorov equations describing a bi-T1D component biophysical model were used to simulate the inhomogeneous magnetization transfer (ihMT) signal from ihMTRAGE sequences at high RF power and low duty-cycle with different switching time values for the dual saturation experiment: Δt = 0.0, 0.8, 1.6, and 3.2 ms. Simulations were compared with experimental signals on the brain gray and white matter tissues of healthy mice at 7T. RESULTS: The lengthening of Δt created ihMT high-pass T1D -filters, which efficiently eliminated the signal from T1D components shorter than 1 ms, while partially attenuating that of longer components (≥ 1 ms). Subtraction of ihMTR images obtained with Δt = 0.0 ms and Δt = 0.8 ms generated a new ihMT band-pass T1D -filter isolating short-T1D components in the 100-µs to 1-ms range. Simulated ihMTR values in central nervous system tissues were confirmed experimentally. CONCLUSION: Long- and short-T1D components were successfully isolated with high RF power and low duty-cycle ihMT filters in the healthy mouse brain. Future studies should investigate the various T1D -range microstructural correlations in in vivo tissues.

Endogenous neural stem cells modulate microglia and protect against demyelination.

In response to corpus callosum (CC) demyelination, subventricular zone-derived neural progenitors (SVZdNPs) are mobilized and generate new myelinating oligodendrocytes (OLG). Here, we examine the putative immunomodulatory properties of endogenous SVZdNPs during demyelination in the cuprizone model. SVZdNP density was higher in the lateral and rostral CC regions, and demyelination was inversely correlated with activated microglial density and pro-inflammatory cytokine levels. Single-cell RNA sequencing showed that CC areas with high levels of SVZdNP mobilization were enriched in a microglial cell subpopulation with an immunomodulatory signature. We propose MFGE8 (milk fat globule-epidermal growth factor-8) and ß3 integrin as a ligand/receptor pair involved in dialogue between SVZdNPs and microglia. Immature SVZdNPs mobilized to the demyelinated CC were found highly enriched in MFGE8, which promoted the phagocytosis of myelin debris in vitro. Overall, these results demonstrate that, in addition to their cell replacement capacity, endogenous progenitors have immunomodulatory properties, highlighting a new role for endogenous SVZdNPs in myelin regeneration.

Myelin Repair: From Animal Models to Humans.

It is widely thought that brain repair does not occur, but myelin regeneration provides clear evidence to the contrary. Spontaneous remyelination may occur after injury or in multiple sclerosis (MS). However, the efficiency of remyelination varies considerably between MS patients and between the lesions of each patient. Myelin repair is essential for optimal functional recovery, so a profound understanding of the cells and mechanisms involved in this process is required for the development of new therapeutic strategies. In this review, we describe how animal models and modern cell tracing and imaging methods have helped to identify the cell types involved in myelin regeneration. In addition to the oligodendrocyte progenitor cells identified in the 1990s as the principal source of remyelinating cells in the central nervous system (CNS), other cell populations, including subventricular zone-derived neural progenitors, Schwann cells, and even spared mature oligodendrocytes, have more recently emerged as potential contributors to CNS remyelination. We will also highlight the conditions known to limit endogenous repair, such as aging, chronic inflammation, and the production of extracellular matrix proteins, and the role of astrocytes and microglia in these processes. Finally, we will present the discrepancies between observations in humans and in rodents, discussing the relationship of findings in experimental models to myelin repair in humans. These considerations are particularly important from a therapeutic standpoint.

Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production.

Myelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS.

Promoting Myelin Repair through In Vivo Neuroblast Reprogramming.

Demyelination is frequently observed in a variety of CNS insults and neurodegenerative diseases. In rodents, adult neural stem cells can generate oligodendrocytes and participate to myelin repair. However, these cells mainly produce migratory neuroblasts that differentiate in the olfactory bulb. Here, we show that, in the demyelination context, a small subset of these neuroblasts can spontaneously convert into myelinating oligodendrocytes. Furthermore, we demonstrate that the contribution of neuroblasts to myelin repair can be improved by in vivo forced expression of two transcription factors: OLIG2 and SOX10. These factors promote directed fate conversion of endogenous subventricular zone neuroblasts into mature functional oligodendrocytes, leading to enhanced remyelination in a cuprizone-induced mouse model of demyelination. These findings highlight the unexpected plasticity of committed neuroblasts and provide proof of concept that they could be targeted for the treatment of demyelinated lesions in the adult brain.

Region and dynamic specificities of adult neural stem cells and oligodendrocyte precursors in myelin regeneration in the mouse brain.

Myelin regeneration can occur in the brain following demyelination. Parenchymal oligodendrocyte progenitors (pOPC) are known to play a crucial role in this process. Neural stem cells (NSC) residing in the ventricular-subventricular zone (V-SVZ) also have the ability to generate oligodendrocytes but their contribution to endogenous myelin repair was so far considered to be negligible. Here, we addressed the relative contribution of pOPC and V-SVZ-derived neural progenitors (SVZdNP) to remyelination in cuprizone mouse models of acute or chronic corpus callosum (CC) demyelination. Using genetic tracing, we uncover an unexpected massive and precocious recruitment of SVZdNP in the anterior CC after acute demyelination. These cells very quickly adopt an oligodendrocytic fate and robustly generate myelinating cells as efficiently as pOPC do. In more posterior areas of the CC, SVZdNP recruitment is less important whereas pOPC contribute more, underlining a regionalization in the mobilization of these two cell populations. Strikingly, in a chronic model when demyelination insult is sustained in time, SVZdNP minimally contribute to myelin repair, a failure associated with a depletion of NSC and a drastic drop of progenitor cell proliferation in V-SVZ. In this context, pOPC remain reactive, and become the main contributors to myelin regeneration. Altogether our results highlight a region and context-dependent contribution of SVZdNP to myelin repair that can equal pOPC. They also raise the question of a possible exhaustion of V-SVZ proliferation potential in chronic pathologies.

Oligodendrogenesis in the normal and pathological central nervous system.

Oligodendrocytes (OLGs) are generated late in development and myelination is thus a tardive event in the brain developmental process. It is however maintained whole life long at lower rate, and myelin sheath is crucial for proper signal transmission and neuronal survival. Unfortunately, OLGs present a high susceptibility to oxidative stress, thus demyelination often takes place secondary to diverse brain lesions or pathologies. OLGs can also be the target of immune attacks, leading to primary demyelination lesions. Following oligodendrocytic death, spontaneous remyelination may occur to a certain extent. In this review, we will mainly focus on the adult brain and on the two main sources of progenitor cells that contribute to oligodendrogenesis: parenchymal oligodendrocyte precursor cells (OPCs) and subventricular zone (SVZ)-derived progenitors. We will shortly come back on the main steps of oligodendrogenesis in the postnatal and adult brain, and summarize the key factors involved in the determination of oligodendrocytic fate. We will then shed light on the main causes of demyelination in the adult brain and present the animal models that have been developed to get insight on the demyelination/remyelination process. Finally, we will synthetize the results of studies searching for factors able to modulate spontaneous myelin repair.

Netrin 1 contributes to vascular remodeling in the subventricular zone and promotes progenitor emigration after demyelination.

Neural stem cells are maintained in the adult brain, sustaining structural and functional plasticity and to some extent participating in brain repair. A thorough understanding of the mechanisms and factors involved in endogenous stem/progenitor cell mobilization is a major challenge in the promotion of spontaneous brain repair. The main neural stem cell niche in the adult brain is the subventricular zone (SVZ). Following demyelination insults, SVZ-derived progenitors act in concert with oligodendrocyte precursors to repopulate the lesion and replace lost oligodendrocytes. Here, we showed robust vascular reactivity within the SVZ after focal demyelination of the corpus callosum in adult mice, together with a remarkable physical association between these vessels and neural progenitors exiting from their niche. Endogenous progenitor cell recruitment towards the lesion was significantly reduced by inhibiting post-lesional angiogenesis in the SVZ using anti-VEGF blocking antibody injections, suggesting a facilitating role of blood vessels for progenitor cell migration towards the lesion. We identified netrin 1 (NTN1) as a key factor upregulated within the SVZ after demyelination and involved in local angiogenesis and progenitor cell migration. Blocking NTN1 expression using a neutralizing antibody inhibited both lesion-induced vascular reactivity and progenitor cell recruitment at the lesion site. We propose a model in which SVZ progenitors respond to a demyelination lesion by NTN1 secretion that both directly promotes cell emigration and contributes to local angiogenesis, which in turn indirectly facilitates progenitor cell emigration from the niche.

Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain.

Ciliary neurotrophic factor (CNTF) has been shown to be expressed after brain lesions and in particular after demyelination. Here, we addressed the role of this cytokine in the regulation of neural progenitor migration in the adult rodent brain. Using an acute model of demyelination, we show that CNTF is strongly re-expressed after lesion and is involved in the postlesional mobilization of endogenous progenitors that participate in the myelin regenerative process. We show that CNTF controls the migration of subventricular zone (SVZ)-derived neural progenitors toward the demyelinated corpus callosum. Furthermore, an ectopic source of CNTF in adult healthy brains changes SVZ-derived neural progenitors' migratory behavior that migrate toward the source by activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. Using various in vitro assays (Boyden chambers, explants, and video time-lapse imaging), we demonstrate that CNTF controls the directed migration of SVZ-derived progenitors and oligodendrocyte precursors. Altogether, these results demonstrate that in addition to its neuroprotective activity and its role in progenitor survival and maturation, CNTF acts as a chemoattractant and participates in the recruitment of endogenous progenitors during myelin repair.

Olesoxime accelerates myelination and promotes repair in models of demyelination.

OBJECTIVE: Multiple sclerosis is a neurodegenerative disease characterized by episodes of immune attack of oligodendrocytes leading to demyelination and progressive functional deficit. One therapeutic strategy to address disease progression could consist in stimulating the spontaneous regenerative process observed in some patients. Myelin regeneration requires endogenous oligodendrocyte progenitor migration and activation of the myelination program at the lesion site. In this study, we have tested the ability of olesoxime, a neuroprotective and neuroregenerative agent, to promote remyelination in the rodent central nervous system in vivo. METHODS: The effect of olesoxime on oligodendrocyte progenitor cell (OPC) differentiation and myelin synthesis was tested directly in organotypic slice cultures and OPC-neuron cocultures. Using naive animals and different mouse models of demyelination, we morphologically and functionally assessed the effect of the compound on myelination in vivo. RESULTS: Olesoxime accelerated oligodendrocyte maturation and enhanced myelination in vitro and in vivo in naive animals during development and also in the adult brain without affecting oligodendrocyte survival or proliferation. In mouse models of demyelination and remyelination, olesoxime favored the repair process, promoting myelin formation with consequent functional improvement. INTERPRETATION: Our observations support the strategy of promoting oligodendrocyte maturation and myelin synthesis to enhance myelin repair and functional recovery. We also provide proof of concept that olesoxime could be useful for the treatment of demyelinating diseases.

Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain.

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.

A2B5 cells from human glioblastoma have cancer stem cell properties.

Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5(+) cells isolated from 11 human GBM. These cells display neurosphere-like, self-renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5(+)-derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5(+)-derived spheres revealed three distinct populations of cells: A2B5(+)/CD133(+), A2B5(+)/CD133(-) and A2B5(-)/CD133(-), with striking proportion differences among GBM. Both A2B5(+)/CD133(+) and A2B5(+)/CD133(-) cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein-cell lines that display--after serum induction--distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5(+) cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumor's behavior.

Cell migration in the normal and pathological postnatal mammalian brain.

In the developing brain, cell migration is a crucial process for structural organization, and is therefore highly regulated to allow the correct formation of complex networks, wiring neurons, and glia. In the early postnatal brain, late developmental processes such as the production and migration of astrocyte and oligodendrocyte progenitors still occur. Although the brain is completely formed and structured few weeks after birth, it maintains a degree of plasticity throughout life, including axonal remodeling, synaptogenesis, but also neural cell birth, migration and integration. The subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus are the two main neurogenic niches in the adult brain. Neural stem cells reside in these structures and produce progenitors that migrate toward their ultimate location: the olfactory bulb and granular cell layer of the DG respectively. The aim of this review is to synthesize the increasing information concerning the organization, regulation and function of cell migration in a mature brain. In a normal brain, proteins involved in cell-cell or cell-matrix interactions together with secreted proteins acting as chemoattractant or chemorepellant play key roles in the regulation of neural progenitor cell migration. In addition, recent data suggest that gliomas arise from the transformation of neural stem cells or progenitor cells and that glioma cell infiltration recapitulates key aspects of glial progenitor migration. Thus, we will consider glioma migration in the context of progenitor migration. Finally, many observations show that brain lesions and neurological diseases trigger neural stem/progenitor cell activation and migration toward altered structures. The factors involved in such cell migration/recruitment are just beginning to be understood. Inflammation which has long been considered as thoroughly disastrous for brain repair is now known to produce some positive effects on stem/progenitor cell recruitment via the regulation of growth factor signaling and the secretion of a number of chemoattractant cytokines. This knowledge is crucial for the development of new therapeutic strategies. One of these strategies could consist in increasing the mobilization of endogenous progenitor cells that could replace lost cells and improve functional recovery.

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum.

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.

Understanding the regulation and function of adult neurogenesis: contribution from an insect model, the house cricket.

Since the discovery of adult neurogenesis, a major issue is the role of newborn neurons and the function-dependent regulation of adult neurogenesis. We decided to use an animal model with a relatively simple brain to address these questions. In the adult cricket brain as in mammals, new neurons are produced throughout life. This neurogenesis occurs in the main integrative centers of the insect brain, the mushroom bodies (MBs), where the neuroblasts responsible for their formation persist after the imaginal molt. The rate of production of new neurons is controlled not only by internal cues such as morphogenetic hormones but also by external environmental cues. Adult crickets reared in an enriched sensory environment experienced an increase in neuroblast proliferation as compared with crickets reared in an impoverished environment. In addition, unilateral sensory deprivation led to reduced neurogenesis in the MB ipsilateral to the lesion. In search of a functional role for the new cells, we specifically ablated MB neuroblasts in young adults using brain-focused gamma ray irradiation. We developed a learning paradigm adapted to the cricket, which we call the "escape paradigm." Using this operant associative learning test, we showed that crickets lacking neurogenesis exhibited delayed learning and reduced memory retention of the task when olfactory cues were used. Our results suggest that environmental cues are able to influence adult neurogenesis and that, in turn, newly generated neurons participate in olfactory integration, optimizing learning abilities of the animal, and thus its adaptation to its environment. Nevertheless, odor learning in adult insects cannot always be attributed to newly born neurons because neurogenesis is completed earlier in development in many insect species. In addition, many of the irradiated crickets performed significantly better than chance on the operant learning task.