Cells Of External Granular Layer Research Articles

Anti-NMDA-Receptor Encephalitis in a Patient with Ovarian Teratoma, Harboring Brain Histology of Varying Developmental Stages and Regions with Chronic Inflammation

Abstract Introduction/Objective Anti-NMDA-receptor encephalitis is a subacute, autoimmune disorder thought to be caused by autoantibodies directed against the N-methyl-D-aspartate (NMDA) receptor. Clinical symptoms of anti-NMDAR encephalitis may mimic schizophrenia and psychotic spectrum disorders or substance-induced psychosis. Although initially described in association with ovarian teratomas in women, anti-NMDAR encephalitis has been reported in individuals without paraneoplastic association, as well as in males. Herein, we report a case of a 29-year-old woman with suicidal ideation and other neuropsychiatric manifestations who was found to have a right ovarian cystic mass by imaging study. Microscopically, the resected ovarian mass is composed of mature skin, fat, cartilage and neural tissues. Nerve, ganglions and multiple brain tissues are present. Cerebellum including external granular cell layer (normally only seen in infants), cerebrum-like, choroid plexus and other neural elements are present. There is peripheral lymphoplasmacytic infiltrates around and within the neuroglial matrix. Cerebral spinal fluid was concurrently tested positive for Anti-NMDAR. The combined clinical, histological, and laboratory findings confirmed the above diagnosis. Although Anti-NMDAR encephalitis is a familiar entity to many clinical psychiatrist and neurologists, it is less commonly reported in the pathology literature. Its resultant relationship to cystic teratoma warrants awareness of this condition by pathologists. Methods/Case Report Case Report Results (if a Case Study enter NA) NA Conclusion Anti-NMDA-receptor encephalitis is related to cystic teratoma, therefore pathologists need to be aware of this condition.

Hydroxyurea Exposure and Development of the Cerebellar External Granular Layer: Effects on Granule Cell Precursors, Bergmann Glial and Microglial Cells.

The current paper presents a histological analysis of the cell death in the cerebellar external granular layer (EGL) following the treatment with a single dose (2mg/g) of hydroxyurea (HU). The rats were examined at postnatal days (P) 5, 10, and 15, and sacrificed at appropriate times ranging from 6 to 48h after treatment administration. Studies were done in each cortical lobe (anterior, central, posterior, and inferior). The quantification of several parameters, such as density of 5-bromo-2'-deoxyuridine, TUNEL, vimentin, and tomato lectin-stained cells, revealed that HU compromises the viability of EGL cells. Our results indicate that P10 is a time of high vulnerability to injury. We also show here that the anterior and central lobes are the cortical regions most susceptible to the action of the HU. Additionally, our data also indicate that from 6 to 24h after HU-exposure is a time-window of high sensibility to this agent. On the other hand, our ultrastructural analysis confirmed that HU administration produces the activation of apoptotic cellular events in the EGL, resulting in a substantial number of dying cells. Different stages of apoptosis can be observed in all cortical lobes at all investigated postnatal ages and survival times. Moreover, we observed that dying neuroblasts were covered by laminar processes of Bergmann glia, and that these unipolar astrocytes presented cytological features of phagocytes engulfing apoptotic bodies and cell debris. The electron microscopy study also revealed the participation of ameboid microglial cells in the phagocytosis of apoptotic cells in the regions of the EGL with extensive cell death.

Towards A Structural Characterization of The Leopard Gecko Cerebellum: Purkinje Cells And Cells of The External Granular Layer

The cerebellum is a major component of the hindbrain, and plays important roles in motor coordination, posture, and cognitive functions. Although the gross anatomy and histological organization of the cerebellum has been documented across many species, less is known about the contributing cell types, especially among non‐mammals. Here we focus on Purkinje cells (PC) and cells of the External Granular Layer (EGL) in a representative lizard, the leopard gecko (Eublepharis macularius). PC serve as the major motor output of the cerebellum. Similar to other vertebrates, gecko PC are large, calbindin‐immunopositive neurons located at the junction between the molecular layer and the granular layer. Golgi‐cox impregnation reveals a dendritic arborization broadly comparable with that of alligators (and distinct from that of mammals). There is typically a single, lengthy primary dendrite that passes towards the molecular layer. This primary dendrite divides into multiple short dendritic branches that conclude in thin spiny branchlets with abundant, closely packed spines. Adult geckos also retain an EGL, a progenitor cell population that characteristically disappears prior to adulthood in mammals. Cells of the gecko EGL are immunopositive for the transcription factor SOX2, a hallmark protein of neural stem/progenitor cells, and the intermediate filament (and radial glia marker) glial fibrillary acid protein, but are immunonegative for NeuN, a marker of mature neurons. Using a bromodeoxyuridine pulse‐chase strategy, we determined that a subset of gecko EGL cells were label retaining after a 140 day chase period. Combined, these data provide important new information about the evolutionary variability of the cerebellum, with implications for functional adaptations in a species capable of tail loss.Support or Funding InformationNatural Sciences and Engineering Research Council Discovery Grant #400358 (MKV), #436190 (CDCB) and The Canada Foundation for Innovation #25371 (MKV) and #30381 (CDCB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Developmental Injury to the Cerebellar Cortex Following Hydroxyurea Treatment in Early Postnatal Life: An Immunohistochemical and Electron Microscopic Study.

Postnatal development of the cerebellar cortex was studied in rats administered with a single dose (2mg/g) of the cytotoxic agent hydroxyurea (HU) on postnatal day (P) 9 and collected at appropriate times ranging from 6h to 45days. Quantification of several parameters such as the density of pyknotic, mitotic, BrdU-positive, and vimentin-stained cells revealed that HU compromises the survival of the external granular layer (EGL) cells. Moreover, vimentin immunocytochemistry revealed overexpression and thicker immunoreactive glial processes in HU-treated rats. On the other hand, we also show that HU leads to the activation of apoptotic cellular events, resulting in a substantial number of dying EGL cells, as revealed by TUNEL staining and at the electron microscope level. Additionally, we quantified several features of the cerebellar cortex of rats exposed to HU in early postnatal life and collected in adulthood. Data analysis indicated that the analyzed parameters were less pronounced in rats administered with this agent. Moreover, we observed several alterations in the cerebellar cortex cytoarchitecture of rats injected with HU. Anomalies included ectopic placement of Purkinje cells and abnormities in the dendritic arbor of these macroneurons. Ectopic granule cells were also found in the molecular layer. These findings provide a clue for investigating the mechanisms of HU-induced toxicity during the development of the central nervous system. Our results also suggest that it is essential to avoid underestimating the adverse effects of this hydroxylated analog of urea when administered during early postnatal life.

Principal Component and Cluster Analysis of Morphological Variables Reveals Multiple Discrete Sub-phenotypes in Weaver Mouse Mutants

The present study evaluates the usefulness of the principal component analysis-based cluster analysis in the categorization of several sub-phenotypes in the weaver mutant by using several morphological parameters from the cerebellar cortex of control, heterozygous (+/wv) and homozygous (wv/wv) weaver mice. The quantified parameters were length of the cerebellar cortex, area of the external granular layer, area of the molecular layer, number of the external granular layer cells (EGL), and number of Purkinje cells (PCs). The analysis indicated that at postnatal day 8, the genotype +/wv presented three sub-phenotypes tagged as +/wv (0), +/wv (1) and +/wv (2), whereas two sub-phenotypes designated as wv (0)/wv (1) and wv (0)/wv (2) were identified in the genotype wv/wv. The number of PCs for the genotype +/wv and the number of EGL cells for the genotype wv/wv were the variables that discriminated the best among sub-phenotypes. Each one of the sub-phenotypes showed specific abnormalities in the cytoarchitecture of the cerebellar cortex as well as in the foliar pattern. In particular, the wv (0)/wv (1) and wv (0)/wv (2) sub-phenotypes had the most altered cytoarchitectonics, followed by the +/wv (2) sub-phenotype and then by the +/wv (1) one. The sub-phenotype +/wv (0) was the less affected one. Apart from reporting for the first time the coexistence of several sub-phenotypes in the weaver mutant, our approach provides a new statistical tool that can be used to assess cerebellar morphology.

Development of an early induction model of medulloblastoma in Ptch1 heterozygous mice initiated withN-ethyl-N-nitrosourea

Mice heterozygous for the ptch1 gene (ptch1 mice) are known as a valuable model of medulloblastoma, a common brain tumor in children. To increase the incidence and reduce the time required for tumor development, allowing for evaluation of modifier effects on medulloblastoma in a short time, we attempted to develop an early induction model of medulloblastoma in ptch1 mice initiated with N-ethyl-N-nitrosourea (ENU). Ptch1 mice and their wild-type littermates received a single intraperitoneal injection of ENU (10, 50 or 100 mg/kg) on postnatal day 1 (d1) or 4 (d4), and histopathological assessment of brains was conducted at 12 weeks of age. The width of the external granular layer (EGL), a possible origin of medulloblastoma, after injection of 100 mg ENU on d1 or d4 was measured in up to 21-day-old mice. Cerebellar size was apparently reduced at the 50 mg dose and higher regardless of genotype. Microscopically, early lesions of medulloblastomas occurred with a high incidence only in ptch1 mice receiving 10 mg on d1 or d4, but a significant increase was not observed in other groups. Persistent EGL cells and misalignment of Purkinje cells were increased dose-dependently. Although EGL was strikingly decreased after ENU injection, strong recovery was observed in mice of the d1-treated group. In summary, neonatal treatment with ENU is available for the induction of medulloblastoma in ptch1 mice, and 10 mg of ENU administered on d1 appeared to be an appropriate dose to induce medulloblastoma.

Expression of Sonic Hedgehog During Cell Proliferation in the Human Cerebellum

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.

Neuropathology of fetal stage Seckel syndrome: A case report providing a morphological correlate for the emerging molecular mechanisms

Seckel syndrome is a rare autosomal recessive disorder characterized by intrauterine growth retardation, dwarfism, microcephaly and mental retardation. Pathological descriptions of fetal stage Seckel syndrome are rare and pre-date the evolving understanding of the genetic and molecular mechanisms involved. The autopsy findings in a case of fetal Seckel syndrome at 30weeks gestation are presented, with detailed description of the neuropathological findings.Severe neurological abnormalities in a male fetus were observed that included microencephaly, cortical neuronal migration disorder, white matter tract hypoplasia/aplasia, premature depletion of the germinal matrix with cystic transformation and patchy absence of the external granular cell layer of the cerebellum. The striking neuropathological finding in this case was evidence of failure of the developing brain’s germinal elements, providing rare morphological insight into the abnormal development of the Seckel syndrome fetal brain. The selective failure of this proliferating cell population correlates with the emerging molecular evidence that Seckel syndrome is caused by defects in ATR-dependent DNA damage signaling with resultant premature death of proliferating cells.

Inherited neuroaxonal dystrophy in dogs causing lethal, fetal‐onset motor system dysfunction and cerebellar hypoplasia

Neuroaxonal dystrophy in brainstem, spinal cord tracts, and spinal nerves accompanied by cerebellar hypoplasia was observed in a colony of laboratory dogs. Fetal akinesia was documented by ultrasonographic examination. At birth, affected puppies exhibited stereotypical positioning of limbs, scoliosis, arthrogryposis, pulmonary hypoplasia, and respiratory failure. Regional hypoplasia in the central nervous system was apparent grossly, most strikingly as underdeveloped cerebellum and spinal cord. Histopathologic abnormalities included swollen axons and spheroids in brainstem and spinal cord tracts; reduced cerebellar foliation, patchy loss of Purkinje cells, multifocal thinning of the external granular cell layer, and loss of neurons in the deep cerebellar nuclei; spheroids and loss of myelinated axons in spinal roots and peripheral nerves; increased myocyte apoptosis in skeletal muscle; and fibrofatty connective tissue proliferation around joints. Breeding studies demonstrated that the canine disorder is a fully penetrant, simple autosomal recessive trait. The disorder demonstrated a type and distribution of lesions homologous to that of human infantile neuroaxonal dystrophy (INAD), most commonly caused by mutations of phospholipase A2 group VI gene (PLA2G6), but alleles of informative markers flanking the canine PLA2G6 locus did not associate with the canine disorder. Thus, fetal-onset neuroaxonal dystrophy in dogs, a species with well-developed genome mapping resources, provides a unique opportunity for additional disease gene discovery and understanding of this pathology.

KDM1A/LSD1 has a functional role in migration of medulloblastoma cells and their neuronal precursors

DNA methylation and histone acetylation are important epigenetic regulators of gene expression in cancer. Histone methylation has recently been proven to be a reversible process regulated by lysine (K)-specific demethylase 1A (KDM1A/LSD1). We previously reported that KDM1A expression in primary medulloblastomas is correlated with adverse patient outcome. Growth and migration of medulloblastoma cells was reduced after siRNA-mediated knockdown of KDM1A in vitro in a modified wound-healing assay. KDM1A inhibition using the MAO inhibitor, tranylcypromine, resulted in increased global H3K4 methylation and inhibited growth of MB cells. Medulloblastoma has been suggested to arise from the external granular cell layer (EGL) of the developing cerebellum. Interestingly, KDM1A mRNA was significantly upregulated in these granular cell precursors (GCPs) derived from various mouse lines at the time when GCPs normally migrate from the EGL to the internal granule layer. Our results suggest that KDM1A is not only over-expressed in medulloblastoma, but may play an important role in migration of medulloblastoma cells and their progenitors.

Antiprion Prophylaxis by Gene Transfer of a Soluble Prion Antagonist

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the normal prion protein PrP(C). Here, we delivered the soluble prion antagonist PrP-Fc(2) to the brains of mice by lentiviral gene transfer. Although naïve mice developed scrapie at 175 +/- 5 days postintracerebral prion inoculation (dpi), gene transfer before inoculation delayed disease onset by 72 +/- 4 days. At 170 days postintracerebral prion inoculation, PrP(Sc) accumulation and prion infectivity in PrPFc-treated brains were reduced by 3.6 and 4.2 logs, respectively. When PrP-Fc(2) was delivered 30 days after prion inoculation, survival of the treated animals was extended by 25 days. We then used tissue-specific recombination to express PrP-Fc(2) in the entire central nervous system, in only astrocytes, or in only oligodendrocytes. Oligodendrocyte-restricted PrP-Fc(2) expression impaired PrP(Sc) deposition and delayed disease even though oligodendrocytes are completely resistant to prion infection, suggesting that PrP-Fc(2) affords protection via noncell autonomous mechanisms. These results suggest that somatic gene transfer of prion antagonists may be effective for postexposure prophylaxis of prion diseases.

Cerebellar granule cell and Bergmann glial cell maturation in the rat is disrupted by pre- and post-natal exposure to moderate levels of ethanol

Migration of the external granular layer cells in the cerebellum of rats was delayed after exposure to moderate levels of ethanol during a pre-gestational period, through gestation and lactation until weaning. After ethanol withdrawal, cell soma and dendrites were observed to be larger in granule cells. Likewise, Bergmann glia showed several cytoarchitectonic features suggesting cell immaturity, as well as some apparent compensatory plastic responses after ethanol withdrawal. These effects may be due to ethanol impairing neurotrophin-mediated processes during cerebellar development that could lead to alterations in Purkinje cell structure and activity, and thereafter in the psychoneural functions in which the cerebellar cortex is involved.

Proliferation and migration of granule cells in the developing rat cerebellum: Cisplatin effects

We evaluated the relationship among proliferation, death and migration of granule cells in lobules VI-VIII of vermis, in comparison with lobule III, during cerebellar development. To this aim, a single injection of cisplatin, i.e., a cytostatic agent that is known to induce death of proliferating granule cells, was given to 10-day-old rats. Histochemical markers of proliferating (PCNA immunoreaction) and apoptotic (TUNEL staining) cells were used; the variations of the external granular layer (EGL) thickness were evaluated in parallel. After PCNA and TUNEL reactions, evident changes of the whole EGL were found on PD11 (1 day after treatment), when a reduction of the thickness of this layer was found in treated rats, mainly in consequence of the high number of apoptotic cells in all the cerebellar lobules. On PD17 (7 days after treatment), a thick layer of proliferating cells was observed in lobules VI-VIII of treated rats, while the peculiar pattern of the normal development showed a thin EGL. At the same time, in treated rats, the number of apoptotic cells in EGL was low. In all developmental stages of treated rats, after GFAP immunoreaction, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet were present. The damage of radial glia suggests an alteration of migratory processes of granule cells, which is also evidenced by the decreased thickness of the premigratory zone of the EGL. Injured radial glia fibers were restricted to lobules VI-VIII and they persisted at PD30, leading to the presence of ectopic granule cells in the molecular layer, as we previously described.

Immunoblockage of 9-O-acetyl GD3 ganglioside arrests the in vivo migration of cerebellar granule neurons.

During development of the cerebellum, radial glial cells guide the migration of granule cell precursors from the external granular cell layer toward the internal granular cell layer. The cellular membranes of migrating neurons and glial fibers organize a specialized migration junction at the site of contact between these cells, and several molecules have been implicated in the control of this glial-guided neuronal migration program. The monoclonal antibody Jones (mAb Jones) recognizes the ganglioside 9-O-acetyl GD3, which is expressed in migratory profiles in the developing and adult CNS. Recently, this ganglioside was suggested to play a role in neuronal migration in cerebellar cultures. In this report, we use antibody perturbation assays to investigate a possible role of 9-O-acetyl GD3 in the neuronal migration program in vivo. The results show that chronic intracerebroventricular administration of mAb Jones arrests neuronal migration in the developing cerebellum of live animals. Proliferating granule cell precursors were labeled with 5-bromo-2'-deoxyuridine (BrdU), and their migratory behavior was analyzed and compared with control groups. Immunoblockage of 9-O-acetyl GD3 arrests 43% of the BrdU-labeled granule precursors in the external granular cell layer. Together with our previous results, this report strongly suggests that the ganglioside 9-O-acetyl GD3 plays a crucial role in the migration of cerebellar granule cells along radial glial fibers in the developing rat cerebellum.

Secretoneurin promotes pertussis toxin-sensitive neurite outgrowth in cerebellar granule cells.

The neuropeptide secretoneurin (SN) is an endoproteolytic product of the chromogranin secretogranin II. We investigated the effects of SN on the differentiation of immature cerebellar granule cells derived from the external granular layer (EGL). Secretoneurin caused concentration-dependent increases in neurite outgrowth, reflecting differentiation. The maximum effect was reached at a concentration of 100 nm SN. Secretoneurin immunoneutralization using specific antiserum significantly decreased neurite outgrowth; however, neurite morphology was altered. An affinity chromatography-purified antibody significantly inhibited the outgrowth response to SN (p < 0.001) without altering the morphology. Binding studies suggest the existence of specific G-protein-coupled receptors on the surface of monocytes that recognize SN. Assuming that SN promotes neurite outgrowth in EGL cells by acting through a similar G-protein-coupled mechanism, we treated SN-stimulated EGL cultures with pertussis toxin. Exposure to pertussis toxin (0.1 micro g/mL) showed a significant inhibition of the SN-induced outgrowth. To establish a second messenger pathway we used the protein kinase C inhibitor staurosporine. We found that EGL cell viability was not enhanced following chronic SN treatment for 24 h. These data indicate that SN is a novel trophic substance that can affect cerebellar maturation, primarily by accelerating granule cell differentiation through a signalling mechanism that is coupled to pertussis toxin-sensitive G-proteins.

Stromal cell-derived factor 1 is secreted by meningeal cells and acts as chemotactic factor on neuronal stem cells of the cerebellar external granular layer

The cerebellar external granular layer (EGL) is an unusually long-lasting neural proliferative zone positioned immediately beneath the pial surface. Its position and stability critically depend on meningeal cells, as their selective destruction leads to its rapid dispersal, creating massive cortical ectopia. Similar ectopias have recently been described as a side effect of deficiency for stromal cell-derived factor 1 (SDF-1), a chemoattractant for haematopoietic precursor cell migration. Here we show that SDF-1 is present in meningeal cells in vivo and in vitro, where it is secreted in functionally relevant concentrations into the medium. Correspondingly, the SDF-1 receptor (termed CXCR4) can be demonstrated on stem cells of the external granular layer, but is absent on postmitotic cells commencing their final inward migration. We show that SDF-1 is concentrated by heparan sulphate proteoglycans highly expressed in the EGL in a laminar fashion, which thus might act to locally restrict SDF-1 action to the EGL in a kind of step gradient. In vitro, SDF-1 chemotactically attracts neuronal cells isolated from the external, but not from the internal granular layer, in a Boyden chamber assay in concentrations found in meningeal cell-conditioned medium. Selective removal of SDF-1 from conditioned media by immunoprecipitation abolishes their chemoattractive action, which can be reconstituted again by the addition of recombinant SDF-1. Meningeal cells are thus an important source for the expression of SDF-1 during brain development, which – comparable to its role in haematopoiesis – appears to be a key factor attracting precursor cells to their proliferative compartment.

Developmental changes of synaptojanin expression in the human cerebrum and cerebellum

Synaptojanin is a highly abundant polyphosphoinositide phosphatase in nerve terminals, and has been thought to play roles in clathrin-mediated synaptic vesicle endocytosis and signaling. In order to determine the broader role of synaptojanin in the central nervous system, we examined synaptojanin expression in the cerebrum and cerebellum from the fetal to the adult period by means of immunohistochemical and Western blot analyses. Immunohistochemistry consistently revealed the localization of synaptojanin in Cajal–Retzius cells, cortical plate neurons, subplate neurons, intermediate neurons, germinal matrix cells and the ventricular neuroepithelium of the fetal cerebrum. In the fetal cerebellum, synaptojanin immunoreactivity was localized in the external granular cell layer, Purkinje cell layer neuropil, cytoplasm of Purkinje cells and internal granular cells. The immunoreactivity in these structures was decreased around birth. After birth, the synaptojanin immunoreactivity of cortical neurons in the cerebrum, Purkinje cell layer neuropil, and internal granular cells and Purkinje cells in the cerebellum increased and reached a plateau after 11 years of age. These results were consistent with the intensity observed on Western blot analysis. These developmental changes of synaptojanin suggest a broader role in not only synaptic vesicle recycling, but also the regulation of neuronal migration and synaptogenesis in the fetal cerebrum and cerebellum.

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ERT) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.

Effect of nicotine on cerebellar granule neuron development

To assess the role of nicotinic cholinergic receptors (nAChR) on neuronal maturation, nAChR expression and the direct effects of nAChR activation were examined in cerebellar external granular layer (EGL) precursors isolated in vitro. Treatment of EGL neuroblasts with nicotine elicited a concentration-dependent increase in DNA content and synthesis, implying an increase in cell numbers. Pretreatment of cultures with the nAChR antagonist dihydro-beta-erythroidine (DHBE) attenuated nicotine-induced changes in DNA abundance and synthesis. Furthermore, chronic nicotine treatment for 4-7 days promoted EGL cell survival. Epibatidine but not cytisine stimulated granule neuroblast DNA synthesis and survival. Survival effects mediated by nicotine and epibatidine were attenuated by pretreating cultures with DHBE. Immunocytochemical analysis revealed that EGL neurons possessed alpha3, but not alpha4, nAChR immunoreactivity. Quantitative autoradiography was used to determine which nAChRs are present during the period of granule cell neurogenesis in vivo. On postnatal day 5, the EGL was intensely labelled by [3H]-epibatidine but virtually devoid of [3H]-A85380 binding, suggesting that a high concentration of alpha3 AChRs is present in granule neuroblasts. The pharmacology of [3H]-epibatidine displacement from EGL neurons also suggested an interaction with the alpha3-nAChR subunits. Together these data provide novel evidence that the activation of nAChRs directly affect the development of primary cerebellar neuroblasts and further suggest that the effects are mediated through the alpha3-nAChR subtype.