Localization of the type 1 corticotropin releasing factor receptor (CRF-R1) in the embryonic mouse cerebellum

Corticotropin releasing factor (CRF) is present in the adult, as well as in the embryonic and postnatal rodent cerebellum. Further, the distribution of the type 1 CRF receptor has been described in adult and postnatal animals. The focus of the present study is to determine the distribution and cellular relationships of the type 1 CRF receptor (CRF-R1) during embryonic development of the cerebellum. Between embryonic day (E)11 and E12, CRF-R1 immunoreactive puncta are uniformly distributed in the ventricular zone, the site of origin of Purkinje cells, nuclear neurons, and GABAergic interneurons, as well as the germinal trigone, the birthplace of the precursors of granule cells. Between E13 and 18, the distribution of immunolabeled puncta decreases in both the ventricular zone and the germinal trigone and increases in the intermediate zone, as well as in the dorsal aspect of the cerebellar plate. Between E14 and 18, antibodies that label specific populations of cerebellar neurons were combined with the antibody for the receptor to determine the cellular elements that expressed CRF-R1. At E14, CRF-R1 immunoreactivity is co-localized in neurons immunolabeled with PAX-2, an antibody that is specific for GABAergic interneurons. These neurons continue to express CRF-R1 as they migrate dorsally toward the cerebellar surface. Between E16 and 18, Purkinje cells, immunolabeled with calbindin, near the dorsal surface of the cerebellum express CRF-R1 in their cell bodies and apical processes. CRF has been shown to have a depolarizing effect on adult and postnatal Purkinje cells. Further, CRF has been shown to contribute to excitability of hippocampal neurons during embryonic development by binding to CRF-R1; depolarization induced excitability appears to be critical for cell survival. The location of the type one CRF receptor and the presence of its primary ligand, CRF, in the germinal zones of the cerebellum and in migrating neurons suggest that this receptor/ligand interaction could be important in the regulation of neuronal survival through cellular mechanisms that lead to depolarization of embryonic cerebellar neurons.     

Parvalbumin and calretinin in the avian thymus

The avian thymic hormone, known to support the maturation of T-lymphocytes, is biochemically similar to parvalbumin. However, its exact cellular distribution in the thymus is unknown. We have therefore studied the occurrence of parvalbumin and other related calcium-binding proteins in this organ by immunohistochemistry during development and after hatching. Parvalbumin immunoreactivity appears in the epithelial cells on embryonic day 9, and is expressed in the cortical reticular cells in the adult. Calretinin is found in the clusters of medullary epithelial cells from embryonic day 11, whereas calbindin D-28k is absent from this organ. Thus, calcium binding-proteins are excellent markers for different compartments of the avian thymus in embryonic life and after hatching, and their expression seems to coincide with their functional maturation.     

Expression of SV2 in the Seveloping Chick Cerebellum: Comparison with Calbindin and AMPA Glutamate Receptors 2/3

The well‐organized cerebellum is an ideal model to investigate the developmental appearance and localization of pre‐ and postsynaptic structures. One of the synaptic proteins abundant in the central nervous system and localized in presynaptic vesicle membranes is the synaptic vesicle protein 2 (SV2). SV2 was shown to be involved in priming and modulating synaptic vesicles and having an effect in epileptic diseases. So far there are no data available describing the developmental localization of this protein in the cerebellum. We followed the expression pattern of SV2 and compared it with the expression of the neuronal calcium‐binding protein Calbindin and the AMPA glutamate receptor subunits 2/3 (GluR 2/3), both shown to be early expressed in the developing chick cerebellum predominantly in Purkinje cells. We detected the expression of SV2 in presynaptic terminals (mainly from climbing and mossy fibers) as soon as they are formed at embryonic day 16 in the inner molecular layer. Purkinje cells express Calbindin and GluR 2/3 in the soma and postsynaptically in the primary dendrites at this stage. With ongoing development, the pattern of SV2 expression follows the development of Purkinje cell dendrites in the molecular layer, suggesting a synaptic refinement of labeled climbing and later parallel fibers. Anat Rec, 291:538–546, 2008. © 2008 Wiley‐Liss, Inc.     

Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) within the developing chick brain.

Doublecortin (DCX) is a microtubule-associated protein widely expressed in the developing mammalian nervous system and important for neuronal migration. DCX is known to belong to a novel protein family defined by sequence homology and the presence of a conserved microtubule-binding domain, but the functions of other members of this family are still undefined. In this study, we describe the cloning of the chick ortholog of doublecortin-like kinase (DCLK), a member of this family, and assess the expression of DCX and DCLK in the layered regions of the developing chick brain. DCX and DCLK are widely expressed in pallial and subpallial structures, including the telencephalon, optic tectum, and cerebellum, in similar distribution patterns. In addition to their expression in migrating cells, both proteins were also detected in the ventricular zone and in postmigratory Purkinje cells. Finally, DCX and DCLK were found to be coexpressed in all areas examined. In postmigratory Purkinje cells, DCX and DCLK both colocalized to the cell membrane, although DCLK was also distributed more generally throughout the cell soma. These data are consistent with multiple roles for DCX and DCLK in the developing chicken brain and suggest that the chick cerebellum will be an intriguing system to explore the effects of DCX and DCLK on postmigratory neuronal function.     

Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3

Plasma membrane calcium ATPase isoforms (PMCAs) are expressed in a wide variety of tissues where cell-specific expression provides ample opportunity for functional diversity amongst these transporters. The PMCAs use energy derived from ATP to extrude submicromolar concentrations of intracellular Ca²⁺ ([Ca²⁺]_i) out of the cell. Their high affinity for Ca²⁺ and the speed with which they remove [Ca²⁺]_i depends upon splicing at their carboxy (C)-terminal site. Here we provide biochemical and functional evidence that a brain-specific, C-terminal truncated and therefore fast variant of PMCA2, PMCA2a, has a role at hippocampal CA3 synapses. PMCA2a was enriched in forebrain synaptosomes, and in hippocampal CA3 it colocalized with the presynaptic marker proteins synaptophysin and the vesicular glutamate transporter 1, but not with the postsynaptic density protein PSD-95. PMCA2a also did not colocalize with glutamic acid decarboxylase-65, a marker of GABA-ergic terminals, although it did localize to a small extent with parvalbumin-positive presumed inhibitory terminals. Pharmacological inhibition of PMCA increased the frequency but not the amplitude of mEPSCs with little effect on mIPSCs or paired-pulse depression of evoked IPSCs. However, inhibition of PMCA activity did enhance the amplitude and slowed the recovery of paired-pulse facilitation (PPF) of evoked EPSCs. These results indicated that fast PMCA2a-mediated clearance of [Ca²⁺]_i from presynaptic excitatory terminals regulated excitatory synaptic transmission within hippocampal CA3.     

Asynchrony in the expression of guanosine 3':5'-phosphate-dependent protein kinase by clusters of Purkinje cells during the perinatal development of rat cerebellum

The early maturation of Purkinje cells was studied by immunocytochemistry in the rat cerebellum. The antiserum against guanosine 3':5'-phosphate-dependent protein kinase used in this study has been shown previously to label specifically all Purkinje cells in the adult rat. Immunoreactive Purkinje cells are first observed at embryonic day 17, 2 days after the end of proliferation of this neuronal population. At this time, most of the labeled cells are situated in the subventricular zone, although some immunoreactive Purkinje cells have already reached the cortex. Between embryonic day 17 and birth, four clusters of immunoreactive Purkinje cells appear in each hemicerebellum. Their time course and their pathways of migration to the cortex were followed. The immunoreactive clusters are tailed by a fibre-like immunostained material. The pattern of the migrating clusters at embryonic day 19 is very similar to the pattern of the corticonuclear projection observed at birth. From comparison between sections of embryos processed either for immunocytochemistry or Cresyl Violet staining, it appears that all the Purkinje cells are not immunoreactive. Positive and negative clusters of Purkinje cells are sharply delineated, their cells never mix. Immunopositive and negative clusters of Purkinje cells coexist until postnatal day 3. However, from birth onwards, negative clusters begin progressively in a caudorostral sequence to express guanosine 3':5'-phosphate-dependent protein kinase and rapidly attain the same level of immunoreactivity as previously labeled clusters. From postnatal day 5 all the Purkinje cells are immunoreactive. It is concluded that a compartmentalization of the cerebellar cortex is present very early and is evidenced by differences in the biochemical maturation of Purkinje cells. The axons of Purkinje cells reach the deep nuclei, following the same pathways as the clusters of Purkinje cells migrating to the cortex. Therefore, the mechanisms regulating the selection of the migratory routes followed by each Purkinje cell cluster are essential for the achievement of the topography of the corticonuclear projection. The level of protein kinase immunoreactivity cannot be taken as an index of the overall maturation of Purkinje cells, because it does not always coincide with the expression of other makers of biochemical and morphological differentiation of these neurons. During the early establishment of the cerebellar maps, an asynchrony in the expression of parts of the same genotype in the Purkinje cells may help in the establishment of ordered connections.     

B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain

Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(DeltaB2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(DeltaB2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(DeltaB2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB.     

Structural and functional analysis of the apoptosis-associated tyrosine kinase (AATYK) family

Apoptosis-associated tyrosine kinase (AATYK) is a protein kinase that is predominantly expressed in the nervous system and is involved in apoptosis and neurite growth of cerebellar granule cells. In this study, we cloned three new members of the mouse AATYK family, AATYK1B, AATYK2 and AATYK3. AATYK1B is a splicing variant of the previously reported AATYK1 (referred to as AATYK1A hereafter). In comparison with AATYK1A, these three AATYK members were characterized by having an extra N-terminal region that consists of a signal peptide-like sequence and a predicted transmembrane (TM) region, which is followed by a kinase domain and a long C-terminal domain. Both TM-containing AATYK isoforms (AATYK(+)TM: AATYK1B, 2, and 3) and TM-lacking isoform (AATYK(-)TM: AATYK1A) were recovered in membrane fractions, suggesting that AATYK(+)TM and AATYK(-)TM are transmembrane- and peripheral-membrane protein kinases, respectively. AATYK1A was recovered in the soluble fraction when the cells were treated with 2-bromo palmitate, suggesting that AATYK1A associates with membrane via palmitoylation. The kinase domain was highly conserved among all AATYK members and was shown to be catalytically active. Three AATYK family members were predominantly expressed in adult mouse brains with almost similar expression profiles: widespread distribution over the various brain regions, especially in the cerebellum and hippocampus, and up-regulated expression during development of the cerebellum. In cultured cerebellar granule cells, AATYK1 was abundantly localized in both soma and axons, AATYK2 distribution was restricted to soma, and AATYK3 was punctately present over the cells. AATYK1 was concentrated in the central domain of growth cones of dorsal root ganglion neurons. Our results indicate that AATYK family members are brain-dominant and membrane-associated kinases with slightly different distribution patterns in the developing and adult mouse brain, which may be involved in fine regulation of neuronal functions including neurite extension and apoptosis.     

Ultrastructural analysis of climbing fiber-Purkinje cell synaptogenesis in the rat cerebellum.

Previous reports have described the transient expression of the neuropeptides calcitonin gene-related peptide and neuropeptide Y in selected subsets of rat olivocerebellar compartments during embryonic and postnatal development. Using these neuropeptides as endogenous markers for olivocerebellar fibers, the aim of this electron microscopic analysis was to reveal the synaptogenetic processes occurring between climbing fibers and their target Purkinje cells, from embryonic day 19 to postnatal day 16, the period during which Purkinje cells undergo intense emission and retraction of dendrites, and climbing fibers translocate their synapses along Purkinje cell membrane surfaces.The present findings provide the first direct evidence that climbing fiber synaptogenesis starts on embryonic day 19 and that these first synapses mainly involve the Purkinje cell embryonic dendrite rather than the Purkinje cell soma. At the same age, the presence of unlabeled synapses resembling calcitonin gene-related peptide-labeled synapses in the Purkinje cell plate makes it possible to conclude that climbing fibers form a major synaptic investment on embryonic Purkinje cells, a finding that strongly supports the hypothesis of an early differentiating role of climbing fibers on cerebellar development. Furthermore, during the period of intense dendritic remodeling of Purkinje cells, 'myelin figures' were often detected in Purkinje cell dendrites suggesting that they may at least in part represent real ultrastructural markers of membrane turnover that identifies the sites where Purkinje cell dendritic rearrangement is taking place. Finally the finding that the climbing fiber terminals apposed to degenerating dendrites did not generally show signs of degeneration leads us to suggests that climbing fiber translocation from a perisomatic to a dendritic location may be driven by the Purkinje cell dendritic remodeling.     

Transmembrane protein 50b (C21orf4), a candidate for Down syndrome neurophenotypes, encodes an intracellular membrane protein expressed in the rodent brain

Transmembrane protein 50b, Tmem50b, previously referred to as C21orf4, encodes a predicted transmembrane protein and is one of few genes significantly over-expressed during cerebellar development in a Down syndrome mouse model, Ts1Cje. In order to assess potential mechanisms by which Tmem50b could contribute to Down syndrome-related phenotypes, we determined the expression patterns of Tmem50b mRNA, as well as Tmem50b protein distribution, expression and subcellular localization. In situ hybridization in mice at embryonic day 14.5 showed cortical plate and spinal cord mRNA expression. By postnatal day 7, strong mRNA expression was seen in the cerebellum, hippocampus and olfactory bulb, with diffuse cortical expression. Quantitative PCR of adult mouse tissue showed Tmem50b mRNA expression in the brain, heart and testis. A rabbit polyclonal antibody was generated against Tmem50b and rat and mouse tissue screening by Western blot, and immunohistochemistry showed that protein expression concurred with mRNA expression. Double immunofluorescence revealed that Tmem50b is highly expressed in rat and mouse glial fibrillary acidic protein-positive cells in vivo and in vitro, but less so in neuronal MAP2- or beta-tubulin II-positive cells in vitro. Tmem50b is invariably expressed in cultured mouse neural precursor cells. In adult mouse cerebellum sections, Tmem50b immunoreactivity was found in Purkinje and Golgi cell somata and in Bergmann glial processes. Electron microscopy confirmed that Tmem50b was present on endoplasmic reticulum (ER) and Golgi apparatus membranes. Results indicate that Tmem50b is a developmentally-regulated intracellular ER and Golgi apparatus membrane protein that may prove important for correct brain development through functions associated with precursor cells and glia.     

Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis

Purkinje cell maturation during thyroxine-induced metamorphosis in premetamorphic bullfrog tadpoles was studied using electron microscopy and Golgi (silver-impregnated) preparations. Cerebella from tadpoles were examined following 1, 2, or 3 weeks of thyroxine treatment. Particular attention was paid to possible differences between the two populations of Purkinje cells previously described, i.e. (i) the smaller population located in the dorsal part of the cerebellum, where the Purkinje cells show dendritic arborization long before the appearance of the external granular layer, and (ii) the larger population located in the middle and ventral regions of the cerebellum, where the Purkinje cells begin to undergo maturation during metamorphosis when the external granular layer is established.Following thyroxine treatment, both populations of Purkinje cells showed rapid maturational change. In the mature (dorsal) group, dendritic growth resumed in the presence of an external granular layer increasing the complexity of their dendritic arbors. Moreover, climbing fiber synapses translocated from contacts on the soma to the thorns of growing dendrites, and somatic processes often disappeared. The immature (ventral) group showed dramatic differentiation of the perikaryon including polarization of cytoplasm with subsequent dendritic outgrowth and formation of somatic processes in the presence of climbing fibers. Stellate cell contacts appeared on the smooth portion of the soma of many Purkinje cells. Dendritic growth during thyroxine-induced metamorphosis was characterized by growth (elongation) with minimal branching, which is initially observed during spontaneous metamorphosis. Typically, these growing dendrites ended in growth cones, some with one or several filopodia. Developing Purkinje cell dendritic spines formed synapses with parallel fibers.The present study has provided an example of the dramatic nature of thyroxine's action in inducing the complex series of detailed maturational changes in the cerebellum 1–2yr ahead of schedule. In addition, the results show that thyroxine-induced Purkinje cell maturation is more rapid and synchronous than that seen during spontaneous metamorphosis. It is concluded that Purkinje cell maturation during metamorphosis is largely dependent on thyroid hormone.     

The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association

Visinin-like protein-3 is a member of the family of intracellular neuronal calcium sensors belonging to the superfamily of EF-hand proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades. To gain insights into the characteristics of visinin-like protein-3, we have generated specific antibodies against visinin-like protein-3 and determined the developmental and tissue distribution of the protein and its exact cellular and subcellular localization. Expression of visinin-like protein-3 protein appeared late in development mainly in the cerebellum. It is strongly expressed in cerebellar Purkinje cells. The protein expression results were further confirmed by in situ hybridization and compared with hippocalcin messenger RNA localization. Native cerebellar visinin-like protein-3 was shown to bind calcium and to associate in a calcium-dependent manner with membrane fractions during subcellular fractionation. Recombinant wild-type visinin-like protein-3 was shown to be N-terminally myristoylated in transfected cells. The membrane association was strongly reduced for the non-myristoylated mutant of visinin-like protein-3 in transfected cells.These results suggest that visinin-like protein-3, which is mainly expressed in Purkinje cells in vivo, shows a calcium-dependent association with cell membranes which is mediated by a calcium-myristoyl switch.     

小鼠小脑皮质的组织发生

目的:探讨小鼠小脑皮质的组织发生过程。方法:应用光镜和电镜技术对胚胎和生后小脑皮质进行形态学观察,对各层厚度和细胞密度进行测量。结果:胚胎12 d(E12)小脑原基有室管膜层、套层和边缘层构成,约出生当日(P0)出现外颗粒层、分子层、Purkinje细胞层和内颗粒层。外颗粒层P6/7达最厚,至P20消失。P0至P30内颗粒层细胞逐步分化发育成熟,Purkinje细胞树突树逐渐形成,约P7时Purkinje细胞排列成单层。结论:E12至P0片层化小脑主要经历了细胞增殖、分化与迁移;P0至P30片层化结构逐渐发育成熟,外颗粒层消亡以细胞迁移和凋亡为主,其他各层细胞主要经历了分化发育与凋亡。     

Calbindin immunoreactivity in the developing and adult human cerebellum

Calbindin (CALB), a calcium-binding protein, is known to be expressed in the embryonic nervous system. In this study, we have examined its distribution in the cerebellum of human fetuses (11–25 weeks of gestation) and adult by immunohistochemistry. At the gestational age of 11–12 weeks, CALB immunoreactivity was present in granule and Purkinje cells throughout the cerebellum. By 16–21 weeks of gestation, immunoreactive Purkinje cells were well-differentiated in the vermis and flocculus, and their axons ran towards the deep cerebellar nuclei area, while the axon collaterals were seen to be distributed into adjacent folia. At the gestational period of 24–25 weeks, most Purkinje cells of the flocculus and vermis were arranged in one to two rows, while those of the hemispheres were still undifferentiated. A few Golgi cells of the vermis showed immunoreactivity. The neurons of the deep nuclei were immunonegative right from the gestational age of 11 weeks although a fine stippled staining of fibers was present throughout the body of all nuclei. The fibers lying close to the hilum of the dentate nucleus were strongly CALB-positive. The vestibulocerebellar fibers, being traced at the level of lower pons and upper medulla oblongata were stained as early as 11 weeks of gestation, whereas the olivocerebellar fibers were stained from 16 weeks onward. In the adult cerebellum, Purkinje cells were moderately immunopositive while granule cells were faintly stained; no other cells, including those of the deep nuclei were stained. In the medulla oblongata, the inferior olivary nucleus and olivocerebellar fibers were strongly CALB-positive. Our results indicate that CALB is expressed in early migratory Purkinje cells, and their maturation occurs in a vermal-to-hemisphere gradient. It is likely that CALB plays a significant role in the regulation of Ca²⁺-dependent activities in the developing cerebellum.     

Heterochronic Protein Expression Patterns in the Developing Embryonic Chick Cerebellum

The advantages of the embryonic chick as a model for studying neural development range from the relatively low cost of fertilized eggs to the rapid rate of development. We investigated in ovo cerebellar development in the chick, which has a nearly identical embryonic period as the mouse (19–22 days). We focused on three antigens: Calbindin (CB), Zebrin II (ZII), and Calretinin (CR), and our results demonstrate asynchronous expression patterns during cerebellar development. Presumptive CB+ Purkinje cells are first observed at embryonic day (E)10 in clusters in posterior cerebellum. At E12, corresponding with global expression of CB across the cerebellum, Purkinje cells began to express ZII. By E14–E16, Purkinje cells disperse into a monolayer and develop a pattern of alternating immunopositive and immunonegative ZII stripes. CR is initially expressed by clusters of presumptive Purkinje cells in the nodular zone at E8. However, this expression is transient and at later stages, CR is largely confined to the granule and molecular layers. Before hatch (E18–E20), Purkinje cells adopt a morphologically mature phenotype with complex dendritic arborizations. Comparing this data to that seen in mice, we found that the sequence of Purkinje cell formation, protein expression, and development is similar in both species, but these events consistently begin ～5–7 days earlier in the precocial chick cerebellum, suggesting an important role for heterochrony in neurodevelopment. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

A longitudinal study of bioelectric activity in the pre- and post-hatch chick.

A technique for embryonic implantation and the subsequent recording of electrocortical, neck muscle, and ocular activity continously from the 20th day of incubation through hatching and the first few days thereafter is demonstrated. The embryonic maturation of the EEG, with a characteristic muscle burst pattern heralding hatching was found, supporting previous reports obtained with acute preparations. The technique for injection into the chorioallantoic membrane (CAM) vessels or direct deposition onto the CAM is also described. The usefulness of the embryonic neurophysiological implantation coupled with the injection at specific stages of development is discusses as an approach to the understanding of the parameters of the maturation of the sleep-wakefulness cycle, neurochemistry, and behavior.     

Intracranial cerebellar grafts: Intermediate filament immunohistochemistry and electrophysiology

Summary Pieces of the developing cerebellar anlage were prepared from 13–15 day old rat embryos and transplanted to the cerebellar region of 5–7 and 13–14 day old rat pups. Approximately two months later, sections showed most grafts to consist of both cerebellar cortex, with a typical trilaminar organization, and white matter areas containing large neuronal perikarya.The astrocytic populations were studied using immunohistochemistry with antisera raised against the intermediate filaments, glial fibrillary acidic protein (GFA), and vimentin. The GFA-antiserum revealed a glial interface along most of the border between host brain and graft. Both antisera stained long, slender, although slightly distorted Bergmann fibers spanning the molecular layer. Using GF-Aantiserum, star-shaped fluorescent astrocytes were seen in the granular layer and in the white matter. Only in the white matter did the amount of GFA-like immunoreactivity suggest an astrocytic gliosis. With vimentin antiserum fluorescent astrocytes in the white matter were seen. There were no signs of increased amounts of vimentin-like immunoreactivity. Taken together, the amount and distribution of GFA-and vimentin-like immunoreactivity suggests a rather normal astrocytic development in the cerebellar grafts.Using an antiserum against the neurofilament (NF) triplet, delicate immunoreactive fibres were seen in both the molecular and the granular layer. No positive cell bodies could be visualized in the cortical areas. Although the Purkinje cells themselves were negative, fibre baskets around them were intensely stained. In the white matter a high density of NF-positive fibres and some positive perikarya were visualized. Thus the distribution of NF-like immunoreactivity in the grafts corresponded well to the normal NF distribution.The functional maturation of the cerebellar grafts was studied electrophysiologically. A spontaneous mean discharge rate of 19.3 + 1.7 Hz was recorded from the Purkinje cells. This compares with a discharge rate of 26.8 + 1.0 Hz for Purkinje neurons in situ. The difference was at least partly ascribable to the absence of climbing fibre bursts in the grafts. Local stimulation of the graft surface caused both decreased and increased Purkinje cell discharge. In conclusion, these experiments suggest that grafts of fetal cerebellar buds to the young cerebellum develop into cerebellar tissue having both morphological and electrophysiological characteristics quite similar to the normal cerebellum.