Phosphorylation of the neurogenic transcription factor SOX11 on serine 133 modulates neuronal morphogenesis.

The intellectual disability gene, Sox11, encodes for a critical neurodevelopmental transcription factor with functions in precursor survival, neuronal fate determination, migration and morphogenesis. The mechanisms regulating SOX11's activity remain largely unknown. Mass spectrometric analysis uncovered that SOX11 can be post-translationally modified by phosphorylation. Here, we report that phosphorylatable serines surrounding the high-mobility group box modulate SOX11's transcriptional activity. Through Mass Spectrometry (MS), co-immunoprecipitation assays and in vitro phosphorylation assays followed by MS we verified that protein kinase A (PKA) interacts with SOX11 and phosphorylates it on S133. In vivo replacement of SoxC factors in developing adult-generated hippocampal neurons with SOX11 S133 phospho-mutants indicated that phosphorylation on S133 modulates dendrite development of adult-born dentate granule neurons, while reporter assays suggested that S133 phosphorylation fine-tunes the activation of select target genes. These data provide novel insight into the control of the critical neurodevelopmental regulator SOX11 and imply SOX11 as a mediator of PKA-regulated neuronal development.

Developmental Changes in Serotonergic Modulation of GABAergic Synaptic Transmission and Postsynaptic GABAA Receptor Composition in the Cerebellar Nuclei.

Outputs from the cerebellar nuclei (CN) are important for generating and controlling movement. The activity of CN neurons is controlled not only by excitatory inputs from mossy and climbing fibers and by γ-aminobutyric acid (GABA)-based inhibitory transmission from Purkinje cells in the cerebellar cortex but is also modulated by inputs from other brain regions, including serotonergic fibers that originate in the dorsal raphe nuclei. We examined the modulatory effects of serotonin (5-HT) on GABAergic synapses during development, using rat cerebellar slices. As previously reported, 5-HT presynaptically decreased the amplitudes of stimulation-evoked inhibitory postsynaptic currents (IPSCs) in CN neurons, with this effect being stronger in slices from younger animals (postnatal days [P] 11-13) than in slices from older animals (P19-21). GABA release probabilities accordingly exhibited significant decreases from P11-13 to P19-21. Although there was a strong correlation between the GABA release probability and the magnitude of 5-HT-induced inhibition, manipulating the release probability by changing extracellular Ca2+ concentrations failed to control the extent of 5-HT-induced inhibition. We also found that the IPSCs exhibited slower kinetics at P11-13 than at P19-21. Pharmacological and molecular biological tests revealed that IPSC kinetics were largely determined by the prevalence of α1 subunits within GABAA receptors. In summary, pre- and postsynaptic developmental changes in serotonergic modulation and GABAergic synaptic transmission occur during the second to third postnatal weeks and may significantly contribute to the formation of normal adult cerebellar function.

Synaptic excitation by climbing fibre collaterals in the cerebellar nuclei of juvenile and adult mice.

KEY POINTS: The inferior olive sends instructive motor signals to the cerebellum via the climbing fibre projection, which sends collaterals directly to large premotor neurons of the mouse cerebellar nuclei (CbN cells). Optogenetic activation of inferior olivary axons in vitro evokes EPSCs in CbN cells of several hundred pA to more than 1 nA. The inputs are three-fold larger at younger ages, 12 to 14 days old, than at 2 months old, suggesting a strong functional role for this pathway earlier in development. The EPSCs are multipeaked, owing to burst firing in several olivary afferents that fire asynchronously. The convergence of climbing fibre collaterals onto CbN cells decreases from ∼40 to ∼8, which is consistent with the formation of closed-loop circuits in which each CbN neuron receives input from 4-7 collaterals from inferior olivary neurons as well as from all 30-50 Purkinje cells that are innervated by those olivary neurons. ABSTRACT: The inferior olive conveys instructive signals to the cerebellum that drive sensorimotor learning. Inferior olivary neurons transmit their signals via climbing fibres, which powerfully excite Purkinje cells, evoking complex spikes and depressing parallel fibre synapses. Additionally, however, these climbing fibres send collaterals to the cerebellar nuclei (CbN). In vivo and in vitro data suggest that climbing fibre collateral excitation is weak in adult mice, raising the question of whether the primary role of this pathway may be developmental. We therefore examined climbing fibre collateral input to large premotor CbN cells over development by virally expressing channelrhodopsin in the inferior olive. In acute cerebellar slices from postnatal day (P)12-14 mice, light-evoked EPSCs were large (> 1 nA at -70 mV). The amplitude of these EPSCs decreased over development, reaching a plateau of ∼350 pA at P20-60. Trains of EPSCs (5 Hz) depressed strongly throughout development, whereas convergence estimates indicated that the total number of functional afferents decreased with age. EPSC waveforms consisted of multiple peaks, probably resulting from action potential bursts in single collaterals and variable times to spike threshold in converging afferents. Activating climbing fibre collaterals evoked well-timed increases in firing probability in CbN neurons, especially in younger mice. The initially strong input, followed by the decrement in synaptic strength coinciding with the pruning of climbing fibres in the cerebellar cortex, implicates the climbing fibre collateral pathway in early postnatal development. Additionally, the persistence of substantial synaptic input at least to P60 suggests that this pathway may function in cerebellar processing into adulthood.

Labeling of neuronal morphology using custom diolistic techniques.

BACKGROUND: Diolistic labeling is increasingly utilized in neuroscience as an efficient, reproducible method for visualization of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery allows for non-toxic fluorescent labeling of multiple neurons in both living and fixed tissue. Since first described, this labeling method has been modified to fit a variety of research goals and laboratory settings. NEW METHOD: Diolistic labeling has traditionally relied on commercially available devices for the propulsion of coated micro-particles into tissue sections. Recently, laboratory built biolistic devices have been developed which allow for increased availability and customization. Here, we discuss a custom biolistic device and provide a detailed protocol for its use. RESULTS: Using custom diolistic labeling we have characterized alterations in neuronal morphology of the lateral/dentate nucleus of the rat cerebellum. Comparisons were made in developing rat pups exposed to abnormally high levels of 5-methyloxytryptamine (5-MT) pre-and postnatally. Using quantitative software; dendritic morphology, architecture, and synaptic connections, were analyzed. COMPARISON WITH EXISTING METHOD(S): The rapid nature of custom diolistics coupled with passive diffusion of dyes and compatibility with confocal microscopy, provides an unparalleled opportunity to examine features of neuronal cells at high spatial resolution in a three-dimensional tissue environment. CONCLUSIONS: While decreasing the associated costs, the laboratory-built device also overcomes many of the obstacles associated with traditional morphological labeling, to allow for reliable and reproducible neuronal labeling. The versatility of this method allows for its adaptation to a variety of laboratory settings and neuroscience related research goals.

Developmental change in the contribution of voltage-gated Ca(2+) channels to the pacemaking of deep cerebellar nuclei neurons.

The activity of the deep cerebellar nuclei (DCN) neurons conveys the bulk of the output of the cerebellum. To generate these motor signals, DCN neurons integrate synaptic inputs with their own spontaneous activity. We have previously reported that N-type voltage-gated Ca(2+) channels modulate the spontaneous activity of the majority of juvenile DCN neurons in vitro. Specifically, pharmacologically blocking N-type Ca(2+) channels increases their firing rate causing DCN cells to burst. Adult DCN neurons however, behaved differently. To further investigate this change, we have studied here the effect of cadmium on the firing rate of DCN neurons in acute cerebellar slices obtained from adult (>2 months old) or juvenile (12-21 days old) rats and mice. Strikingly, and in contrast to juvenile DCN cells, cadmium did not affect the pacemaking of adult DCN cells. The activity of Purkinje cells (PCs) however was transformed into high-frequency bursting, regardless the age. Further, we questioned whether these findings could be due to an artifact associated with the added difficulty of preparing adult DCN slices. Hence we proceeded to examine the spontaneous activity of DCN neurons in anesthetized juvenile and adult rats and mice in vivo. When cadmium was injected into the DCN in vivo no significant change in firing rate was observed, conversely to most juvenile DCN neurons which showed high-frequency bursts after cadmium injection. In these same animals, PCs pacemaking showed no developmental difference. Thus our results demonstrate a remarkable age-dependent functional modification in the regulation of DCN neurons pacemaking.

Effects of developmental hyperserotonemia on the morphology of rat dentate nuclear neurons.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social cognition, disordered communication, restricted interests and repetitive behaviors. Furthermore, abnormalities in basic motor control, skilled motor gestures, and motor learning, are common in ASD. These characteristics have been attributed to a possible defect in the pre- and postnatal development of specific neural networks including the dentate-thalamo-cortical pathway, which is involved in motor learning, automaticity of movements, and higher cognitive functions. The current study utilized custom diolistic labeling and unbiased stereology to characterize morphological alterations in neurons of the dentate nucleus of the cerebellum in developing rat pups exposed to abnormally high levels of the serotonergic agonist 5-methyloxytryptamine (5-MT) pre-and postnatally. Occurring in as many as 30% of autistic subjects, developmental hyperserotonemia (DHS) is the most consistent neurochemical finding reported in autism and has been implicated in the pathophysiology of ASD. This exposure produced dramatic changes in dendritic architecture and synaptic features. We observed changes in the dendritic branching morphology which did not lead to significant differences (p>0.5) in total dendritic length. Instead, DHS groups presented with dendritic trees that display changes in arborescence, that appear to be short reaching with elaborately branched segments, presenting with significantly fewer (p>0.001) dendritic spines and a decrease in numeric density when compared to age-matched controls. These negative changes may be implicated in the neuropathological and functional/behavioral changes observed in ASD, such as delays in motor learning, difficulties in automaticity of movements, and deficits in higher cognitive functions.

Maturation of membrane properties of neurons in the rat deep cerebellar nuclei.

Patch clamp recordings of neurons in the adult rat deep cerebellar nuclei have been limited by the availability of viable brain slices. Using a new slicing technique, this study was designed to explore the maturation of membrane properties of neurons in the deep cerebellar nuclei (DCN)-an area involved in rat eyeblink conditioning. Compared to whole-cell current-clamp recordings in DCN in rat pups at postnatal day 16 (P16) to P21, recordings from weanling rats at P22-P40 revealed a number of significant changes including an increase in the amplitude of the afterhyperpolarization (AHP)-an index of membrane excitability which has been shown to be important for eyeblink conditioning-a prolonged interval between the first and second evoked action potential, and an increase in AHP amplitude for hyperpolarization-induced rebound spikes. This is the first report of developmental changes in membrane properties of DCN which may contribute to the ontogeny of eyeblink conditioning in the rat.

Distribution of SNAP25, VAMP1 and VAMP2 in mature and developing deep cerebellar nuclei after estrogen administration.

Synaptosomal-associated protein of 25kDa (SNAP25), vesicle-associated membrane protein 1 (VAMP1) and 2 (VAMP2) are components of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNARE) complex which is involved in synaptic vesicle exocytosis, a fundamental step in neurotransmitter release. SNARE expression in cerebellum correlates with specific neurotransmitter pathways underlying synaptic diversification and defined synaptic properties. In this study we firstly characterized the distribution of SNAP25, VAMP1 and VAMP2 in the nerve terminals of a defined cerebellar region, the deep cerebellar nuclei (DCN), of adult and newborn rats. Then, given the pivotal role of estradiol (E2) in the synaptic organization of the cerebellar circuitry in early postnatal life, we examined whether administration of E2 in the newborn DCN affected synaptic density and changed the distribution of the presynaptic proteins SNAP25, VAMP1 and VAMP2, together with post synaptic density protein 95 (PSD95). Results showed that: (1) distribution of SNAP25, VAMP1 and VAMP2 in adult DCN differs significantly from that found in newborn DCN; (2) administration of E2 in the newborn DCN affected synaptic density and also changed the distribution of the pre- and postsynaptic proteins. The differential distribution of SNAP25, VAMP1 and VAMP2 in nerve terminals of adult and newborn rats may correlate with specific stages of neuronal phenotypic differentiation. The effects of E2 on SNAP25, VAMP1, VAMP2, PDS95 and synaptic density suggest that pre- and postsynaptic proteins are under estrogenic control during development and that synaptic maturation can also be related with the activity of this steroid.

Sequence of synaptogenesis in the fetal and neonatal cerebellar system - part 1: Guillain-Mollaret triangle (dentato-rubro-olivo-cerebellar circuit).

Precise temporal and spatial sequences of synaptogenesis occur in the cerebellar system, as in other synaptic circuits of the brain. In postmortem brain sections of 172 human fetuses and neonates, synaptophysin immunoreactivity was studied in nuclei of the Guillain-Mollaret triangle: dentato-olivo-rubro-cerebellar circuit. Synaptophysin demonstrates not only progressive increase in synaptic vesicles in each structure, but also shows the development of shape from amorphous globular neuronal aggregates to undulated nuclei. Intensity of synaptophysin reactivity is strong before the mature shape of these nuclei is achieved. Accessory olivary and deep cerebellar nuclei are intensely stained earlier than the principal olivary and dentate nuclei. The dorsal blades of both form earlier than the ventral, with reactivity initially peripheral. Initiation of synaptophysin reactivity is at 13 weeks in the inferior olive (r6, r7) and at 16 weeks in the dentate (r2). Initial synaptic vesicles are noted at 13 weeks in the red nucleus (r0); synapses form initially on the small neurons at 13 weeks but thereafter simultaneously on small and large neurons. Form and reactivity follow caudorostral, dorsoventral and mediolateral gradients in the axes of the rhombencephalon. This study provides control data to serve as a basis for interpreting aberrations in synaptogenesis in malformations of the cerebellar system, genetic disorders and acquired insults to the cerebellum and brainstem during fetal life, applicable to tissue sections and complementing biochemical and molecular techniques.

[Quantitative analysis of the change in neuronal numerical density of the human nucleus dentatus within development].

BACKGROUND/AIM: The role of the dentate nucleus is to coordinate input information coming from the lower olivary complex and various parts of the brainstem of the spinal marrow with the output information from the cerebellar cortex. To better understand functions and relations of the dentate nucleus it is highly important to study its development process. The aim of this study was to determine a possible mathematical model of decrease in neuronal numerical density of the human nucleus dentatus at different stages of development. METHODS: This study included 25 fetal brains of different age (12.5-31 weeks of gestational age and one brain of a 6-day-old newborn). The brains were fixed in 10% formalin-alcohol solution and embedded in paraffin. Sections were cut at a thickness of 6, 15, and 30 microm and stained with cresyl violet. Each fifth section was analyzed using a light microscope, and numerical density of dentate nucleus neurons was established using the M42 Weibel's grid system. RESULTS: The obtained results revealed a constant decrease in numerical density value. The changes of numerical densities at different stages of development correspond with Boltzmann function principles. The first, almost perpendicular part of Boltzmann function corresponds with the development of the dorsomedial lamina and the appearance of ventrolateral lamina primordium. The second, more or less horizontal part of Boltzmann function corresponds with the development of both laminae. CONCLUSION. The obtained results indicate that Boltzmann function can be considered a mathematical model of change in neuronal numerical density of dentate nucleus at different stage of development.

Extracerebellar progenitors grafted to the neurogenic milieu of the postnatal rat cerebellum adapt to the host environment but fail to acquire cerebellar identities.

Stem or progenitor cells acquire specific regional identities during early ontogenesis. Nonetheless, there is evidence that cells heterotopically transplanted to neurogenic regions of the developing or mature central nervous system may switch their fate to adopt host-specific phenotypes. Here, we isolated progenitor cells from different germinative sites along the neuraxis where GABAergic interneurons are produced (telencephalic subventricular zone, medial ganglionic eminence, ventral mesencephalon and dorsal spinal cord), and grafted them to the prospective white matter of the postnatal rat cerebellum, at the time when local interneurons are generated. The phenotype acquired by transplanted cells was assessed by different criteria, including expression of region-specific transcription factors, acquisition of morphological and neurochemical traits, and integration in the cerebellar cytoarchitecture. Regardless of their origin, all the different types of donor cells engrafted in the cerebellar parenchyma and developed mature neurons that shared some morphological and neurochemical features with local inhibitory interneurons, particularly in the deep nuclei. Nevertheless, transplanted cells failed to activate cerebellar-specific regulatory genes. In addition, their major structural features, the expression profiles of type-specific markers and the laminar placement in the recipient cortex did not match those of endogenous interneurons generated during the same developmental period. Therefore, although exogenous cells are influenced by the cerebellar milieu and show remarkable capabilities for adapting to the foreign environment, they essentially fail to switch their fate, integrate in the host neurogenic mechanisms and adopt clear-cut cerebellar identities.

Purkinje cell input to cerebellar nuclei in tottering: ultrastructure and physiology.

Homozygous tottering mice are spontaneous ataxic mutants, which carry a mutation in the gene encoding the ion pore of the P/Q-type voltage-gated calcium channels. P/Q-type calcium channels are prominently expressed in Purkinje cell terminals, but it is unknown to what extent these inhibitory terminals in tottering mice are affected at the morphological and electrophysiological level. Here, we investigated the distribution and ultrastructure of their Purkinje cell terminals in the cerebellar nuclei as well as the activities of their target neurons. The densities of Purkinje cell terminals and their synapses were not significantly affected in the mutants. However, the Purkinje cell terminals were enlarged and had an increased number of vacuoles, whorled bodies, and mitochondria. These differences started to occur between 3 and 5 weeks of age and persisted throughout adulthood. Stimulation of Purkinje cells in adult tottering mice resulted in inhibition at normal latencies, but the activities of their postsynaptic neurons in the cerebellar nuclei were abnormal in that the frequency and irregularity of their spiking patterns were enhanced. Thus, although the number of their terminals and their synaptic contacts appear quantitatively intact, Purkinje cells in tottering mice show several signs of axonal damage that may contribute to altered postsynaptic activities in the cerebellar nuclei.

Comparative analysis of cadherin expression and connectivity patterns in the cerebellar system of ferret and mouse.

The cerebellum shows remarkable variations in the relative size of its divisions among vertebrate species. In the present study, we compare the cerebella of two mammals (ferret and mouse) by mapping the expression of three cadherins (cadherin-8, protocadherin-7, and protocadherin-10) at similar postnatal stages. The three cadherins are expressed differentially in parasagittal stripes in the cerebellar cortex, in the portions of the deep cerebellar nuclei, in the divisions of the inferior olivary nucleus, and in the lateral vestibular nucleus. The expression profiles suggest that the cadherin-positive structures are interconnected. The expression patterns resemble each other in ferret and mouse, although some differences can be observed. The general resemblance indicates that cerebellar organization is based on a common set of embryonic divisions in the two species. Consequently, the large differences in cerebellar morphology between the two species are more likely caused by differential growth of these embryonic divisions than by differences in early embryonic patterning. Based on the cadherin expression patterns, a model of corticonuclear projection territories in ferret and mouse is proposed. In summary, our results indicate that the cerebellar systems of rodents and carnivores display a relatively large degree of similarity in their molecular and functional organization.

Gray matter injury associated with periventricular leukomalacia in the premature infant.

Neuroimaging studies indicate reduced volumes of certain gray matter regions in survivors of prematurity with periventricular leukomalacia (PVL). We hypothesized that subacute and/or chronic gray matter lesions are increased in incidence and severity in PVL cases compared to non-PVL cases at autopsy. Forty-one cases of premature infants were divided based on cerebral white matter histology: PVL (n = 17) with cerebral white matter gliosis and focal periventricular necrosis; diffuse white matter gliosis (DWMG) (n = 17) without necrosis; and "Negative" group (n = 7) with no abnormalities. Neuronal loss was found almost exclusively in PVL, with significantly increased incidence and severity in the thalamus (38%), globus pallidus (33%), and cerebellar dentate nucleus (29%) compared to DWMG cases. The incidence of gliosis was significantly increased in PVL compared to DWMG cases in the deep gray nuclei (thalamus/basal ganglia; 50-60% of PVL cases), and basis pontis (100% of PVL cases). Thalamic and basal ganglionic lesions occur almost exclusively in infants with PVL. Gray matter lesions occur in a third or more of PVL cases suggesting that white matter injury generally does not occur in isolation, and that the term "perinatal panencephalopathy" may better describe the scope of the neuropathology.

Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum.

Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so-called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin-R (TN-R), hyaluronan (HA), and link proteins, such as cartilage link protein 1 (Crtll). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN-R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and link proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin-binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtll, and HASs mRNAs. However, Crtll is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other link protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtll might be crucial elements for the initial assembly of PNNs.

Delayed development of adult-generated granule cells in dentate gyrus.

A substantial fraction of adult-generated granule cells in the dentate gyrus survive and integrate into the existing neuronal network. These newborn neurons must navigate the environment of the adult brain, a setting that is presumably less optimized for neuronal maturation compared with that in the developing brain. We used EGFP (enhanced green fluorescent protein) expression in newborn granule cells to compare the maturation of adult-generated granule cells to those generated in neonates. Labeled newborn granule cells had indistinguishable physiological properties in adults and neonates, indicating they were at the same functional stage. However, the maturation of adult-generated granule cells was slower than neonatal-generated granule cells. Depolarizing GABAergic network activity and transcription factor activation were reduced in adults relative to neonates, suggesting a role for neural activity in the maturation of newborn granule cells. Consistent with this idea, maturation was altered in mice lacking the GABA synthetic enzyme GAD65 (glutamic acid decarboxylase 65). Together, these results provide evidence that activity-dependent processes in the local environment influence the maturation of newborn granule cells.

Synaptic destabilization by neuronal Nogo-A.

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and beta-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules.

Activity-mediated shift in reversal potential of GABA-ergic synaptic currents in immature neurons.

Gamma-aminobutyric acid (GABA), which is inhibitory in the adult central nervous system, can be excitatory in the developing brain. The change from excitatory to inhibitory action of GABA during development is caused by a negative shift in its reversal potential. Here, we report a presynaptic activity-mediated negative shift in the reversal potential of the GABA-mediated synaptic currents in immature deep cerebellar nuclei neurons. This shift appears to be due to an increased expression and activation of the K+-Cl- co-transporter type 2 (KCC-2) through the activation of protein kinase A, protein synthesis and activation of protein phosphatases. Thus, maturation of the GABA response may rely on an activity-dependent up-regulation of KCC-2.

Postnatal cerebellar granule cells of the white rat (Rattus norvegicus): a quantitative study, using design-based stereology.

A stereological study was carried out on postnatal cerebellar granule cells of rats aged 6 and 10 days, for detecting whether and how much they would differ from those of young adult rats. The following parameters were estimated: number-weighted mean volume of the nucleus and of the soma; mean total surface area of the soma; mean absolute volumes per cell of total cytoplasm, mitochondria, Golgi apparatus, and cytosol; mean surface density of the rough endoplasmic reticulum (RER); mean total surface area of the RER. These values were compared between the two postnatal ages. In addition, those values were also analysed in comparison to the ones depicted in young adult rats (60 days), already published by our team, in order to detect similarities between them. It was noticed that, between 6 and 10 days, the mean surface density of the RER was the only parameter that did not change significantly. The comparison of each of the postnatal ages with 60 days revealed that, with the exception of the absolute volume of Golgi apparatus, significant differences were displayed concerning other organelles and cellular compartments. It was concluded that, although fine structural differences have been disclosed, from the stereological point of view postnatal granule cells at 10 days were practically similar to the young adult ones at 60 days. Some potential physiological implications have been considered.

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum.

Previous physiological and pharmacological studies have shown that the serotonin2A (5-HT2A) receptor is involved in cerebellar functions. However, the expression of 5-HT2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT2A receptors during the cerebellar development.