Changes in expression of P2X purinoceptors in rat cerebellum during postnatal development

Changes in expression of P2X receptors (P2X1-7) during postnatal development of the rat cerebellum are described. At P3, immunoreactivity (ir) to all the P2X receptors, except for P2X3 receptors, was found in Purkinje cells and deep cerebellar nuclei, P2X5-ir being most prominent. Granular and microglial cells were labeled for P2X5 (weakly) and P2X4 receptors, respectively. At P7, expression of all the P2X receptors (with the exception of P2X3) was up-regulated, P2X5 and P2X6 receptors being most prominent. Scattered P2X receptor-ir in unipolar brush cells in the granular cell layer and P2X1- and P2X7-ir of microglial cells was also present. At P14, the dendritic trees of Purkinje cells were intensely labeled by P2X1-7 receptor antibodies, except for P2X3, while P2X1, P2X4 and P2X7 receptor immunostaining in microglial cells and P2X5 receptor immunostaining in granular cells was up-regulated. At P21, expression of all P2X receptors (except P2X3) was down-regulated in the Purkinje cells and deep cerebellar nuclei; P2X1, P2X4 and P2X7 receptors-ir was present in microglial cells. In contrast, expression of P2X5-ir in granular cells was up-regulated. At P60, expression levels of all the P2X receptors (except P2X3) were similar with those at P21. In double-labeling experiments, almost all the P2X-ir Purkinje cells were immunoreactive for calbindin-D28k, while 60-80% of P2X-ir cells in the granular cell layer were immunoreactive for calretinin. The possible short- and long-term functional significance of the changes in expression of P2X receptors during postnatal development is discussed.     

Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development

Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERβ mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.     

Functional synaptic circuits in the subplate during fetal and early postnatal development of cat visual cortex

Among the first postmitotic cells of the cerebral cortex is a special population located below the cortical plate: the subplate neurons. These neurons reach a high degree of morphological maturity during fetal life, well before the neurons of the cortical layers have matured, yet nearly all of these cells die after birth in the cat. Subplate neurons are also known to receive synaptic contacts. Here we have investigated whether these contacts are functional by making intracellular recordings from subplate neurons in cortical slices maintained in vitro. Subplate neurons were identified based on their location and morphology by injecting them with biocytin following the intracellular recordings. At all ages studied between embryonic day 50 and postnatal day 9, electrical stimulation of the optic radiations elicited EPSPs and synaptic and antidromic spikes in subplate neurons, indicating that some of the synapses seen at the ultrastructural level are indeed capable of synaptic transmission. The spiking patterns of 39 morphologically identified subplate neurons were examined by injecting depolarizing current, which revealed that a large majority gave only a single spike or a brief train of spikes in response to maintained depolarization, in contrast to the regular spiking pattern found in many neurons of adult cortex. Biocytin injections into subplate neurons revealed that they are a morphologically heterogeneous population with respect to their dendritic branching patterns; roughly half were inverted pyramids, the classic subplate neuron morphology. The axonal processes of subplate neurons were remarkable in that many not only arborized within the subplate, but also entered the cortical plate and terminated in the marginal zone. At early postnatal ages, these axons also gave off collaterals within cortical layer 4. The results of this study indicate that subplate neurons participate in synaptic microcircuits during development. While the presynaptic identity of the input to subplate neurons is not known conclusively, it is likely that geniculocortical axons, which wait in close proximity to subplate neurons, contribute significantly. The pattern of axonal branching of subplate neurons also implies that information conferred to subplate neurons may be relayed, in turn, to the neurons of cortical layer 4. Finally with the death of subplate neurons, the geniculocortical axons leave the subplate and invade the cortical plate to innervate directly the neurons of layer 4. Thus, subplate neurons may function as a crucial, but transient synaptic link between waiting geniculocortical axons and their ultimate target cells in the cortex.     

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.     

Morphology and quantitative changes of transient NPY-ir neuronal populations during early postnatal development of the cat visual cortex

The early postnatal development of neuropeptide Y-containing neurons in the visual cortex of the cat was analyzed. Immunohistochemistry reveals several stages of morphological differentiation and degeneration. Completely undifferentiated neurons have very small somata with nuclei surrounded by a thin rim of cytoplasm and processes unclearly differentiated into dendrites and axons. Processes bear growth cones. Differentiating neurons show an increase in soma size and complexity of processes. Axons are recognizable. Fully differentiated neurons have well-defined axonal and dendritic patterns. Degenerating neurons are identified by thick, heavily beaded processes covered by hairy appendages and vacuolar inclusions in the somata. Cell death is expressed by shrunken somata and lysed, fragmented processes. According to their postnatal time course of differentiation and/or degeneration, NPY-immunoreactive neurons, which form several morphologically distinct cell types, are grouped into 3 neuronal populations. (1) Pseudopyramidal cells, bitufted "rectangular" cells with wide dendritic fields, unitufted cells, and small multipolar cells are located in the gray matter and have a rather primitive morphology resembling cell types found in lower vertebrate cortex and tectum. They constitute a first transient neuronal population, because all neurons are fully differentiated at birth and become largely eliminated by postnatal day (P) 12. (2) Axonal loop cells are mainly located in the white matter. Their most prominent feature is an often long hairpin loop formed by either the main axon itself or by a major collateral. The axonal branches pass through the cortex to connect the white matter and layer I. Axons do not form local plexusses and terminal elements in the gray matter. Neurons differentiate perinatally, form a first peak from P6 to P10, followed by a decrease in cell number and innervation density at P12, followed by a second peak from P15 to P20. After P20 the number of axonal loop cells steadily decreases, and they become eliminated by P48. (3) A third population consists of neurons with a higher degree of axonal ramification and a variety of axonal patterns. Early members are located mainly at the layer VI/white matter border, differentiate during the first postnatal week, and give rise to a diffuse innervation of the gray matter without forming specific terminal elements. Some of the early axonal patterns persist into adulthood, whereas others are not found in the adult brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Astrocytes in cat visual cortex studied by GFAP and S-100 immunocytochemistry during postnatal development.

A monoclonal antibody to glial fibrillary acidic protein (GFAP) and a polyclonal antiserum to the S-100 protein were used to study the expression of these astrocytic proteins in the postnatal visual cortex of the cat. Three changes in antigen expression of these astroglial markers could be distinguished over development. First, the density of cells in the white matter, which are heavily labelled with both antibodies from birth until adulthood, diminishes after the third postnatal weeks. By intracellular filling with Lucifer Yellow the reduction of the cell density can be attributed to the disappearance of large astrocytes with a morphology of transforming radial glia, present only in early postnatal development. Second, heavily labelled, large cells present in the grey matter at the seventh postnatal day have disappeared by the fifth postnatal week. On the basis of their morphology these cells can also be classified as radial glial cells. Finally, astroglial cells of the adult-like stellate form appear to be labelled in the cortical layers between the third and seventh postnatal weeks. While the density of these cells and the S-100 immunoreactivity of the cell bodies is adult-like at the fourth postnatal week, there is a gradual increase of the staining intensity with the GFAP antibody up to the seventh postnatal week. This developmental period is paralleled by the appearance of S-100-positive astrocytic processes. The gradual expression of GFAP immunoreactivity and the increased expression of S-100 is interpreted as reflecting the time course of astrocytic maturation. A possible relation of the maturation of astrocytes and cortical development, both of which are prominent in the time period between the third and seventh postnatal week, is discussed.     

Down-regulation of 14-3-3 eta gene expression by IGF-I in mouse cerebellum during postnatal development

Insulin-like growth factor I (IGF-I) overexpression in the postnatal cerebellum of transgenic (Tg) mice results in remarkable cerebellar overgrowth characterized by a near doubling of granule cell number that is predominantly due to inhibition of apoptosis. Using this Tg model we set out to investigate IGF-I anti-apoptotic mechanisms by defining the influence of IGF-I on gene expression. Using a cDNA array technique, we screened a total of 243 mouse apoptosis-related genes, and found that 14-3-3 eta gene expression was significantly reduced in the cerebella of Tg mice compared with their wild-type (Wt) littermates. Using Northern blot analysis to corroborate our microarray finding, we showed that 14-3-3 eta mRNA abundance was decreased from postnatal day P5 through P17. Nonetheless, the expression pattern of 14-3-3 eta in Tg mice followed the same pattern observed in Wt mice, and was indistinguishable from that in Wt mice at P20 and P23. 14-3-3 eta protein abundance, as determined by Western immunoblot analyses, showed similar decreases in the cerebella of Tg mice. In situ hybridization demonstrated that 14-3-3 eta was predominantly, if not exclusively, expressed and regulated in Purkinje cells. 14-3-3 proteins have multiple functions, including participation in pathways that favor cell survival. Our finding of IGF-I-induced down-regulation of 14-3-3 eta expression in Purkinje cell at a time when IGF-I promotes granule cell survival leads us to speculate that down-regulation of 14-3-3 eta may: (a) serve a negative feedback role to modulate Purkinje cell survival, i.e. limit Purkinje cell number, and/or (b) function as part of a distinct signaling mechanism, perhaps one that augments the capacity of Purkinje cells to promote granule cell survival.     

Alterations in the laminar distribution of pentagastrin binding sites in cat visual cortex during postnatal development

The distribution and characteristics of [3H]pentagastrin (CCK-5) binding sites were examined in the visual cortex of cats of varied age. CCK-5-labelled binding sites in a highly age-dependent, laminar-specific manner. In young kittens, CCK-5 binding sites were found dominantly in layers IV-VI. During the first 3 months of postnatal development the laminar pattern of binding changed so that by 95 days postnatal, layers I-III and VI were the most densely labelled. CCK-5 binding sites appear to be members of a class of receptors which exhibit laminar alterations in their distribution during early postnatal development.     

The muscarinic agonist xanomeline increases monoamine release and immediate early gene expression in the rat prefrontal cortex.

BACKGROUND: The muscarinic agonist xanomeline has been shown to reduce antipsychotic-like behaviors in patients with Alzheimer's disease. Because atypical antipsychotic agents increase dopamine release in prefrontal cortex and induce immediate early gene expression in prefrontal cortex and nucleus accumbens, the effect of xanomeline was determined on these indices. METHODS: The effect of xanomeline on extracellular levels of monoamines in brain regions was determined using a microdialysis technique, and changes in expression of the immediate early genes c-fos and zif/268 in brain regions were evaluated using in situ hybridization histochemistry. RESULTS: Xanomeline increased extracellular levels of dopamine in prefrontal cortex and nucleus accumbens but not in striatum. Xanomeline increased expression of c-fos and zif/268 in prefrontal cortex and nucleus accumbens. There was no change in immediate early gene expression in striatum. CONCLUSIONS: Xanomeline increased extracellular levels of dopamine, which is similar to the effects of the atypical antipsychotics clozapine and olanzapine. The regional pattern of immediate early gene expression induced by xanomeline resembled that of atypical antipsychotic agents. Based on the antipsychotic-like activity of xanomeline in Alzheimer's patients and the similarity to atypical antipsychotic agents, we suggest that xanomeline may be a novel antipsychotic agent.     

Morphometric analysis of phrenic motoneurons in the cat during postnatal development.

The dendritic geometry of 20 phrenic motoneurons from four postnatal ages (2 weeks, 1 and 2 months, and adult) was examined by using intracellular injection of horseradish peroxidase. The number of primary dendrites (approximately 11-12) remained constant throughout postnatal development. In general, postnatal growth of the dendrites resulted from an increase in the branching and in the length and diameter of segments at all orders of the dendritic tree. There was one exception. Between 2 weeks and 1 month, the maximum extent of the dendrites increased in parallel with the growth of the spinal cord; however, there was no increase in either combined dendritic length or total membrane surface area. In addition, there was a significant decrease in the number of dendritic terminals per cell (59.8 +/- 9.3 vs. 46.4 +/- 7.4 for 2 weeks and 1 month, respectively). The distance from the soma, where the peak number of dendritic terminals per cell occurred, ranged from 700-900 microns at 2 weeks and 2 months to 1,300-1,700 microns in the adult. The diameter of dendrites as a function of distance from the soma along the dendritic path increased with age. The process of maturation tended to increase the distance from the soma over which the surface area and dendritic trunk parameter (sigma d1.5/D1.5) remained constant. The three-dimensional distribution of dendrites was analyzed by dividing space into six equal volumes or hexants. This analysis revealed that the postnatal growth in surface area in the rostral and caudal hexants was proportionately larger than that in either the medial, lateral, dorsal, or ventral hexants. Strong linear correlations were found between the diameter of the primary dendrite and the combined length, surface area, volume, and number of terminals of the dendrite at all ages studied.     

Differential expression of the estrogen receptors alpha and beta during postnatal development of the rat cerebellum

Estrogen receptor (ER) beta is a dominant ER subtype in the adult cerebellum. However, it is not known if this is also the case for the developing cerebellum. In the present study, quantitative real-time RT-PCR demonstrated that levels of cerebellar ERalpha mRNA in neonatal pups were significantly higher than in adults. In contrast, expression levels of cerebellar ERbeta mRNA remained significantly unchanged during postnatal development. In situ hybridization and immunohistochemistry demonstrated that ERalpha mRNA and protein were predominantly expressed by Purkinje cells at all ages examined. ERalpha-expressing Purkinje cells were confined to the anterior lobes at postnatal day 7 (P7) but distributed in most lobes at P14 and P21. In the adult cerebellum, however, only a few ERalpha-immunoreactive Purkinje cells were observed. Thus, ERalpha expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation proceed, suggesting that a role for ERalpha in Purkinje cell differentiation. ERbeta expression occurred in Golgi type neurons in the granular layer at P7, Purkinje cells at P14, and basket cells in the molecular layer at P21 and was detected in all the cell types in the adult cerebellum, suggesting a role for ERbeta associated with neuronal differentiation and maintenance. Furthermore, double-labeled immunofluorescence for ERalpha and ERbeta demonstrated their colocalization in Purkinje cells at P14, suggesting a possibility of their interaction. The discrete expression profiles for ERalpha and ERbeta in the developing cerebellum suggest the two ERs play distinct roles in cerebellar development.     

SMI-32 immunoreactivity in human striate cortex during postnatal development.

SMI-32, an antibody which recognizes the non-phosphorylated epitopes on the neurofilament proteins was used to study the morphological changes in the human striate cortex during postnatal development. Striate cortices from 12 autopsied patients with ages ranging from 1 day to 70 years were obtained. Using the avidin-biotin-peroxidase method, the first SMI-32 immunoreactive neurons were identified at sublayers Vb/VIa on the first postnatal day. At 5 months, the next group of neurons to develop immunoreactivity were in IVb. By 15 months, SMI-32 immunoreactive neurons were observed at III, IVa, IVb, V and VI. The changes in SMI-32 immunoreactivity (ir) were stabilized from 3 years and after. The SMI-32 ir in the striate cortex could be a useful morphological correlate for studying developmental diseases affecting the neocortex.     

Quantitative analysis of the postnatal development of Purkinje neurons in the cerebellum of the cat

We have studied the postnatal quantitative changes of cortical Purkinje neurons in the cerebellum of the cat at the following postnatal groups of age: P0, P42, P72 and adults. An unbiased counting method, the optical fractionator was used for the estimation of Purkinje cell numbers. A significant increase of Purkinje cell number was found between P0 (1.097 x 10(6)) and P42/P72 (1.805 x 10(6) and 1.895 x 10(6)) declining to 1.429 x 10(6) in the adult, still 30% higher than in the newborn. It was also observed that during the first few postnatal weeks large "gaps" were present in the Purkinje monolayer as revealed by Nissl staining and metabotropic glutamate receptor 1alpha immunocytochemistry. These Purkinje cell gaps were observed most frequently in well-definable areas, especially in the intermediate zone of the neocerebellum. Simultaneously with the numerical increase of Purkinje neurons between the P0 and P72 age groups, these gaps disappeared after the third postnatal week resulting in the completion of the Purkinje monolayer in the whole cerebellum.     

Differential effects of quinolinic acid lesions on muscarinic acetylcholine receptors in cat visual cortex during postnatal development

Quinolinic acid (QA) lesions of neurons in cat visual cortex were combined with conventional in vitro autoradiographic methods in order to define the cellular locus of the muscarinic acetylcholine receptor (mAChR). Animals of various postnatal ages had QA unilaterally injected into the visual cortex. Four to fourteen days later they were sacrificed and processed for electron microscopy (EM) or in vitro autoradiography. QA lesions at the various postnatal ages were found to eliminate intrinsic cortical neurons and their processes while leaving intact glia, fibers of passage and axon terminals from outside the lesion zone. Autoradiograms of visual cortex labelled with [3H]QNB (which labels M1 and M2 subtypes) showed an age-dependent loss of binding sites, with the greatest decreases occurring after 65 days postnatal. Examined separately, only the M1 mAChRs labelled with [3H]pirenzepine exhibited these age-dependent alterations. The results indicate a differential distribution of the M1 mAChRs during postnatal development. The loss of receptors late in postnatal life following QA suggests a dominantly neuronal locus; the relatively small loss early in postnatal life suggests a locus on other cellular elements.     

Region-specific gene expression in early postnatal mouse thalamus

Previous studies in the developing mouse thalamus have demonstrated that regional identity is established during early stages of development (Suzuki-Hirano et al. J. Comp. Neurol. 2011;519:528-543). However, the developing thalamus often shows little resemblance to the anatomical organization of the postnatal thalamus, making it difficult to identify genes that might mediate the organization of thalamic nuclei. We therefore analyzed the expression pattern of genes that we have identified as showing regional expression in embryonic thalamus on postnatal days (P) 6-8 by using in situ hybridization. We also identified several genes expressed only in the postnatal thalamus with restricted expression in specific nuclei. We first demonstrated the selective expression of neurotransmitter-related genes (vGlut2, vGAT, D2R, and HTR2C), identifying the neurotransmitter subtypes of cells in this region, and we also demonstrated selective expression of additional genes in the thalamus (Steel, Slitrk6, and AI852580). In addition, we demonstrated expression of genes specific to somatosensory thalamic nuclei, the ventrobasal posterior nuclei (VP); a visual thalamic nucleus, the dorsal lateral geniculate nucleus (dLGN); and an auditory thalamic nucleus, the medial geniculate body (MGB) (p57Kip, Nr1d1, and GFRα1). We also identified genes that are selectively expressed in multiple different nuclei (Foxp2, Chst2, and EphA8). Finally, we demonstrated that several bone morphogenetic proteins (BMPs) and their inhibitors are expressed in the postnatal thalamus in a nucleus-specific fashion, suggesting that BMPs play roles in the postnatal thalamus unrelated to their known role in developmental patterning. Our findings provide important information for understanding the mechanisms of nuclear specification and connectivity during development, as well as their maintenance in adult thalamus.     

Early postnatal development of cholecystokinin-immunoreactive structures in the visual cortex of the cat

The early postnatal development of cholecystokinin-immunoreactive (CCK-ir) neurons was analyzed in visual areas 17 and 18 of cats aged from postnatal day 0 to adulthood. Neurons were classified mainly by axonal criteria. According to their chronology of appearance neurons are grouped into three neuronal populations. The first population consists of five cell types which appear perinatally in areas 17 and 18. Four of them have axons terminating in layer VI. Neurons with columnar dendritic fields of layers IV and V display a conspicuous dendritic arborization with the long dendrites always arranged parallel to each other. This way they form a vertically oriented dendritic column. The neurons differentiate at around P 2 and are present until the end of the second postnatal week. They disappear possibly by degeneration and cell death. Multipolar neurons of layer VI have long dendrites and axonal domains of up to 800 micron in diameter. Three percent of these neurons send out two axons instead of only one. Neurons differentiate at P 0 and the cell type persists into adulthood. Bitufted to multipolar neurons of layer V constitute a frequent type; 10% of these cells issue two axons. They differentiate at P 2 and the type survives into adulthood. Bitufted to multipolar neurons of layers II/III appear at P 2 and send their axons into layer VI. So, early postnatally an axonal connection from superficial cortical layers to layer VI is established. The cell type persists into adulthood. The fifth cell type of the first population is constituted by the neurons of layer I with intralaminar axons which differentiate at P 2. Although they derive from the early marginal zone, the cell type survives into adulthood. The second population consists of two cell types which appear around the end of the second and during the third postnatal week in areas 17 and 18. Multipolar neurons of layer II have horizontally or obliquely arranged basket axons which, during the second postnatal month, form patches of high fiber and terminal density along the layer I/II border. Neurons with descending main axons issuing horizontal and oblique collaterals of layers II-IV form broad axonal fields. The third population in area 17 is constituted by three cell types: Bitufted neurons with axons descending in form of loose bundles of layers II/III differentiate during the fifth postnatal week. Small basket cells of layers II/III with locally restricted axonal plexuses and somewhat larger basket cells of layer IV appear during the sixth and seventh week.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early postnatal development of functional ocular dominance columns in cat primary visual cortex

During postnatal development of the visual cortex the thalamocortical afferents serving the two eyes segregate into alternating patches called ocular dominance (OD) columns. Interested in the dynamics of this segregation process we studied the appearance of functional OD columns in the primary visual cortex of normally raised and strabismic kittens aged 2-6 weeks using 2-deoxyglucose labelling in awake animals. In both experimental groups, OD columns covering the entire area 17 and spanning all cortical laminae are first visible at 3 weeks and appear already adult-like at 4 weeks, much earlier than thought on the basis of previous anatomical studies. We hypothesize that a small and anatomically undetectable imbalance between the afferents from the two eyes is amplified by intracortical interactions so that their activity patterns become different and may guide the segregation process of the afferents in cortical layer IV.