Yulin Liu

The correlation between cortical neuron maturation and neurofilament phosphorylation: a developmental study of phosphorylated 200 kDa neurofilament protein in cat visual cortex.

Neurofilament proteins are major constituents of the neuronal cytoskeleton. When phosphorylated, neurofilament proteins are thought to be involved in slowing down the slower component of axonal transport, thereby increasing neurofilament stability and conferring resistance to proteolysis. In order to investigate the correlation between phosphorylated neurofilament and plasticity of the cat visual cortex, we applied a monoclonal antibody, which recognizes only the phosphorylated form of neurofilament protein, to kitten visual cortex. This antibody appeared to recognize only axons. There was a progressive increase of phosphorylated neurofilament-immunoreactive axons in the cortical layers and white matter during development. In the visual cortex of young kittens, phosphorylated neurofilament immunoreactive processes were mostly long, fine, and continuous axons. However, in kittens older than 2 months of age, phosphorylated neurofilament immunoreactivity was characterized by short, punctae-like staining. The immunoreactive axons were most dense in deep cortical layers V and VI initially, followed at later ages by layers IVa and IVb, and finally in layer IVc and in the superficial cortical layers of adult animals. This developmental laminar pattern of distribution was not affected by early input restriction and coincides with electrophysiological evidence of early neuronal maturation. In addition, the phosphorylated neurofilament immunoreactivity can also be found in the dendrites and perikaya of degenerating neurons. These results suggest that phosphorylated neurofilament can be used as an index of functional maturity in the kitten visual cortex and also as a marker for neurodegeneration.

Involvement of muscarinic acetylcholine receptors in regulation of kitten visual cortex plasticity

Receptor autoradiographic methods specific for M3 and M1 muscarinic acetylcholine receptors were used to investigate the development and input-dependent laminar redistribution of these receptor populations during the critical period for kitten visual cortex plasticity. Analysis of the binding curves of [3H]4-diphenylacetyl-N-methyl-piperidine (4-DAMP) and [3H]pirenzepine (PZ) indicated that these two ligands bound heterogeneously to muscarinic acetylcholine receptors with different affinities. While [3H]4-DAMP showed a high affinity for M3 receptors and much lower affinities for M1 and M2 receptors, [3H]PZ displayed higher affinity for M1 receptors. By carefully choosing concentrations of labelled and unlabeled ligands, the patterns of laminar distribution for both receptor subtypes within visual cortex were obtained. Both receptors were most concentrated in cortical layer IV immediately after birth and during the most sensitive period of visual cortex plasticity. The binding density for both receptor subtypes thinned out progressively in this layer to concentrate in more superficial layers as plasticity waned with age. Moreover, interruption of visual or spontaneous input to visual cortex induced either by lesion or by tetrodotoxin infusion into lateral geniculate nucleus prevented the developmental redistribution of these receptors from layer IV to superficial layers, that is, the pattern of laminar distribution remained that of the age at which the lesion or tetrodotoxin infusion into the lateral geniculate nucleus was performed. The results indicate that the developmental expression of M3 and M1 muscarinic acetylcholine receptors in kitten visual cortex depends on cortical inputs.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and distribution of neurons and glial cells expressing ß-adrenergic receptors in developing kitten visual cortex

The morphology and distribution of cells expressing beta-adrenergic receptors has been studied in developing kitten visual cortex using a monoclonal antibody which recognizes both beta-1 and beta-2 adrenergic receptors. We found specific populations of neurons and glial cells which express beta-adrenergic receptor immunoreactivity in the kitten visual cortex. In adult animals, the receptors are most concentrated in the superficial and deep cortical layers (layers I, II, III and VI). About 50% of the stained neural cells in adult cat visual cortex are glial cells. Most of the immunoreactive neurons in layers III and V are pyramidal cells while those in layers II and IV are more likely to be nonpyramidal cells. In neonatal kittens, staining is weaker than that in adult cats and it appears to be concentrated in neurons of the deep cortical layers and in the subcortical plate and white matter. Only a few immunoreactive glial cells were found at this age. Receptor numbers increase after birth and by 24 days of age, the laminar distribution of beta-adrenergic receptors approaches that of adult animals. Immunoreactive glial cells in the white matter show a progressive increase in number throughout postnatal development.

Nerve growth factor induced modification of presynaptic elements in adult visual cortex in vivo

Nerve growth factor (NGF) has been shown to play important roles in neuronal survival, growth and differentiation. Recently, we have found that intracortical infusion of NGF into adult cat visual cortex can recreate ocular dominance plasticity, suggesting that NGF is also involved in activity-dependent modification of synaptic connectivity in the adult brain. To further explore the mechanisms of NGF-induced plasticity in adult visual cortex, we studied two presynaptic markers: GAP-43 and synaptophysin. Immunocytochemical staining showed that NGF-treatment of adult visual cortex selectively increased the level of the phosphorylated form of GAP-43, while the total level of GAP-43 was not changed. These results demonstrate that NGF-treatment stimulates phosphorylation processes of GAP-43 in vivo. In addition, NGF-treatment of adult visual cortex increased the level of synaptophysin immunoreactivity. Since the phosphorylated form of GAP-43 is known to be enriched in the membrane skeleton of growth cones and of developing synapses, and the phosphorylation of GAP-43 has been linked with events that underlie synaptic plasticity, and since synaptophysin is a major component of presynaptic vesicles, our results suggest that NGF-treatment of adult visual cortex modulates presynaptic terminals, possibly by inducing axonal sprouting and formation of new synapses, and that these changes may play a role in the NGF-induced functional plasticity.

Development and regulation of β adrenergic receptors in kitten visual cortex: An immunocytochemical and autoradiographic study

The developmental pattern and laminar distribution of beta 1 and beta 2 adrenergic receptor subtypes were studied in cat visual cortex with autoradiography using [125I]iodocyanopindolol as a ligand and also with immunocytochemistry using a monoclonal antibody directed against beta adrenergic receptors. In the primary visual cortex of adult cats, the laminar distributions of both beta 1 and beta 2 adrenergic receptors revealed by autoradiography were very similar, with concentrations in layers I, II, III and VI. In young kittens (postnatal days 1 and 10), fewer beta adrenergic receptors were present, and they were concentrated in the deep cortical layers (V-VI) and subcortical white matter. Between postnatal days 15 and 40, beta adrenergic receptors increased in density more quickly in the superficial layers than they did in the deep and middle cortical layers. By postnatal day 40, the adult pattern was achieved, with two bands of intense binding in the superficial and deep cortical layers and a lower density in layer IV. Immunocytochemical techniques applied to adult cat cortex showed that beta adrenergic receptor-like immunoreactivity was found in different populations of neurons and glial cells. The immunoreactive neural cells were most dense in layers II, III and VI. About 50% of these immunoreactive neural cells were glial cells, primarily astrocytes. Immunoreactive pyramidal cells were mostly located in layers III and V. In layer IV, many stellate cells were stained. Immunoreactive astrocytes in the subplate and white matter progressively increased in number during development until adulthood. The pattern of laminar distribution and the developmental process was not affected by interrupting noradrenergic innervation from locus coeruleus either before or after the critical period. However, when visual input was interrupted by lesions of the lateral geniculate nucleus in young kittens (postnatal day 10), the density of both beta adrenergic receptor subtypes decreased significantly in the deep cortical layers. Lateral geniculate nucleus lesions in adult cats resulted in a pronounced decrease in beta adrenergic receptor density in layer IV.

Development and regulation of alpha adrenoceptors in kitten visual cortex

Alpha-1 and alpha-2 adrenergic receptors were localized in developing cat visual cortex by using [3H]prazosin and [3H]rauwolscine, respectively as selective ligands. The effects of neuronal input on the development of the two receptor subtypes were also studied in animals with lesions at various sites within the central visual pathways. Binding densities for both ligands increased during the first few postnatal weeks and declined thereafter. For both receptor subtypes, the highest concentration of binding sites was found in the subplate zone of the cortex in neonatal animals. Both ligands showed their highest concentrations in cortical layer IV beginning at postnatal day 30 and in the superficial cortical layers in adulthood. However, the developmental redistribution of alpha-1 receptors began at earlier ages than that of the alpha-2 sites. The alpha-1 sites were still concentrated in the subplate zone up to 60 days postnatal, while the alpha-2 sites in this region disappeared much earlier. Receptor binding densities were also examined in animals with quinolinic acid lesions within cortex, lesions of the lateral geniculate nucleus and lesions of the optic tract. The results indicate that both alpha-adrenoceptor subtypes were mainly located on cortical cells, and that the absence of neuronal activity during development resulted in a reduction of the binding density for both subtypes in the visual cortex. An additional major reduction in alpha-2 but not alpha-1 binding sites was observed following the lateral geniculate nucleus lesion, suggesting that the development of alpha-2 receptors is also dependent on input from the lateral geniculate nucleus. Removal of the lateral geniculate nucleus early in life resulted in a significant increase in alpha-1 receptors in the subplate region, indicating that receptor densities in this zone may be negatively regulated by the lateral geniculate nucleus afferents. These results show that adrenergic receptors reorganize during postnatal cortical development with a strong temporary concentration in the subplate zone. The reorganization process is heavily influenced by cortical inputs.

An improved staining technique for cytochrome C oxidase.

Cytochrome C oxidase (CO) has been shown to be an indicator of neuronal activity in the brain. In the primate visual cortex, CO staining also differentiates cell populations encoding visual properties such as color, contrast, ocularity, and movement. We have developed a modified method which dramatically enhances the intensity and contrast of CO staining. This method can be applied to both fixed and non-fixed tissues. The sensitivity of this method is sufficiently high that, even after years of storage, tissues can still be well stained for CO activity. Such tissue is poorly stained with current methods. This CO staining technique may also be useful for double labeling of CO with other anatomical markers.

Postnatal development of inositol 1,4,5-trisphosphate receptors: a disparity with protein kinase C

Ligand-stimulated phosphoinositide hydrolysis activates a bifurcating second messenger system, releasing inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG), which activates protein kinase C (PKC). Yet, in developing cat visual cortex and hippocampus, high levels of [3H]PDBu binding (labelling PKC) appear much earlier than do [3H]IP3 labelled sites. Binding distributions for the two ligands also appear to be complimentary in both brain regions. Moreover, early surgical removal of input to the visual cortex increases [3H]PDBu binding without affecting that of [3H]IP3. Our results suggest that, (1) at certain developmental stages, IP3 and PKC may act individually or complimentarily rather than synergistically in the visual cortex and hippocampus; (2) in neonatal cortex, IP3 metabolites rather than IP3 itself may act as second messengers; (3) although both IP3 receptors and PKC are localized in intracortical cells, their expression is regulated by different mechanisms during development.

Postnatal development and laminar distribution of noradrenergic fibers in cat visual cortex

Previous studies have indicated that adrenergic receptors show significant changes either in laminar distribution or in number during the critical period of kitten visual cortex development. In order to further investigate the postnatal development of this neurotransmitter system, especially in relation to the critical period, we used a polyclonal antibody against dopamine-beta-hydroxylase to localize noradrenaline-containing afferents in visual cortex of kittens of various ages from birth to adulthood. In young kittens, less than 2 weeks of age, noradrenergic fibers were sparse, short and randomly oriented, and were concentrated in layer I and in deep cortical layers V and VI. By postnatal day 40, the fibers were present throughout all cortical layers and exhibited higher densities in layers I, II, III, V and VI, with a band of lower staining in layer IV. While tangential fibers predominated in layers I, V and VI, relatively straight radial fibers traversed layers II and III. After postnatal day 40, we did not find major changes in the laminar distribution of adrenergic fibers. This developmental laminar distribution pattern of adrenergic fibers resembles that of the beta-adrenergic receptors that we and others have studied in kitten visual cortex, but differs from that of alpha-adrenergic receptors.

A study of tachykinin-immunoreactivity in the cat visual cortex

The localization of tachykinin-immunoreactivity in the cat visual cortex (area 17) was investigated using immunohistochemical methods. Strong laminar specificity was observed, with immunoreactivity highest in layer V, followed by layers I, VI, II and III, and the lowest density in layer IV. Most of the immunoreactive product was localized in neuronal processes. A few immunopositive cell bodies were also present. The immunopositive neurons were non-pyramidal, multipolar, or bipolar in shape, and mostly found in layer V. There were particularly dense immunopositive fibers and varicosities around somata in layer V. These may represent tachykinin-containing presynaptic terminals (boutons). The results provide anatomical evidence that tachykinins may primarily affect layer V neurons in the cat visual cortex.