Gene expression patterns during enhanced periods of visual cortex plasticity.

During a critical period in its postnatal development the mammalian visual cortex displays susceptibility to experience-dependent alterations of neuronal response properties. Plasticity represents an integrated set of developmental processes controlled by a transcriptional hierarchy that coordinates the action of many genes. To illuminate the expression of these critical genes, we examined gene expression patterns of 18371 non-redundant cDNAs in the visual cortex of cats at birth, at eye opening, at the peak of the critical period of eye dominance plasticity and in the adult cat using filter-based cDNA arrays and software-based hierarchical cluster analysis. We identified a small set of genes that were selectively expressed during the peak of the critical period for plasticity. We further examined the patterns of expression of these genes by analyzing the gene expression pattern of dark-reared chronologically older animals that are known to retain this ocular dominance plasticity beyond the chronologically defined critical period. This additional cluster assessment allowed us to separate age-related changes in the patterns of gene expression from plasticity-related changes, thus identifying a subset of genes that we define as plasticity candidate genes. Those plasticity candidate genes that have previously characterized functions include participants in second messenger systems, in cell adhesion, in transmitter recycling and cytokines, among others. Comparison of cDNA array quantitation with reverse transcription-polymerase chain reaction showed almost identical expression profiles for three genes that we examined. The expression pattern of one identified gene, opioid binding cell adhesion molecule, from the cDNA array analysis, is also in agreement with immunocytochemical results. We conclude that the approach of high-density cDNA array hybridization can be used as a useful tool for examining a complex phenomenon of developmental plasticity since it is amenable to multiple developmental stage gene expression comparisons.

Heme oxygenase-1 plays an important protective role in experimental autoimmune encephalomyelitis.

Increasing evidence shows that oxidative stress plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of the human disease, multiple sclerosis (MS). Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes heme to the antioxidant bilirubin and carbon monoxide, and represents a powerful endogenous defensive mechanism against free radicals in many diseases. However, the role of this important enzyme in EAE remains unknown. In this study, we showed high expression of HO-1 in lesions of EAE, and demonstrated that hemin, an inducer of HO-1, inhibited EAE effectively. In contrast, tin mesoporphyrin, an inhibitor of HO-1, markedly exacerbated EAE. Our results suggest that endogenous HO-1 plays an important protective role in EAE, and that targeted induction of HO-1 overexpression may represent a new therapy for the treatment of multiple sclerosis.

Pyruvate released by astrocytes protects neurons from copper-catalyzed cysteine neurotoxicity.

We have found previously that astrocytes can provide cysteine to neurons. However, cysteine has been reported to be neurotoxic although it plays a pivotal role in regulating intracellular levels of glutathione, the major cellular antioxidant. Here, we show that cysteine toxicity is a result of hydroxyl radicals generated during cysteine autoxidation. Transition metal ions are candidates to catalyze this process. Copper substantially accelerates the autoxidation rate of cysteine even at submicromolar levels, whereas iron and other transition metal ions, including manganese, chromium, and zinc, are less efficient. The autoxidation rate of cysteine in rat CSF is equal to that observed in the presence of approximately 0.2 microm copper. In tissue culture tests, we found that cysteine toxicity depends highly on its autoxidation rate and on the total amount of cysteine being oxidized, suggesting that the toxicity can be attributed to the free radicals produced from cysteine autoxidation, but not to cysteine itself. We have also explored the in vivo mechanisms that protect against cysteine toxicity. Catalase and pyruvate were each found to inhibit the production of hydroxyl radicals generated by cysteine autoxidation. In tissue culture, they both protected primary neurons against cysteine toxicity catalyzed by copper. This protection is attributed to their ability to react with hydrogen peroxide, preventing the formation of hydroxyl radicals. Pyruvate, but not catalase or glutathione peroxidase, was detected in astrocyte-conditioned medium and CSF. Our data therefore suggest that astrocytes can prevent cysteine toxicity by releasing pyruvate.

Laminar distribution of cholinergic- and serotonergic-dependent plasticity within kitten visual cortex.

Both cholinergic and serotonergic modulatory projections to mammalian striate cortex have been demonstrated to be involved in the regulation of postnatal plasticity, and a striking alteration in the number and intracortical distribution of cholinergic and serotonergic receptors takes place during the critical period for cortical plasticity. As well, agonists of cholinergic and serotonergic receptors have been demonstrated to facilitate induction of long-term synaptic plasticity in visual cortical slices supporting their involvement in the control of activity-dependent plasticity. We recorded field potentials from layers 4 and 2/3 in visual cortex slices of 60--80 day old kittens after white matter stimulation, before and after a period of high frequency stimulation (HFS), in the absence or presence of either cholinergic or serotonergic agonists. At these ages, the HFS protocol alone almost never induced long-term changes of synaptic plasticity in either layers 2/3 or 4. In layer 2/3, agonist stimulation of m1 receptors facilitated induction of long-term potentiation (LTP) with HFS stimulation, while the activation of serotonergic receptors had only a modest effect. By contrast, a strong serotonin-dependent LTP facilitation and insignificant muscarinic effects were observed after HFS within layer 4. The results show that receptor-dependent laminar stratification of synaptic modifiability occurs in the cortex at these ages. This plasticity may underly a control system gating the experience-dependent changes of synaptic organization within developing visual cortex.

Identification of differentially expressed genes in the visual structures of brain using high-density cDNA grids.

The hybridization patterns of 18,371 high-density-grid-arrayed non-redundant complementary DNA (cDNA) clones were examined using three different sources of cDNA probes. The first set of probes was synthesized from mRNA isolated from visual brain areas MT and V4 of Vervet monkey. The second set of probes was derived from cDNA libraries constructed from two micro dissected sets of layers of the monkey Lateral Geniculate Nucleus layers within the visual pathway, namely the magnocellular and parvocellular layers. The third set of cDNA probes was synthesized from the subtracted fractions of the cDNAs enriched for either the magnocellular or the parvocellular layers of the Lateral Geniculate Nucleus. Software, linked directly to the Genbank database, was developed to aid in the rapid identification of both expressed and differentially expressed genes. Our results indicate that both the cDNA probes synthesized from mRNA and cDNA libraries can identify similar fractions of expressed genes. However, the subtracted cDNA probes improve the efficiency of detection for those genes that are expressed at much lower abundance. Analyses of these results for the differential expression patterns of these genes were validated by semi-quantitative PCR on the DNA derived from the whole tissue cDNA libraries. A list of some known genes that are statistically differentially expressed within the magnocellular layers of the LGN and area MT in the primate visual areas is derived.

The auditory motion aftereffect: its tuning and specificity in the spatial and frequency domains.

In this paper, the auditory motion aftereffect (aMAE) was studied, using real moving sound as both the adapting and the test stimulus. The sound was generated by a loudspeaker mounted on a robot arm that was able to move quietly in three-dimensional space. A total of 7 subjects with normal hearing were tested in three experiments. The results from Experiment 1 showed a robust and reliable negative aMAE in all the subjects. After listening to a sound source moving repeatedly to the right, a stationary sound source was perceived to move to the left. The magnitude of the aMAE tended to increase with adapting velocity up to the highest velocity tested (20 degrees/sec). The aftereffect was largest when the adapting and the test stimuli had similar spatial location and frequency content. Offsetting the locations of the adapting and the test stimuli by 20 degrees reduced the size of the effect by about 50%. A similar decline occurred when the frequency of the adapting and the test stimuli differed by one octave. Our results suggest that the human auditory system possesses specialized mechanisms for detecting auditory motion in the spatial domain.

Confocal imaging of N-methyl-D-aspartate receptors in living cortical neurons.

The fluorescence-conjugated N-methyl-D-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl-D-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17-20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10-15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl-D-aspartate receptor-associated polyamine site. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl-D-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl-D-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of fluorescently labeled clusters compared with the control group, neurons in the high potassium group exhibited a higher number of fluorescently labeled receptor clusters. These results suggest that more active neurons may tend to form more N-methyl-D-aspartate synapses during early development.

Astrocytes provide cysteine to neurons by releasing glutathione.

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.

Columnar distribution of serotonin-dependent plasticity within kitten striate cortex.

Recent studies have identified the potential for an important role for serotonin (5-HT) receptors in the developmental plasticity of the kitten visual cortex. 5-HT(2C) receptors are transiently expressed in a patchy fashion in the visual cortex of kittens between 30-80 days of age complementary to patches demarcated by cytochrome oxidase staining. 5-HT, operating via 5-HT(2C) receptors, increases cortical synaptic plasticity as assessed both in brain slices and in vivo. Herein, we report that bath application of 5-HT substantially increases the probability of long-term potentiation within 5-HT(2C) receptor-rich zones of cortex, but this effect is not observed in the 5-HT(2C) receptor-poor zones. Instead, in these zones, 5-HT application increases the probability of long-term depression. These location-specific effects of 5-HT may promote the formation of compartment-specific cortical responses.

Effects of astrocytes on neuronal attachment and survival shown in a serum-free co-culture system.

In order to study the neurosupportive effects of glial cells, we optimized a glial-neuron non-contact co-culture method. Astrocyte conditioned medium (ACM) and an astrocyte feeder layer were used to promote neuronal attachment and neuronal survival respectively. Neuron-enriched cultures were prepared from cortices of E-18 day rat embryos. Instead of plating cells in serum-supplemented medium, as an indispensable first-step procedure for many serum-free culture protocols, we found that coating the coverslips briefly with ACM was sufficient for the healthy attachment and neurite outgrowth of the dissociated neurons in serum-free medium. A high survival rate of the low density (4x10(4) cells/cm(2)) neuronal cultures was achieved by co-culturing primary neurons with an astrocyte feeder layer. This non-contact co-culture method could be easily implemented with ordinary culture dishes. Our serum-free chemically defined medium was MEM supplemented with insulin, transferrin, selenium and pyruvate. In this serum-free medium, glial cells did not proliferate and a neuron-enriched population was obtained without the need for mitotic inhibitors. Our experimental results reveal a critical role for astrocytes in neuronal attachment and growth. This method can be used to study glial-neuron interactions as well as culturing low-density population of pure neurons.

Axonal cytoskeleton changes in experimental optic neuritis.

Axonal loss and degeneration in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE) have been suggested by brain imaging, pathological and axonal transport studies. Further elucidation of the processes and mechanisms of axonal degeneration in demyelinating diseases is therefore of potential importance in order to alleviate the permanent disabilities of MS patients. However, detailed studies in this area are impeded by the small number of reliable models in which the onset and location of demyelination can be well-controlled. In this study, microinjection of polyclonal rabbit anti-galactocerebroside (anti-Gal C) antibody and guinea pig complement was used to induce local demyelination in the rat optic nerve. We found that treatment with appropriate volumes of the antibody and complement could induce local demyelination with minimal pressure- or trauma-induced damage. Local changes in neurofilaments (NFs) and microtubules (MTs) were examined with both immunohistochemistry (IHC) and electron microscopy (EM). On day 1 after microinjection, we observed moderate NF and MT disassembly in the local demyelinated area, although in most cases, no apparent inflammatory cell infiltration was seen. The NF and MT changes became more apparent on days 3, 5, 7 after microinjection, along with gradually increased inflammatory cell infiltration. These results suggested that acute demyelination itself may induce local cytoskeleton changes in the demyelinated axons, and that the ensuing local inflammation may further enhance the axonal damage. When the lesions were stained with specific antibodies for T lymphocytes, macrophages, and astrocytes, we found that most of the cells were macrophages, suggesting that macrophages may play a greater role in inflammation-related axonal degeneration and axonal loss. These results were confirmed and further characterized on the ultrastructural level.

Dichotic pitch: a new stimulus distinguishes normal and dyslexic auditory function.

Two patterns of appropriately filtered acoustic white noise can be binaurally fused by the human auditory system to extract pitch and location information that is not available to either ear alone. This phenomenon is called dichotic pitch. Here we present a new method for generating more effective and useful dichotic pitch stimuli. These novel stimuli allow the psychophysical assessment of dichotic pitch detection thresholds. We show that dichotic pitch detection is significantly impaired in individuals with developmental dyslexia, as compared to average readers. These results suggest a low-level auditory deficit associated with dyslexia and also demonstrate the potential value of our new dichotic pitch stimuli for assessment of auditory processing.

Histochemical mapping of NADPH-diaphorase in monkey and human eyes.

PURPOSE: To localize nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in normal human and monkey eyes. METHODS: NADPH-d activity in ocular tissue sections was revealed by histochemical staining. RESULTS: NADPH-d staining was found in the anterior and posterior ocular segment of both species. In the anterior segment, reaction product was present in the corneal epithelium and endothelium, iris dilator and sphincter, lens capsule, ciliary muscle, and non-pigmented epithelial cells and stroma of the ciliary processes. Staining density in the longitudinal ciliary muscle was higher than in the other ciliary muscle regions in the human; such regionalization was less apparent in the monkey. The trabecular meshwork stained minimally if at all in both species. In the posterior segment, staining was seen around choroidal blood vessels and choroidal nerves, and in the retinal pigment epithelial, photoreceptor, bipolar, inner plexiform and ganglion cell layers. CONCLUSIONS: NADPH-d is widespread in human and monkey eyes, indicating significant and presumably functionally relevant NO production in the various ocular structures.

Effects of tetrodotoxin treatment in LGN on neuromodulatory receptor expression in developing visual cortex.

The expression and distribution patterns of transmitter receptors change dramatically during pre- and post-natal development of the visual cortex, but the factors that control these processes are largely unknown. We have tested the hypothesis that input activity from the lateral geniculate nucleus (LGN), one major input source to visual cortex, may contribute to the processes underlying transmitter receptor redistributions in the visual cortex during development. We found that a short period of tetrodotoxin (TTX) treatment in LGN retarded the developmental expression and age-dependent reorganization of neuromodulatory receptors, including muscarinic, serotonergic and adrenergic receptors, in kitten primary visual cortex. The visual cortices ipsilateral to the TTX infusion site displayed a 'younger' receptor pattern than that of their contralateral control counterparts in the same animals. The results suggest that active input from LGN regulates the expression profile of a broad range of receptors in the developing visual cortex.

Developmental expression and regulation of alpha 1 adrenergic receptors in cultured cortical neurons.

The expression and distribution of alpha 1 receptors in cultured neurons derived from rat visual cortex were studied with confocal microscopy using the fluorescently labeled alpha 1 adrenergic receptor selective antagonist BODIPY FL prazosin. The receptors were found to be clustered on neuronal somata and on proximal dendrites. We found that expression of the alpha 1 receptor is regulated both by neuronal excitability and by the usage of the receptor itself. The specificity of receptor regulation to the blockade of a particular receptor class was also studied. We approached this by comparing the effects of treatment with the alpha 1 adrenoreceptor antagonist prazosin and M1 muscarinic receptor antagonist pirenzepine on the expression of alpha 1 and M1 receptors in cultured cortical neurons. The results showed that blockade of muscarinic receptors with pirenzepine up-regulated muscarinic receptor expression selectively without changing alpha 1 receptor expression. Conversely blockade of alpha 1 receptors up-regulated alpha 1 expression but not muscarinic receptor expression. This implies that the expression levels of M1 and alpha 1 receptors are both regulated through specific signal transduction pathways. The interactions between neuronal activity and receptor activation (or blockade) on receptor expression were studied as well. In these experiments we compared the effects of high K+, tetrodotoxin (TTX), prazosin, and noradrenaline on the expression of alpha 1 and M1 receptors in cultured neurons. The results show that high K+ exposure increased both alpha 1 and M1 receptor expression regardless of the presence of receptor agonists or antagonists. On the other hand, TTX exposure reduced both alpha 1 and M1 receptor expression regardless of the presence of the receptor agonists or antagonists. This implies that regulation of receptor number is predominantly regulated by neuronal activity rather than by receptor occupancy.

Serotonin facilitates synaptic plasticity in kitten visual cortex: an in vitro study.

We have addressed the role of serotonin-2C (5-HT2C) receptors in the development and maintenance of synaptic plasticity in the kitten visual cortex. In visual cortical slices, taken from 40- to 80-day-old kittens, bath application of serotonin markedly facilitated the induction of both long-term depression (LTD) and long-term potentiation (LTP). Field potential responses to white matter stimulation were recorded from layer IV after a regime of low frequency stimulation (LFS; 1 Hz, 15 min), which reliably induced LTP or LTD in younger kittens (less than 30 days of age). At 40-80 days, this protocol almost never induced LTD or LTP in layer IV. However, in 50% of the visual cortical slices studied in 40-80-day-old kittens, LTD or LTP was induced, if serotonin (1 or 10 microM) was co-applied with LFS. No such serotonin facilitation of long-term plasticity was ever detected in > 120-day-old animals, indicating that serotonin facilitates synaptic plasticity within a defined period of visual cortical development. Serotonergic 5-HT2C receptors are likely to contribute to the synaptic plasticity observed in layer IV, since mesulergine, an antagonist of the 5-HT2C receptor, completely blocked synaptic modifications induced by the combination of low frequency stimulation and serotonin application.

Blockade of serotonin-2C receptors by mesulergine reduces ocular dominance plasticity in kitten visual cortex.

We have investigated the role of serotonin-2C (5-HT2C) receptors in modulation of ocular dominance plasticity in kitten visual cortex. A small quantity of the 5-HT2C receptor blocker, mesulergine, was infused into the visual cortex of one hemisphere of 5- to 7-week-old kittens using osmotic minipumps, while the control hemisphere received vehicle solution. At the same time, one eyelid of the experimental animals was sutured shut. The ocular dominance distributions in the visual cortex (area 17) were assessed using extracellular recording methods after 1 week of combined mesulergine infusion and monocular deprivation. We found that the majority of the neurons remained binocularly responsive in the mesulergine-treated hemisphere, while most of the neurons recorded were either unresponsive or only weakly responsive to the deprived eye in the control hemisphere. Local infusion of mesulergine into the kitten visual cortex thus reduced the shift of ocular dominance that normally occurs in animals of these ages following monocular deprivation. The blocking effect seems to be distance-dependent and therefore dose-dependent: the farther away the recording sites were from the injection site, the fewer binocularly responsive cells were found. These results are relevant to previous findings indicating transient overexpression of 5-HT2C receptor in visual cortex of kittens at these ages. The data suggest that the 5-HT2C receptor system may be involved in the formation and modification of ocular dominance columns in the developing visual cortex.

Glaucomatous optic nerve cupping as an optic neuropathy.

Intraocular pressure (IOP), which causes the lamina cribrosa to bulge backward, produces a pressure gradient along the axoplasm of exiting optic nerve axons, and challenges the circulation, interacts with presently unknown physiologic or anatomic factors to harm the optic nerve and causes loss of vision. Present treatment of glaucoma is limited to efforts to lower IOP. Future treatments may be directed at other contributing anatomic or physiologic abnormalities that permit IOP to be harmful, or at some step in the pathway from insult to mortal injury of the axon or cell body.

Visualization of muscarinic cholinergic receptors on chick cardiomyocytes and their involvement in phosphatidylcholine hydrolysis.

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.