Bidirectional regulation of Munc13-3 protein expression by age and dark rearing during the critical period in mouse visual cortex.

Rearing in darkness slows the time course of the visual cortical critical period, such that at 5 weeks of age normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic [Mower GD (1991) The effect of dark rearing on the time course of the critical period in cat visual cortex. Dev Brain Res 58:151-158]. Thus, genes that are important for visual cortical plasticity should show differences in expression between normal and dark-reared visual cortex that are of opposite direction in young versus older animals. Previously, we showed by differential display polymerase chain reaction and northern blotting that mRNA for Munc13-3, a mammalian homologue of the C. elegans uncoordinated (unc) gene, shows such bidirectional regulation in cat visual cortex [Yang CB, Zheng YT, Li GY, Mower GD (2002) Identification of Munc13-3 as a candidate gene for critical period neuroplasticity in visual cortex. J Neurosci 22:8614-8618]. Here, the analysis is extended to Munc13-3 protein in mouse visual cortex, which will provide the basis for gene manipulation analysis. In mice, Munc13-3 protein was elevated 2.3-fold in dark-reared compared with normal visual cortex at 3.5 weeks and 2.0-fold in normal compared with dark-reared visual cortex at 9.5 weeks. Analysis of variance of protein levels showed a significant interaction, indicating that the effect of dark rearing depended on age. This bidirectional regulation was restricted to visual cortex and did not occur in frontal cortex. Bidirectional regulation was also specific to Munc13-3 and was not found for other Munc13 family members. Munc13 proteins serve a central priming function in synaptic vesicle exocytosis at glutamatergic and GABAergic synapses and this work contributes to the growing evidence indicating a role of Munc13 genes in synaptic plasticity.

Bidirectional regulation of mitochondrial gene expression during developmental neuroplasticity of visual cortex.

The first several months of life are a critical period for neuronal plasticity in the visual cortex, during which anatomical and physiological development depends upon visual experience. Rearing in darkness slows the time course of this critical period, such that at 5 weeks normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic. This study reports the identification of a subset of mitochondrial genes that are regulated in this manner. Opponent patterns of bidirectional expression were found: several genes (ATPase 6, cytochrome b, NADH dehydrogenase subunits 4 and 2) showed elevation in normal cats at 5 weeks and in dark-reared cats at 20 weeks ("plasticity" genes); others (NADH dehydrogenase subunits 3 and 5) showed the opposite ("anti-plasticity" genes). These findings add a new dimension to the growing evidence that changes in mitochondrial gene expression are involved in the neuroplastic response.

Developmental changes in the expression of GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3)) in the cat visual cortex and the effects of dark rearing.

The present study used Western blots and Northern slot blots to determine changes in the level of expression of GABA(A) receptor subunits alpha(1), alpha(2), and alpha(3), in relation to the "critical period" in cat visual cortex. Levels of the GABA(A) alpha(1) subunit were lowest at 1 week, increased four-fold to a maximum at 10 weeks, and declined slightly (35%) into adulthood. Levels of the GABA(A) alpha(2) and alpha(3) subunits were highest at 1 week of age, decreased two-fold by 10 weeks of age and were constant thereafter. Comparison between visual cortex from normal and dark-reared cats at 5 weeks and 20 weeks showed that alpha(1) and alpha(3) subunit expression was elevated in dark-reared animals by approximately 50% at both ages. alpha(2) expression was not affected. These results implicate the importance of a shift from putative immature to mature GABA(A) receptor subunits during the critical period of visual cortex and in conjunction with parallel analysis of NMDA receptor subunit maturation, further support the notion that a changing excitatory/inhibitory balance is critical for neuronal plasticity.

Comparison of the expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) in the visual cortex of normal and dark-reared cats.

In normal development, there are dramatic changes in both the level and the laminar pattern of expression of the two forms of glutamate decarboxylase (GAD67, GAD65), the synthetic enzyme for gamma-aminobutyric acid (GABA). We have used antibodies to determine whether these normal postnatal changes in the expression of the two GADs depend on visual input by comparing normal and dark-reared cat visual cortex. Western blot analysis showed no significant differences in the levels of expression of the two enzymes between rearing conditions at either 5 or 20 weeks. Immunohistochemistry was used to compare the laminar distribution of the GADs in the two rearing conditions. At 1 week of age, both GAD67 and GAD65 immunoreactivity is concentrated in deep layers of visual cortex. At 5 and 20 weeks in both rearing conditions, GAD67-stained cells bodies were distributed rather uniformly across all cortical layers. GAD65 primarily labeled puncta (synaptic terminals) and these were also distributed rather uniformly across all visual cortical layers in both rearing conditions. Counts of GAD67-positive cell bodies and GAD65-positive puncta also revealed no differences between the rearing conditions. Thus, both GAD67, which produces the basal pool of GABA, and GAD65, which is specialized to respond to short-term increases in demand in synaptic terminals, developed normal levels of expression and normal intracellular and laminar distributions in the absence of visual input. Physiological studies suggest immaturity in the GABA system of dark-reared visual cortex. The present results indicate that such abnormalities are not due to presynaptic alterations in GABA synthetic enzymes.

Developmental changes in the expression of NMDA receptor subunits (NR1, NR2A, NR2B) in the cat visual cortex and the effects of dark rearing.

The present study used Western blots to determine changes in the level of expression of the three major NMDA receptor subunits, NR1, NR2A, and NR2B, in relation to the 'critical period' in cat visual cortex. NR2A rose dramatically (10-fold) from very low levels at 1 week to a peak at 5 weeks and gradually declined into adulthood (twofold). NR2B showed a similar time course to NR2A, but the rise from 1 to 5 weeks was of lesser magnitude (twofold). NR1 was expressed at comparable levels at 1, 5, 10 weeks and declined markedly (fivefold) in older animals. No significant effects of dark rearing on the levels of NR2B and NR1 were found. However, NR2A expression was significantly elevated in normal compared to dark reared visual cortex (twofold) at 5 weeks and significantly elevated in dark reared compared to normal visual cortex at 20 weeks (twofold). The close agreement between NR2A expression and both the time course of the critical period and the effects of dark rearing on that time course further indicates a role of this subunit in visual cortical critical period plasticity.

Fos expression during the critical period in visual cortex: differences between normal and dark reared cats.

The present study tested further the notion that immediate early gene expression is involved in neuronal plasticity during the critical period of visual cortex (VC) by comparing Fos induction in normal and dark reared (DR) cats. Western blots indicated that the level of induced Fos expression was higher in normal than DR cat VC at 5 weeks of age, comparable at 10 weeks, and higher in DR than normal cat VC at 20 weeks. Immunohistochemistry indicated that at 5 weeks Fos was induced in cells of all VC layers in both rearing conditions, but to a greater extent in normal than DR cats. At 20 weeks, Fos was largely restricted to cells above and below layer IV in both rearing conditions, but was induced to a greater extent in DR than normal cats. Thus, dark rearing appears to have very similar effects on Fos expression as it has on neuronal plasticity during the postnatal critical period.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of rat somatosensory barrel cortex.

The postnatal development of glutamic acid decarboxylase (GAD; GAD67 and GAD65) expression was studied in the rat somatosensory cortex. Delineation of barrels in layer IV by GAD67 immunoreactivity occurred between postnatal days P3 and P6 and remained evident into adulthood. At birth, a band of GAD67-positive elements was already present in superficial layer V. This band was prominent until P6 and gradually disappeared after P9. In parallel, there was a gradual appearance of GAD67-immunoreactive cells neuropil and puncta, which began in layer VI/subplate at P1 and achieved the adult laminar pattern by about P13. This later GAD67 immunoreactivity was responsible for the demarcation of barrels in layer IV. Development of GAD65 immunoreactivity was delayed relative to GAD67. GAD65 immunoreactivity, which was in little evidence before P6, increased markedly in density and in delineation of cell bodies over the next several weeks. During this prolonged developmental process, GAD65 first showed a negative image of the barrels compared with the septae and the surrounding cortex. Subsequently, there was a filling in of the barrels resulting in rather uniform GAD65 immunoreactivity across the barrel field and surrounding cortex. These results suggest that the development of the gamma-aminobutyric acid (GABA) synthetic system in the barrel cortex involves several processes: the disappearance of a precocious GAD67 system in layer V, the temporally overlapping maturation of the mature GAD67 system in an inside-outside manner, and the delayed and prolonged development of the GAD65 system.

Molecular cloning and analysis of Ca2+/calmodulin-dependent protein kinase II from the chicken brain.

The goals of this study were to identify specific mRNA for isoforms of calmodulin-dependent protein kinase II in chicken forebrain, prepare a cDNA expression library, and perform a sequence analysis of the kinase cDNA. Specific mRNAs for alpha- and beta-subunits of the kinase were identified in Northern blots. The mRNA for the alpha-subunit is larger in the chicken that in the rat, and for the beta-subunit is smaller in the chicken than the rat. Nucleotide sequencing of selected clones demonstrated the presence of an alpha-subunit with a 33 nucleotide insert known as the alpha-B-isoform. Clones of the beta-subunit showed it to contain a deletion of six nucleotides relative to previously described sequences. Variability in the mRNAs of calmodulin kinase II, as shown here, reflect the presence of species-dependent variability in gene structure as well as the presence of different functional isoforms.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of the cat visual cortex.

The postnatal development of GAD67 and GAD65 protein expression and of GAD67 positive neurons and GAD65 containing axon terminals in cat visual cortex was studied. Western blot analysis showed that the expression of both GAD67 and GAD65 increased to approximately two-thirds of the adult level during the first 5 postnatal weeks and gradually increased thereafter. In adult cats, immunohistochemistry showed that GABA and GAD67 containing neurons were found in all cortical layers. Faint cell body staining was seen with the antibody to GAD65, but it densely labeled puncta. In neonates, GABA and GAD67 immunoreactivity was most intense in two distinct bands, one superficial (Layer 1/Marginal zone), another deep (Layer VI/Subplate). Unlike in adults, GAD65 positive cell bodies were clearly evident in neonates and distributed similarly to, but less frequently than, GABA and GAD67. These GAD65 positive cells frequently had morphologies suggestive of embryonic cells and largely disappeared in older animals. During postnatal development, the neurochemical differentiation of GAD67 positive neurons and GAD65 positive axon terminals across visual cortical laminae followed an inside-outside developmental pattern, which reached adult levels after 10 weeks of age. These results suggest that postnatal development of the visual cortical GABA system involves three distinct processes: (A) a dying off of embryonic GABA cells which could play a role in formation of the cortical plate; (B) a period of relative quiescence of the VC GABA system in the first 5 postnatal weeks which could maximize excitatory NMDA effects during the rising phase of the critical period; (C) the prolonged postnatal maturation of the adult GABA system which could be involved in the crystallization of adult physiological properties and the disappearance of neural plasticity.

Immediate early gene expression in cat visual cortex during and after the critical period: differences between EGR-1 and Fos proteins.

Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. The present study used immunohistochemical methods to address two issues regarding IEG expression in response to visual input. One was to define the differential response of distinct IEG families by comparing EGR-1 (also termed zif-268, NGFI-A, and Krox-24) and Fos proteins. The second was to determine whether IEG expression, in addition to reflecting neural activity, is related to the state of plasticity by comparing young and adult visual cortex. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there were marked differences in the expression of the two IEG proteins. EGR-1 responded to visual input with sustained changes in its level of expression. It showed high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos showed a markedly different profile. It had very low basal expression which was not demonstrably affected by darkness and its principal response was a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off' signaling mechanism. There were marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers showed high levels of EGR-1 and Fos proteins. In adults, immunostaining was largely specific to cells located above and below layer IV and only very faint labeling occurred within layer IV. These differences in laminar distribution between ages are inconsistent with a simple explanation of IEG expression in terms of neural activity level; rather, they suggest a relation between IEG expression and the state of plasticity in visual cortex.

Developmental expression of the immediate early gene EGR-1 mirrors the critical period in cat visual cortex.

Immediate early gene (IEG) expression in the central nervous system is thought to play a role in coupling extracellular stimulation with the transcriptional events responsible for long-term functional changes in neurons. The goal of the present study was to determine the postnatal developmental profile of EGR-1 protein (also termed zif268, Krox-24, NGFI-A) expression across the layers of cal visual cortex and relate it to the state of visual cortical development and plasticity. Using a polyclonal antibody, EGR-1 immunoreactivity was studied in animals of various postnatal ages (from 0.5 week to adult). In very young animals (0.5 weeks), EGR-1 positive cells were restricted to deep cortical layers (layer VI/Subplate). With the increasing age, EGR-1 immunoreactivity spread across layers of the visual cortex in an inside-outside manner, and by 5 weeks of age, EGR-1 protein was highly expressed in all layers. EGR-1 expression remained high until approximately 10 weeks of age and then gradually began to decline in layer IV with little change in supra- and infragranular layers. In adult animals, EGR-1 was located predominantly in the layers above and below layer IV. This pattern of EGR-1 expression in developing cat visual cortex has both temporal and laminar similarities with the development of visual cortical connectivity, with the development of orientation selective receptive field properties, and with the level of visual cortical plasticity, suggesting an involvement of EGR-1 expression in these processes.

Localization of cystatin mRNA in chicken brain by in situ hybridization.

The cystatins are a superfamily of proteins that inhibit the lysosomal cysteine proteinases, cathepsins B, H, and L. Members of this superfamily have been found in all tissues and biological fluids analyzed. Previous studies have shown that chicken cystatin mRNA is abundant in brain tissue. In this study, a definitive localization of chicken cystatin mRNA in chicken brain was determined by in situ hybridization. Chicken cystatin mRNA was heavily concentrated in the secretory epithelial cells of the choroid plexus. The rest of the brain failed to show a hybridization signal above that of the control sense strand probe, even after long exposures. We conclude that chicken cystatin is synthesized predominantly by the specialized secretory epithelial cells of the choroid plexus and secreted into the cerebrospinal fluid (CSF). We postulate that chicken cystatin functions to regulate proteinase activity in the CSF and therefore may function as a protective factor for the cellular elements of the central nervous system.

Differences in the induction of Fos protein in cat visual cortex during and after the critical period.

The aim of this study was to compare the inducibility of Fos protein, in terms of magnitude and laminar distribution in visual cortex, between young (5-week-old) and adult cats. Immunohistochemical methods were used to detect Fos protein in visual and frontal cortex of young and adult cats who experienced brief (1 or 4 h) visual experience after a 1 week period of total darkness. In the 5-week-old kittens, densely stained Fos-immunoreactive neurons were found throughout all visual cortical layers as a result of 1 h of visual experience. In the adults, immunoreactive cells were concentrated in supra- and infragranular layers and only very faint labeling was found in layer IV. Immunoreactivity in young kittens persisted at much greater dilutions of primary antibody than in adults, suggesting a difference in the magnitude of induction between ages. The inductions were markedly greater in visual than in frontal cortex, where only scattered immunopositive cells were seen at the highest antibody concentration. The induction of Fos protein in visual cortex was transient and largely disappeared by 4 h at both ages. The differences between ages, both in the magnitude and laminar distribution of immunoreactivity, are inconsistent with a simple explanation of Fos inductions in terms of neural activity level. Rather, these results suggest a relation between Fos inducibility and the level of plasticity in visual cortex.

Developmental and environmental changes in GAP-43 gene expression in cat visual cortex.

Northern/slot blot analysis was used to determine postnatal developmental and environmentally induced changes in the level of expression of GAP-43 mRNA in visual and frontal cortex. Both structures showed a precipitous decline during the first 5 weeks and a slight further decline to adult levels. Dark rearing resulted in a significant elevation of GAP-43 mRNA which was eliminated by brief visual experience. This effect was specific to visual cortex and did not occur in frontal cortex. The effect also did not occur in normal adult cats placed in prolonged darkness, indicating that GAP-43 mRNA levels are not simply activity dependent and are altered by visual input only during early postnatal life. These results are consistent with a role for GAP-43 in the state of visual cortical plasticity.

Differences in c-fos immunoreactivity due to age and mode of seizure induction.

The aim of this study was to determine whether the regional distribution and time course of immunoreactivity to the c-fos protein varies with maturation and method of seizure induction. The effect of the two chemical convulsants, pentylenetetrazol (PTZ) and flurothyl, on the spatial and temporal pattern of c-fos-like immunoreactivity in immature (postnatal day (P) 10) was compared to that in adult rats. Patterns of c-fos-like immunoreactivity following O2 deprivation were also evaluated at the 2 ages because hypoxia is acutely epileptogenic in immature animals but not adults. C-fos-like immunoreactivity was examined at 2, 4, and 6 h after onset of chemically induced seizures or O2 deprivation at both ages. After PTZ or flurothyl seizures, both ages exhibited similar patterns of IR in amygdala, pyriform cortex, and hypothalamus. Age-dependent regional differences were most prominent in cortex: superficial layers of retrosplenial, cingulate, and neocortex stained in adults; staining was confined to deep layers of neocortex in P10 rats. Intense staining of dentate gyrus and hippocampus occurred with more prolonged seizures, but not brief seizures. PTZ administration resulted in staining at 2 h after seizure onset and was reduced by 4 h in adults, but immunoreactivity was not seen until 4 and 6 h after seizure onset in immature rats, indicating an age effect on the time course of IR. In immature rats, immunoreactivity patterns after hypoxia were markedly different from PTZ or flurothyl: staining was confined to layer VI of neocortex in these animals, and rarely involved limbic structures. These differences in the pattern of c-fos immunoreactivity suggest that the neuronal populations involved in epileptogenesis are influenced by age as well as seizure phenotype and intensity.

Brief visual experience induces immediate early gene expression in the cat visual cortex.

Brief visual experience causes rapid physiological changes in the visual cortex during early postnatal development. A possible mediator of these effects is the immediate early genes whose protein products are involved in the rapid response of neurons to transsynaptic stimulation. Here we report evidence that the levels of immediate early gene mRNAs in the visual cortex can be altered by manipulating the visual environment. Specifically, we find that brief (1 h) visual experience in dark-reared cats causes dramatic transient inductions of egr1, c-fos, and junB mRNAs in the visual cortex but not in the frontal cortex. Levels of c-jun and c-myc mRNAs are unaffected. These results suggest that select combinatorial interactions of immediate early gene proteins are an important step in the cascade of events through which visually elicited activity controls visual cortical development.

Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum.

Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their protein products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated from visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal development patterns of mRNA expression for 5 of these genes, c-fos, erg-1, c-jun, jun-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given gene, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development.

Comparison of serotonin 5-HT1 receptors and innervation in the visual cortex of normal and dark-reared cats.

The visual cortical serotoninergic system was compared in normal and dark-reared cats to determine whether visual experience is necessary for its normal development. In vitro receptor binding of [3H]5-HT indicated an increase in 5-HT1 receptor number in dark-reared cats with no change in affinity. This elevation was specific to the visual cortex and no changes were found in the frontal cortex as a result of dark rearing. Autoradiographic histology revealed that in the normal cat visual cortex, 5-HT1 receptors were present in all cortical layers and were slightly more dense in supragranular and infragranular layers. In dark-reared cats, there was a marked elevation in receptor density in supragranular and infragranular layers and little change within layer IV. Immunohistochemical techniques (anti-5-HT) were used to compare serotoninergic innervation in the visual cortex of normal and dark-reared cats. In normal cat visual cortex, serotonin fibers were most dense in the superficial layers (I-III), least dense in layers IV and VI, and intermediate in layer V. No differences were found between normal and dark-reared cats in the laminar distribution or density of serotoninergic innervation. These results indicate that visual experience is necessary for the normal development of the visual cortical serotonin system. The findings that the effects of dark rearing were specific to the visual cortex and that within the visual cortex these effects were specific to supra- and infragranular layers are consistent with a possible role for serotonin in the prolonged physiological plasticity that occurs in the visual cortex of dark-reared cats.

The effect of dark rearing on the time course of the critical period in cat visual cortex.

The effects of dark rearing on the time course of the postnatal critical period for monocular deprivation (MD) in visual cortex were determined in cats who experienced 2 days of MD at various postnatal ages. In normal development, plasticity (susceptibility to MD) was very low at 3 weeks, rose sharply at 6 weeks, and gradually declined over the next 10 weeks. This developmental profile was dramatically altered by dark rearing which slowed the entire time course of the critical period. In dark reared cats, plasticity rose steadily over the first 12 weeks, and was maintained at 16 weeks. There was a crossover of the two profiles of the critical period such that at young ages (6 weeks) normal cats were more plastic than dark reared cats while at later ages (after 9 weeks) dark reared cats were more plastic. A second experiment indicated that dark rearing slowed down the progression of the critical period even after it had been initiated by a period of normal vision. MD produced substantial effects after the normal critical period in cats who were reared normally for the first 6-8 weeks of life and then placed in darkness until 5 months of age. The results are discussed in terms of a simple model of the accelerating effect of visual input and the decelerating effect of total darkness on the time course of the critical period.

Evidence for an enhanced role of GABA inhibition in visual cortical ocular dominance of cats reared with abnormal monocular experience.

The effects of microiontophoretic bicuculline, a gamma-aminobutyric acid (GABA) antagonist, on the ocular dominance of visual cortical neurons were compared in normal cats and cats reared with abnormal monocular visual experience (monocular deprivation, surgical strabismus, and monocular deprivation after dark rearing). Cells that were monocular prior to drug application showed disinhibitory effects on ocular dominance far more frequently than originally binocular cells in all rearing conditions. When the total population of neurons was considered there were marked differences among rearing conditions: only 17% of cells showed change in ocular dominance in normal cats whereas over 50% showed changes in cats reared with abnormal monocular visual experience. These results indicate that GABA inhibition plays an enhanced role in cats with abnormal cortical ocular dominance. The results are interpreted in the context that the GABA system is passively biased by alterations in the pattern of excitatory input to contribute to abnormal patterns of ocular dominance.