Gene expression profile in cerebrum in the filial imprinting of domestic chicks (Gallus gallus domesticus)

In newly hatched chicks, gene expression in the brain has previously been shown to be up-regulated following filial imprinting. By applying cDNA microarrays containing 13,007 expressed sequence tags, we examined the comprehensive gene expression profiling of the intermediate medial mesopallium in the chick cerebrum, which has been shown to play a key role in filial imprinting. We found 52 up-regulated genes and 6 down-regulated genes of at least 2.0-fold changes 3 h after the training of filial imprinting, compared to the gene expression of the dark-reared chick brain. The up-regulated genes are known to be involved in a variety of pathways, including signal transduction, cytoskeletal organization, nuclear function, cell metabolism, RNA binding, endoplasmic reticulum or Golgi function, synaptic function, ion channel, and transporter. In contrast, fewer genes were down-regulated in the imprinting, coinciding with the previous data that the total RNA synthesis increased associated with filial imprinting. Our data suggests that the filial imprinting involves the modulation of multiple signaling pathways.     

MAP2d mRNA is expressed in identified neuronal populations in the developing and adult rat brain and its subcellular distribution differs from that of MAP2b in hippocampal neurones

The brain microtubule-associated protein MAP2 family is composed of high-molecular-weight (MAP2a and MAP2b) and low-molecular-weight (MAP2c and MAP2d) isoforms. The common C-terminal region of HMW MAP2 and MAP2c contains three repeated microtubule-binding domains while MAP2d comprises four repeats. MAP2c mRNA is known to be expressed at high levels in the immature brain. We show that in the brains of rat pups, MAP2c mRNAs are indeed expressed at high levels compared with MAP2d. However, in adult rat brains, MAP2d mRNA levels are higher than MAP2c. In order to identify the neural cells expressing MAP2d, we used in situ hybridization. In vivo, we show that MAP2d mRNA is expressed in well-identified neuronal populations of the brain. In primary cultures of hippocampal neurones, double-labelling experiments confirm that MAP2d is clearly expressed in neurones. We also evaluated in this study the subcellular distribution of the MAP2d mRNAs in cultured hippocampal neurones and we report that in contrast with MAP2b mRNAs, mostly localized in dendrites, MAP2d mRNAs are essentially located in neuronal cell bodies.     

Quantifying mRNA in postmortem human brain: influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance

The quantification of mRNA in postmortem human brain is often made complicated by confounding factors. To assess the importance of potential confounders TaqMan real-time RT-PCR was used to measure seven mRNAs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin, microtubule-associated protein (MAP) 2, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), amyloid precursor protein (APP) isoform 770) in cortical samples taken from 90 Alzheimer's disease (AD) and 81 control brains. Demographic data for the brain samples were assessed for interaction between factors and amounts of mRNA. Gender was found to play a role in that females had lower levels of mRNA relative to males; this was consistent in both the AD and control brains. Age at death had inconsistent but significant correlations to amounts of mRNA; male and female controls both had negative correlations, female AD a positive correlation and male AD no correlation. Positive correlations were found between brain pH and amount of mRNA in all genes except glial fibrillary acidic protein (GFAP); correlations were consistent across all groupings of pathology and gender. Mean brain pH was significantly lower in AD (6.4) than in control subjects (6.5, ANOVA, p<0.01), though there was no difference between male and females of either group. No correlation was found between brain pH and age at death. Postmortem interval was correlated with brain pH in Alzheimer's disease brains but not controls. Agonal state was generally a poor predictor of mRNA levels whilst inter-lobe variance of mRNA was found to be non-significant in control brains. Given that gender, age at death and brain pH all have significant effects upon mRNA levels it is recommended that these factors be taken into account when quantifying gene expression in postmortem human brain.     

In-vivo gene transfer into newly hatched chick brain by electroporation

Newly hatched domestic chicks serve as ideal models for studies of the neural basis of behavioral plasticity, particularly for understanding the mechanisms of learning such as filial imprinting. To elucidate the molecular basis and gene functions involved in learning, we developed an in-vivo gene-transfer system in the brain of a living chick using electroporation. When green fluorescent protein-encoding plasmids were transfected to a chick brain, green fluorescence was clearly observed, and expression at the protein level was confirmed by immunoblotting. Most of the transfected brain cells were neuronal cells with dendrites. This neuron-selective electroporation system will facilitate the analysis of gene functions in the living chick brain and provide further clues as to the molecular mechanisms of avian learning.     

Activation of brain-derived neurotrophic factor/tropomyosin-related kinase B signaling accompanying filial imprinting in domestic chicks (Gallus gallus domesticus)

Newly hatched domestic chicks serve as an important model for experimental studies of neural and behavioral plasticity. Brain-derived neurotrophic factor (BDNF) has been shown to play a critical role in synaptic plasticity, including long-term potentiation, which underlies learning and memory in rodents. Here we show that BDNF mRNA levels increased in the intermediate medial hyperpallium apicale (IMHA), which is the caudal area of the visual Wulst, of imprinted chick brains, and the upregulation of gene expression correlated with the strength of the learned preference to the training object. In addition, activation of tropomyosin-related kinase B (TrkB)/phosphatidylinositol 3-kinase signaling was associated with filial imprinting. However, pharmacological deprivation of TrkB phosphorylation in IMHA did not impair memory formation, suggesting that activation of BDNF/TrkB signaling in IMHA is not involved in memory acquisition in filial imprinting.     

Dlx-2 homeobox gene controls neuronal differentiation in primary cultures of developing basal ganglia

Homeodomain-containing genes of theDlx family are expressed in the developing basal ganglia. To investigate the role ofDlx genes during development, we studied their cellular localization in primary cultures of embryonic basal telencephalon, and examined the changes in cellular phenotypes resulting from blockade ofDlx-2 expression. Cells containingDlx-1, Dlx-2, andDlx-5 mRNAs are immature cells of the neuronal lineage expressing the microtubule-associated proteins (MAPs) MAP1B and MAP2, but not glial fibrillary acidic protein (GFAP). Treatment of these cells with antisense oligonucleotides targeted toDlx-2 caused a specific decrease ofDlx-2 mRNA and protein. This decrease in theDlx-2 gene product was associated with a decrease in the expression of MAP2, a protein localized in neuronal dendrites, along with a smaller decrease in the 200-kDa neurofilament subunit (NF-H). Proteins expressed preferentially in axons were unchanged. This reduction in MAP2 expression was associated with a decrease in dendrite outgrowth and an increased level of cell proliferation. None of these changes were elicited by antisense oligonucleotides targeted toDlx-1. We suggest that theDlx-2 gene product regulates two interrelated aspects of neuronal differentiation: the exit from the mitotic cycle and the capability to grow MAP2-positive dendrites. As such, this gene product may be important for the establishment of neuronal polarity, setting the stage for afferent synaptic connectivity.     

MARCKS and protein F1/GAP-43 mRNA in chick brain: effects of imprinting

The phosphorylation of MARCKS, but not protein F1/GAP-43, is increased in the intermediate and medial portion of the hyperstriatum ventrale (IMHV) after chick imprinting. Here we investigated if MARCKS, but not F1/GAP-43, gene expression would also be altered after imprinting. We first investigated the constitutive mRNA distribution of MARCKS and F1/GAP-43 in chick brain. MARCKS mRNA was expressed in most cells and exhibited a relatively homogeneous distribution. In contrast, F1/GAP-43 mRNA levels were elevated in discrete brain regions, as we had observed in mammals. The highest F1/GAP-43 mRNA levels in the chick brain were in sensory and associational structures such as the hyperstriatal complex and neostriatum, and lower levels were in structures involved in motor control, such as paleostriatum. These results in chick are consistent with the previously drawn generalization that F1/GAP-43 mRNA is expressed in those brain regions which exhibit synaptic plasticity. After imprinting, MARCKS mRNA levels in IMHV were higher in good learners than poor learners. In contrast, analysis of F1/GAP-43 mRNA levels revealed no differences related to training in any brain region sampled. These selective results for MARCKS but not F1/GAP-43 parallel the prior findings on their phosphorylation, and are consistent with our hypothesis that the very same proteins that are post-translationally modified in association with learning and memory also undergo alterations in their gene expression.     

Retinal removal up-regulates cannabinoid CB(1) receptors in the chick optic tectum

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration and neuroprotection. The aim of this study was to evaluate the effects of unilateral retinal ablation on the expression of the cannabinoid receptor subtype 1 (CB(1)) at both protein and mRNA levels in the optic tectum of the adult chick brain. After different survival times postlesion (2-30 days), the chick brains were subjected to immunohistochemical, immunoblotting, and real-time PCR procedures to evaluate CB(1) expression. TUNEL and Fluoro-Jade B were used to verify the possible occurrence of cell death, and immunostaining for the microtubule-associated protein MAP-2 was performed to verify possible dendritic remodeling after lesions. No cell death could be observed in the deafferented tectum, at least up to 30 days postlesion, although Fluoro-Jade B could reveal degenerating axons and terminals. Retinal ablation seems to generate an increase of CB(1) protein in the optic tectum and other retinorecipient visual areas, which paralleled an increase in MAP-2 staining. On the other hand, CB(1) mRNA levels were not changed after retinal ablation. Our results reveal that CB(1) expression in visual structures of the adult chick brain may be negatively regulated by the retinal innervation. The increase of CB(1) receptor expression observed after retinal removal indicates that these receptors are not presynaptic in retinal axons projecting to the tectum and suggests a role of the cannabinoid system in plasticity processes ensuing after lesions.     

Altered levels of microtubule proteins in brains of Alzheimer's disease patients

Summary The amount of microtubule protein present in the total soluble protein from brains of Alzheimer's disease patients and from brains of non-Alzheimer age-matched controls, were determined by radioimmunoassay. No differences were found in the amount of tubulin or microtubule-associated protein MAP2 present in either group. However, the amount of tau protein or MAP1 from the brains of Alzheimer's disease patients was about half of that present in their control counterparts.Supported by Grants from the Comisión Asesora para el Desarrollo Tecnológico y Científico and Fondo de Investigaciones Sanitarias     

The distribution of expression of doublecortin (DCX) mRNA and protein in the zebra finch brain

Using in situ hybridization, we measured the distribution of expression of doublecortin (DCX), a microtubule-associated protein, in zebra finch adult and nestling (P9-11) brains. In adult brain, DCX mRNA was detected mainly in the mesopallium (M), medial striatum (MSt), septum, Area X, diencephalon, telencephalic subventricular zone (SVZ), and Purkinje cells in the cerebellum. The expression at posthatch day 9 (P9) was heavy in almost the entire telencephalon and showed heavier expression in SVZ and song regions such as the high vocal center (HVC) and the robust nucleus of arcopallium (RA). Outside of the telencephalon at P9, we found distinct label in nucleus ovoidalis (OV), nucleus spiriformis lateralis (SpL), and nucleus subpretectalis (SP) in the midbrain, almost the entire diencephalon including nucleus dorsomedialis posterior thalami (DMP), stratum griseum et fibrosum superficiale (SGF) in optic tectum, and Purkinje cells in cerebellum. Most of the heavily labeled areas by in situ hybridization overlapped with immunohistochemical staining for DCX, indicating that DCX mRNA is probably translated into protein in those regions. No sex difference was found in DCX expression at P9 or in the adult except that Area X was labeled only in the adult male. The intensity of expression in the adult was significantly lower than that at P9, which suggests a particular role for DCX in early song bird brain development. If DCX is predominantly expressed in migrating neurons, as suggested from studies in mammals, the present results offer no evidence for a sex difference in neuronal migration.     

Up-regulation of peripherin is associated with alterations in synaptic plasticity in CA1 and CA3 regions of hippocampus

Peripherin is a type III intermediate filament protein normally undetectable in most brain neurons. Here, we report a similar pattern of peripherin expression in the brains of both mice treated with systemic injections of kainic acid (KA) and in peripherin transgenic mice (Per mice) over-expressing the normal peripherin gene under its own promoter. Double-immunofluorescence labeling revealed a partial co-localization of peripherin with the microtubule-associated protein MAP2, but not with neurofilament proteins. Electrophysiological studies revealed that synaptic plasticity was markedly altered in Per mice: in CA1, long-term potentiation (LTP) was decreased in Per slices (+29 +/- 2.0%, vs. +58 +/- 5.4%, in WT); while in CA3, LTP was increased in Per (+63 +/- 3.5% vs. +43 +/- 2.4.0%). In the hippocampus of Per mice, the levels of MAP2 were decreased, though synaptophysin and PSD95 remained unchanged. These intriguing findings suggest a role of peripherin in the alteration of hippocampal synaptic plasticity.     

Temperature effect on immunostaining of microtubule-associated protein 2 and synaptophysin after 30 minutes of forebrain ischemia in rat

Summary The regional distribution of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein synaptophysin was investigated by immunohistochemistry in brains of rats submitted to 30-min forebrain ischemia by four-vessel occlusion. The following brain temperature profiles during ischemia were compared: (1) constant brain temperature of 36°C (normothermia; n=5); (2) spontaneous temperature decline from 36° to 31°C (spontaneous hypothermia; n=5) and (3) constant temperature of 30°C (induced hypothermia; n=5). Normothermia was produced by exposing the ischemic head to an external heat source, and induced hypothermia by cooling the head with liquid nitrogen vapours. Sham-operated animals were either kept at ambient temperature or exposed to the same heat source, as required for maintaining normothermia during ischemia. Seven days after sham operation or ischemia, brains were fixed by perfusion and processed for immunohistochemistry using monoclonal antibodies against MAP2 and synaptic vesicle-specific protein (synaptophysin). Normothermic ischemia resulted in complete loss of MAP2 immunostaining in the whole hippocampus, spontaneous hypothermic ischemia in complete loss of MAP2 in CA1 sector, and induced hypothermic ischemia only in variable loss of MAP2 in CA1 sector. Post-ischemic immunostaining of synaptophysin revealed a temperature-dependent increase in stratum lacunosum-moleculare of CA1 sector, the density of which correlated inversely with MAP2 staining. Comparison with morphological alterations showed a close relationship between loss of MAP2 staining and histological injury. The post-ischemic activation of synaptophysin may reflect regenerative processes associated with synaptic remodelling and, therefore, is an indirect marker of the severity of ischemic injury.     

Immunohistological study on brains of Alzheimer's disease using antibodies to fetal antigens,C-series gangliosides and microtubule-associated protein 5

Summary An immunohistological study of Alzheimer's brains was performed using antibodies to C-series gangliosides and microtubule-associated protein 5 (MAP5), and their staining patterns were compared with those of antibodies to tau and -amyloid precursor protein. Antibodies to C-series gangliosides and MAP5, both of which are known to preferentially expressed in the fetal brains, immunostained dystrophic neurites of senile plaques, neurofibrillary tangles and neuropil threads abundant in 3rd and 5th layers in the cerebral cortex, all of which are considered to be pathological hallmarks of Alzheimer's disease. The immunostaining patterns of these structures by antibodies to C-series gangliosides and MAP5 were similar to those by the antibody to tau. These three antibodies also immunostained some neurons in Alzheimer's brain, although their staining patterns were slightly different from one another; i.e., both diffuse and granular patterns were seen by the antibody to tau, but only granular pattern by the antibodies to C-series gangliosides and MAP5. These neurons immunostained by these three types of antibodies appeared to be the precursors of the classical neurofibrillary tangles, as positively stained neurons were not seen in the brains of non-demented cases. The presence of fetal antigens such as the C-series gangliosides and MAP5 in Alzheimer's brain may suggest that regeneration or sprouting of neurons is ongoing in association with the re-induction of gene expression characteristic for the brain in the early stage of development.     

Spatial and temporal expression profiles of urocortin 3 mRNA in the brain of the chicken (Gallus gallus)

Urocortin 3 (UCN3) is a neuropeptide believed to regulate stress‐coping responses by binding to type 2 corticotropin‐releasing hormone receptors. Here, we report the cloning and brain distribution of UCN3 mRNA in a sauropsid—the chicken, Gallus gallus. Mature chicken UCN3 is predicted to be a 40‐amino acid peptide showing high sequence similarity to human (93%), mouse (93%), and Xenopus (88%) UCN3. During the last third of embryonic development, UCN3 mRNA levels changed differentially in the various brain parts. In all brain parts, UCN3 mRNA levels tended to increase toward hatching, except for caudal brainstem, where a gradual decrease was observed during the last week of embryonic development. In cerebellum, a rapid increase in gene expression occurred between embryonic days 17 and 19. Using in situ hybridization, UCN3 mRNA was found to be expressed predominantly in the hypothalamus, pons, and medulla of posthatch chick brains, but not in some areas that are among the main expression sites in rodents, such as the brain areas where in mammals the median preoptic nucleus and the medial amygdala are located. This suggests that the roles of UCN3 in chicken, and perhaps sauropsids in general, are not all identical to those in rodents.     

Antipsychotics increase microtubule-associated protein 2 mRNA but not spinophilin mRNA in rat hippocampus and cortex

Antipsychotic (neuroleptic) drugs induce structural alterations in synaptic terminals and changes in the expression of presynaptic protein genes. Whether there are also changes in corresponding postsynaptic (dendritic) markers has not been determined. We describe the effect of 14-day treatment with typical (haloperidol, chlorpromazine) or atypical (clozapine, olanzapine, risperidone) antipsychotics on the expression of two dendritic protein genes, microtubule-associated protein 2 (MAP2) and spinophilin, using in situ hybridization, in the rat hippocampus, retrosplenial, and occipitoparietal cortices. MAP2 mRNA was increased modestly in the dentate gyrus and retrosplenial cortex by chlorpromazine, risperidone, and olanzapine and in the occipitoparietal cortex by chlorpromazine, haloperidol, and risperidone. None of the antipsychotics affected spinophilin mRNA in any area. Overall, these results show a modulation of MAP2 gene expression, likely reflecting functional or structural changes in the dendritic tree in response to some typical and atypical antipsychotics. The lack of change in spinophilin mRNA suggests that dendritic spines are not affected selectively by the drugs. The data provide further evidence that antipsychotics regulate genes involved in synaptic structure and function. Such actions may underlie their long-term effects on neural plasticity in areas of the brain implicated in the pathology of schizophrenia.     

MAP2 immunostaining in thick sections for early ischemic stroke infarct volume in non-human primate brain

The delineation of early infarction in large gyrencephalic brain cannot be accomplished with triphenyl-tetrazolium chloride (TTC) due to its limitations in the early phase, nor can it be identified with microtubule-associated protein 2 (MAP2) immunohistochemistry, due to the fragility of large thin sections. We hypothesize that MAP2 immunostaining of thick brain sections can accurately identify early ischemia in the entire monkey brain.Using ischemic brains of one rat and three monkeys, a thick-section MAP2 immunostaining protocol was developed to outline the infarct region over the entire non-human primate brain. Comparison of adjacent thick and thin sections in a rat brain indicated complete correspondence between ischemic regions (100.4 mm³ ± 1.2%, n = 7, p = 0.44). Thick sections in monkey brain possessed the increased structural stability necessary for the extensive MAP2 immunostaining procedure permitting quantification of the ischemic region as a percent of total monkey brain, giving infarct volumes of 11.4, 16.3, and 19.0% of total brain. Stacked 2D images of the intact thick brain tissue sections provided a 3D representation for comparison to MRI images. The infarct volume of 16.1 cm³ from the MAP2 sections registered with MRI images agreed well with the volume calculated directly from the stained sections of 16.6 cm³.Thick brain tissue section MAP2 immunostaining provides a new method for determining infarct volume over the entire brain at early time points in a non-human primate model of ischemic stroke.     

微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化

目的 探讨脆性X智能低下蛋白（fragile X mental retardation protein,FMRP）对微管相关蛋白1B（microtubuleassociated protein 1B,MAP1B）是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型（KO）和同龄野生型（WT）小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 ：6周龄KO小鼠各个脑区MAP1B的平均光密度值（MOD）值均显著低于同龄WT小鼠（P 〈 0.05）,1周龄KO小鼠仅在小脑和海马显著降低（P 〈 0.01） ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低（P 〈 0.05）。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低（P 〈0.05）。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP可能正性调节MAP1B的表达。     

微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化(英文)

目的探讨脆性X智能低下蛋白(fragile X mental retardation protein,FMRP)对微管相关蛋白1B(microtubuleassociated protein 1B,MAP1B)是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型(KO)和同龄野生型(WT)小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 :6周龄KO小鼠各个脑区MAP1B的平均光密度值(MOD)值均显著低于同龄WT小鼠(P < 0.05),1周龄KO小鼠仅在小脑和海马显著降低(P < 0.01) ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低(P < 0.05)。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低(P <0.05)。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP 可能正性调节MAP1B的表达。     

Co-expression of Fos immunoreactivity in protein kinase (PKCγ)-positive neurones: quantitative analysis of a brain region involved in learning

The expression of the gamma protein kinase C isoenzyme (PKCγ) and of the c-fos immediate early gene protein product Fos in the intermediate and medial hyperstriatum ventrale (IMHV) of day-old chicks was determined immunocytochemically. Previous research has shown that (a) there is a learning-related increase in the expression of Fos in the IMHV of the chick after imprinting; (b) PKCγ is expressed in neurones in most regions of the chick forebrain, including the IMHV. In the present study it was found that in imprinted chicks, 96.5% of neurones in the IMHV that expressed Fos also stained positively for PKCγ. These results raise the possibility of a functional connection between PKCγ activation and c-fos expression in neurones in general, and in particular in neurones in the IMHV that are involved in learning.