Localized changes in immediate-early gene regulation during sensory and motor learning in zebra finches.

A complex neural system controls birdsong learning, but its organization is not understood, nor is it known why learning only occurs during a critical period in adolescence. Here, we analyzed developmental regulation in zebra finches of zenk, an immediate-early gene (IEG) implicated in memory consolidation. Basal expression was elevated within auditory telencephalon (specifically, within the caudomedial neostriatum [NCM]) during song acquisition. Expression could be further induced by song playbacks 30 days after hatching but not at 20 days nor in juveniles reared in severe isolation. Singing itself induced zenk in song production nuclei, including Area X, even in adults. Within a compartment of the robust nucleus of the archistriatum (RA), however, this response dwindled as singing matured. These results suggest that the onset of sensory memory storage may be regulated in part at NCM, and motor plasticity may be regulated at RA.

Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.

A male zebra finch learns a song by listening to a tutor, but song learning is normally restricted to a critical period in juvenile development. Here we identify an RNA whose expression in the song control circuit is altered during this critical period. The RNA encodes a soluble presynaptic protein that forms a predicted amphipathic alpha helix typical of the lipid-binding domain in apolipoproteins. We show this protein, which we call synelfin, to be the homolog of the human non-A beta component (and its precursor) recently purified from Alzheimer's disease amyloid. We suggest this highly conserved protein may serve a novel function critical to the regulation of vertebrate neural plasticity.

Probes for rare mRNAs reveal distributed cell subsets in canary brain.

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.

Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro.

Sexual differentiation of the brain is determined in part by steroids such as estrogen, which are generally assumed to arise from the gonads. Here we show that estrogens are produced autonomously in cultured juvenile male zebra finch brain slices, and this brain-derived estrogen is both necessary and sufficient to trigger formation in vitro of a key male-specific synaptic connection in the telencephalic song control circuit. Male-like development was stimulated in female slices cultured with male slices or exposed to estrogen, and estrogen antagonists inhibited song circuit development in slices of either sex. These results reveal a new mode of sex-specific neural development, induced not by differential exposure to gonadal steroids, but rather by differential synthesis of steroids in the brain.

Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes.

Liver-specific mRNA sequences were examined in primary cultures of mouse hepatocytes. After cell disaggregation by collagenase treatment and for at least 24 h in culture, little change in liver-specific mRNA concentrations was noted. Gradually over a period of 140 h, liver-specific mRNAs declined. In contrast, transcriptional assays in which liver cell nuclei were used to produce 32P-labeled nuclear RNA showed that liver-specific gene transcription was greatly diminished within 24 h, while polymerase II transcription of "common" genes and transcription of tRNA and rRNA did not decline. Thus, a prompt differential transcriptional effect seems to underlie the gradual loss of tissue specificity of the primary cultures.

Structure and expression of canary myc family genes.

We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.

Dependence of liver-specific transcription on tissue organization.

When the liver is disaggregated and hepatocytes are cultured as a cellular monolayer for 24 h, a sharp decline (80 to 99% decrease) in the transcription of most liver-specific mRNAs, but not common mRNAs, occurs (Clayton and Darnell, Mol. Cell. Biol. 2:1552-1561, 1983). A wide variety of culture conditions involving various hormones and substrates and cocultivation with other cells failed to sustain high rates of liver-specific mRNA synthesis in cultured hepatocytes, although they continued to synthesize common mRNAs at normal or elevated rates. In contrast, when slices of intact mouse liver tissue were placed in culture, the transcription of liver-specific genes was maintained at high levels (20 to 100% of normal liver). Furthermore, we found that cells in the liver could be disengaged and immediately reengaged in a tissue-like structure by perfusing the liver with EDTA followed by serum-containing culture medium. Slices of reengaged liver continued to transcribe tissue-specific mRNA sequences at significantly higher rates after 24 h in culture than did individual cells isolated by EDTA perfusion followed by culturing as a monolayer. Therefore we conclude that a mature tissue structure plays an important role in the maintenance of maximum tissue-specific transcription in liver cells.

Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels.

The transcription rate and abundance of several liver-specific mRNAs as well as mRNAs common to many cell types were compared in a series of rodent hepatoma cell lines, normal liver cells, and primary hepatocyte cultures. The rat hepatoma cell line, Fao, which displays a liver-specific phenotype, contained eight of eight liver-specific mRNAs examined. However, the transcription rates of most liver-specific mRNAs were found to be low (1 to 30%) compared with normal liver in this and other differentiated cell lines. This low rate is similar to the transcription rates of liver-specific mRNA sequences measured in primary cultures of hepatocytes. Several variant cell lines that had lost differentiated traits contained few or none of the liver-specific mRNAs; clonal descendents which had regained differentiated function regained the tissue-specific mRNAs as a group, but at various concentrations. Because all of the changes observed in mRNA levels were not accompanied by parallel changes in transcription of the same sequences, differential posttranscriptional stabilization of the liver-specific mRNAs must also occur in the different cell lines. These results qualify the utility of cultured cell lines in the study of tissue-specific transcriptional control, but raise the possibility that posttranscriptional mechanisms act in cooperation with transcriptional controls to bring the level of tissue-specific mRNAs closer to those found in liver cells.

Cellular promoters incorporated into the adenovirus genome: cell specificity of albumin and immunoglobulin expression.

Recombinant adenoviruses were constructed in which the viral E1A gene was deleted and the E1B promoter was replaced by the rat albumin, mouse beta-major globin, or mouse immunoglobulin heavy-chain promoter. After infection of human or rat hepatoma cells, E1B-containing mRNAs could be detected only from the virus containing the albumin promoter. Conversely, only the immunoglobulin promoter was active in virus-infected myeloma cells. However, in hepatoma cells transcription from the albumin promoter in the virus was much less than that of the endogenous cellular albumin gene or of other viral genes. In primary mouse hepatocytes endogenous albumin gene transcription was high immediately after plating but declined within 24 h. Expression of the albumin promoter in the virus paralleled that of the cellular albumin gene. From these results it appears that cell-specific expression of albumin depends on the presence of tissue-specific trans-acting factors, but the presence of such factors does not suffice for a maximal rate of transcription, a conclusion that requires direct comparison within a differentiated cell of a newly introduced and preexisting active cell gene.

Posttranscriptional modulation of gene expression in cultured rat hepatocytes.

The maintenance of high levels of two liver-specific mRNAs in cultured hepatocytes was achieved in a serum-free hormonally defined cell culture medium. However, this maintenance of liver-specific mRNA levels did not correlate with the level of transcription of the genes but was apparently due to increased stabilization of the tissue-specific mRNAs. The mRNA stabilization did not occur in serum-supplemented medium. In both defined and serum-supplemented medium, actin and tubulin mRNAs were also greatly increased, in both cases predominantly if not entirely due to increased mRNA stability.

The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease.

Synuclein proteins are produced, in vertebrates, by three genes. They share structural resemblance to apolipoproteins, but are abundant in the neuronal cytosol and present in enriched amounts at presynaptic terminals. Synucleins have been specifically implicated in three diseases:Alzheimer's (AD), Parkinson's (PD) and breast cancer. In AD, a peptide derived from alpha-synuclein forms an intrinsic component of plaque amyloid. In PD, an alpha-synuclein allele is genetically linked to several independent familial cases, and the protein appears to accumulate in Lewy bodies. In breast cancer, increased expression of gamma-synuclein correlates with disease progression. In songbirds, alpha-synuclein expression is correlated with plasticity in the developing song control system. Although the normal function of synucleins is unknown, a role in membrane plasticity seems likely.

Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences.

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.

The opportunities and challenges of large-scale molecular approaches to songbird neurobiology.

High-throughput methods for analyzing genome structure and function are having a large impact in songbird neurobiology. Methods include genome sequencing and annotation, comparative genomics, DNA microarrays and transcriptomics, and the development of a brain atlas of gene expression. Key emerging findings include the identification of complex transcriptional programs active during singing, the robust brain expression of non-coding RNAs, evidence of profound variations in gene expression across brain regions, and the identification of molecular specializations within song production and learning circuits. Current challenges include the statistical analysis of large datasets, effective genome curations, the efficient localization of gene expression changes to specific neuronal circuits and cells, and the dissection of behavioral and environmental factors that influence brain gene expression. The field requires efficient methods for comparisons with organisms like chicken, which offer important anatomical, functional and behavioral contrasts. As sequencing costs plummet, opportunities emerge for comparative approaches that may help reveal evolutionary transitions contributing to vocal learning, social behavior and other properties that make songbirds such compelling research subjects.

Characterization of the precursor protein of the non-A beta component of senile plaques (NACP) in the human central nervous system.

A novel and highly conserved presynaptic protein has been independently described in rodents (synuclein/SYN-1), songbirds (synelfin), and humans (the precursor protein of the non-A beta component of senile plaques, NACP); a fragment of the latter has been detected in senile plaques in Alzheimer's disease (AD). We characterized the expression of NACP in human AD and non-AD brain. A subcellular fractionation study demonstrated that NACP was mainly localized to cytosolic fractions of human temporal cortex. NACP was also detectable in various membrane and vesicular fractions, suggesting that the protein was associated with membrane structures including synaptic vesicles. Pericellular immunostaining of the neuropil was observed in neocortical and limbic regions, supporting a synaptic localization. Senile plaques in AD brains were not immunoreactive, and confocal microscopy suggested a loss of NACP immunoreactivity in cored plaques. No difference was found in the amount of protein in AD and control frontal cortex, as measured by immunoblotting. PCR analysis showed that the full-length mRNA product was the major splice form in both AD and control human brains. Thus, despite the association of a hydrophobic fragment of NACP with senile plaques, our data suggest that the precursor itself is not a significant component of plaques and NACP synthesis is not substantially altered in AD. Nevertheless, the protein is an abundant component of synaptic regions prone to degeneration in AD, and may have a role in the expression or advancement of the disease.

Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson's disease and cortical Lewy body disease contain alpha-synuclein immunoreactivity.

A mutation in the alpha-synuclein gene has recently been linked to some cases of familial Parkinson's disease (PD). We characterized the expression of this presynaptic protein in the midbrain, striatum, and temporal cortex of control, PD, and dementia with Lewy bodies (DLB) brain. Control brain showed punctate pericellular immunostaining. PD brain demonstrated alpha-synuclein immunoreactivity in nigral Lewy bodies, pale bodies and abnormal neurites. Rare neuronal soma in PD brain were immunoreactive for alpha-synuclein. DLB cases demonstrated these findings as well as alpha-synuclein immunoreactivity in cortical Lewy bodies and CA2-3 neurites. These results suggest that, even in sporadic cases, there is an early and direct role for alpha-synuclein in the pathogenesis of PD and the neuropathologically related disorder DLB.

Seasonal and tissue-specific regulation of canary androgen receptor messenger ribonucleic acid.

Natural seasonal fluctuations in androgen levels appear to cause changes in physiology and reproductive behavior, such as singing, in canaries. Little is known, however, about the cellular mechanisms underlying these changes. Because androgens act principally through nuclear receptors in other species, we have isolated and sequenced a cDNA likely to encode the canary androgen receptor and used this cDNA to examine the regulation of AR mRNA levels in the testis, kidney, and liver of the canary. The sequence corresponds to most of the coding portion of seven of the eight exons found in the homologous mammalian gene, including the domains that bind to DNA and androgen and affect transcription. Its mRNA is approximately 8 kilobases in length and is encoded by a single gene. In the testis, the transcript is expressed specifically in the Sertoli cells. The androgen receptor antagonist flutamide represses AR mRNA levels in kidney, but induces them in liver, indicating that androgen regulates its receptor, but does so in a tissue-specific manner, as is seen for the estrogen receptor in rodents. In addition, there are natural seasonal fluctuations in AR mRNA levels in testis and liver correlated with seasonal differences in the levels of circulating androgens. This is the first evidence of natural feedback regulation of AR mRNA levels.

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity.

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.

Differential regulation in the avian song control circuit of an mRNA predicting a highly conserved protein related to protein kinase C and the bcr oncogene.

An RNA identified by differential cDNA cloning (HAT-2) is highly enriched in canary forebrain in areas associated with the control of complex learned behaviors and higher perceptual processes. The nucleotide sequence predicts a protein that is 96% identical to the product of the n-chimaerin gene isolated from human brain and contains two identifiable domains suggesting a novel role in signal transduction processes. One domain is similar to the sequence in protein kinase C which mediates diacylglycerol binding and regulation. The second domain is similar to a portion of BCR, a GTPase-activating protein encoded by the breakpoint cluster region gene. In male canaries examined during the song season, HAT-2 RNA shows variable expression within the song control circuit, and is notably less abundant in the three nuclei which concentrate androgens (HVC, RA and L-MAN). A fundamental function in the vertebrate forebrain and a possible role in the regulation of neural plasticity are suggested by the conserved structure and pattern of expression of this gene in the brain.

Forebrain-enriched RNAs of the canary: a population analysis using hybridization kinetics.

Canaries and other songbirds offer unique advantages for analyzing the relationship between specific gene regulation and neural plasticity. To establish a quantitative profile of the population of RNAs potentially involved in this regulation, we analyzed the solution hybridization kinetics of canary forebrain cytoplasmic polyadenylated RNA. Hybridization of forebrain cDNA to forebrain RNA provides evidence for RNA species at individual concentrations ranging from less than 10(-6) to about 10(-3) (by fractional mass). Cross-hybridization to RNA from the rest of the brain, together with other studies, defines a subpopulation of about 5000 rare RNAs that are enriched in the forebrain compared to the rest of the brain. Some of these forebrain-enriched RNAs are likely to play a role in regulating the neural plasticity characteristic of the canary forebrain.

Immediate-early gene responses in the avian song control system: cloning and expression analysis of the canary c-jun cDNA.

Previous studies have shown that song presentation results in a rapid rise in mRNA levels for the ZENK gene (the avian homologue of zif-268, Egr-1, NGFI-A, and Krox-24) in specific parts of the songbird forbrain. Metrazole-induced seizures also cause an increase in ZENK mRNA, even more widely throughout the telencephalon. Surprisingly, however, little or no ZENK induction by either stimulus was observed in several forebrain areas involved in auditory processing and song production. To learn whether this pattern of regulation is specific to ZENK, we examined the response of another 'immediate-early' gene, c-jun. Here we first describe the identification, cloning and sequence analysis of a canary cDNA encoding c-jun. Then, by in situ hybridization we show that c-jun is also induced by song or seizure, and in a pattern mostly similar to ZENK. As with ZENK, no induction of c-jun is observed in the androgen receptor-containing song nuclei or within the primary thalamo-recipient auditory area of the forebrain. Thus common immediate early gene responses appear to be selectively uncoupled from physiological activation in these specific forebrain regions, which are also characterized by tight developmental, hormonal and seasonal regulation.