Profiles of novel diurnally regulated genes in mouse hypothalamus: expression analysis of the cysteine and histidine-rich domain-containing, zinc-binding protein 1, the fatty acid-binding protein 7 and the GTPase, ras-like family member 11b.

Gene expression profiling of suprachiasmatic nucleus, ventrolateral preoptic area and the lateral hypothalamus was used to identify genes regulated diurnally in the hypothalamus of Mus musculus. The putative transcription regulator, cysteine and histidine-rich domain-containing, zinc binding protein 1, which had not been previously described in brain, was found to cycle diurnally in hypothalamus and forebrain with peak levels of mRNA expression during the dark phase. mRNA for the brain-type fatty acid binding protein 7 was found to change rhythmically in hypothalamic and extra-hypothalamic brain regions reaching peak levels early in the light phase suggesting that lipid metabolism is under circadian regulation in astrocytes. Rhythmically expressed genes in suprachiasmatic nucleus identified here were compared with previous reports in a meta-analysis. Genes held in common included fabp7, and the period gene, Per2. Also identified were genes implicated in guanosine-mediated signaling pathways that included dexamethasone-induced ras-related protein one (dexras1), regulator of G-protein signaling (rgs) 16, and ras-like family member 11b. Northern blotting confirmed diurnal changes in mRNA expression in the hypothalamus for these genes. Ras-like family member 11b was examined in more detail using in situ hybridization and antiphase diurnal changes in expression in suprachiasmatic nucleus and arcuate nucleus were identified implicating the gene in circadian-related, guanosine-mediated signaling. The transcription transactivator protein, CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain, which had not been previously identified in brain, was enriched in suprachiasmatic nucleus and discrete regions of the hypothalamus and forebrain. The potential regulatory role of CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain in the transcription of genes like TGF-alpha implicates the protein in diurnal activity rhythms. These results demonstrate the ability of gene expression profiling to identify potential candidates important in circadian or homeostatic processes.

Differential expression of arc mRNA and other plasticity-related genes induced by nicotine in adolescent rat forebrain.

Relatively little attention has been focused on mechanisms related to neural plasticity and drug abuse in adolescence, compared with abundant research using adult animal models. As smoking is typically initiated in adolescence, an important question to address is whether the adolescent brain responds differently to nicotine compared with the adult. To investigate this question, we examined the expression of a number of early response genes (arc, c-fos and NGFI-B) that have been implicated in synaptic plasticity and addiction, following acute nicotine in adolescent and adult rats. Baseline expression of arc and c-fos was higher in adolescent brains compared with adults. Following acute nicotine treatment (0.1, 0.4mg/kg), we found a marked induction of arc mRNA in the prefrontal cortex of nicotine-treated adolescents compared with a less pronounced increase of arc in the adult. c-fos and NGFI-B were also upregulated by nicotine, but not in an age-related manner. In contrast, nicotine induced less arc, c-fos, and NGFI-B expression in the somatosensory cortex of adolescents compared with adults. A fourth gene, quinoid dihydropteridine reductase was expressed at lower levels in white matter of the adolescent forebrain compared with the adult, but was not affected by nicotine. These results suggest that in adolescence, the activity of specific early response genes is higher in brain regions critical for emotional regulation and decision-making. Further, nicotine affects key plasticity molecules in these areas in a manner different from the adult. Thus, adolescence may represent a neurobiologically vulnerable period with regard to nicotine exposure.

Identification of genes in the oligodendrocyte lineage through the analysis of conditionally immortalized cell lines.

The mouse oligodendrocyte cell lines, N19 and N20.1, were used as sources of potential stage-specific RNA in order to construct a subtraction library enriched in cDNAs expressed early in the oligodendrocyte (OL) lineage. From this library, 23 clones were examined and three were examined in most detail. The mRNAs of the three library clones were preferentially expressed in the N19 (progenitor) compared to the N20.1 (immature) OL line. One of these corresponded to the intermediate filament protein cytokeratin K19, which has not been reported to be expressed in OLs previously. Another was identified as the mouse homolog of T-cadherin, previously reported not to be present in OLs. Antisera raised against a T-cadherin peptide indicated the protein colocalized with the OL lineage markers A(2)B(5), A007, and 01 in mouse primary glial cultures. However, small round cells resembling OL precursors labeled intensely with T-cadherin, but were negative for the other markers, suggesting that this gene might be expressed earlier in the lineage. In early postnatal brain, in addition to the expected neuronal tracts, the T-cadherin antibody labeled small bipolar cells, approximately 8-10 microm in diameter, in white matter tracts. These cells had the morphology of OLs or their precursors and were identified within the cerebellar white matter and the corpus callosum, regions rich in OLs. The third clone, 3g5, was homologous to the P8 clone isolated from rat pancreas. It encoded an 80-amino-acid polypeptide with a protein kinase C domain suggesting a possible role in signal transduction. Antisera to this peptide also colocalized 3g5 with cells expressing A(2)B(5), A007, and 01 in culture and in cells within white matter tracts which had the same morphology as those labeled by T-cadherin in these regions. In addition to these, beta(10) thymosin and mevalonate kinase clones were also isolated from the screen.

Morphine-associated environmental cues elicit conditioned gene expression.

Drug-associated contextual cues can exert a powerful influence on behavior through associative pairing between the drug and the environment. However, the anatomical and molecular substrates for these effects are not well characterized. Using a drug-conditioning paradigm, we examined the expression of the immediate early gene product, Fos, within specific brain circuits using immunocytochemical detection. Rats were given either morphine (5 mg/ml/kg) or saline once a day for 10 days. The drug administration was always paired with a specific environment (activity monitors) different from the home cage. Following this treatment, the rats were returned to the cages at various times thereafter, with only a mock injection. Conditioned behavioral activation was observed in rats at 3, 5, and 7 days following treatment with morphine. In rats showing the conditioned motor response, several cortical and limbic areas showed substantial increases in the number of Fos positive cells, indicating that these regions were more active during exposure to the drug-paired environment. Areas that were most activated included prefrontal cortex, cingulate cortex, nucleus accumbens, and preoptic area. Further analysis showed that this increase in Fos expression was not directly related to the increase in motor activity, and that the drug-associated conditioning and Fos expression was lessened at 7 days and absent by 14 days post-treatment. These results are discussed in terms of their relevance to the problem of relapse in drug addiction.

Embryonic expression of the myelin basic protein gene: identification of a promoter region that targets transgene expression to pioneer neurons.

The myelin basic protein (MBP) gene produces two families of structurally related proteins from three different promoters-the golli products, generated from the most upstream promoter, and the MBPs, produced from the two downstream promoters. In this report we describe the expression of golli proteins within some of the earliest neuronal populations of the brain, including Cajal-Retzius cells and preplate neurons of the forebrain, representing a new marker for these cells. To identify elements responsible for neuronal expression of the golli products, we generated transgenic animals from constructs containing different portions of the upstream promoter. A construct containing 1.1 kb immediately upstream of the golli transcription start site targeted expression of beta-galactosidase to preplate neurons and a subset of Cajal-Retzius cells in transgenic mice-the first reported genetic element to target expression to these pioneer cortical populations. Although expression in Cajal-Retzius cells declined with embryonic development, preplate cells continued to express the transgene after arriving at their final destination in the subplate. Interestingly, expression persisted in subplate neurons found within a distinct layer between the white matter and cortical layer VI well into postnatal life. Birth dating studies with bromodeoxyuridine indicated that these neurons were born between E10.5 and E12.5. Thus, the transgene marked subplate neurons from their birth, providing a fate marker for these cells. This work suggests a role for the MBP gene in the early developing brain long before myelination and especially in the pioneer cortical neurons important in the formation of the cortical layers.

Expression of oligodendrocytic mRNAs in glial tumors: changes associated with tumor grade and extent of neoplastic infiltration.

We examined the expression of glial- and neuronal-specific mRNAs within human gliomas using in situ hybridization. We found that low-grade astrocytomas contained a high number of proteolipid protein (PLP) mRNA-positive cells and that the number of PLP-stained cells decreased markedly with increasing tumor grade. Interestingly, the ratio of PLP mRNA-stained cells:myelin basic protein (MBP) mRNA-stained cells in normal white matter and low-grade astrocytoma was about 2:1 but approached 1:1 with increasing tumor grade. This parameter appeared to be a good indicator of tumor infiltration in astrocytomas, so we tested this in the analysis of other gliomas. Unlike astrocytomas, oligodendrogliomas were found consistently to contain few PLP mRNA- or MBP mRNA-expressing cells. In contrast, gemistocytic astrocytomas, typically highly invasive tumors, contained high numbers of PLP-positive cells and a ratio of PLP mRNA:MBP mRNA-stained cells of about 1.5:1, similar to low-grade astrocytomas. Nonradioactive in situ hybridization also enabled the morphological identification of specific cells. For example, gemistocytic astrocytes, which were found to be strongly vimentin mRNA positive, contained little glial fibrillary acidic protein mRNA and did not stain for PLP or MBP mRNAs. Neuronal mRNAs, such as neurofilament 68, were observed in small numbers of entrapped neurons within gliomas but were uninformative with respect to predicting tumor grade. Our results suggest that oligodendrocytes survive low-grade tumor infiltration and that glial tumor cells, unlike cell lines derived from them, do not express oligodendrocyte or neuronal mRNAs. In addition, the expression of mRNAs for the two major myelin protein genes, PLP and MBP, could be used to predict the grade and extent of tumor infiltration in astrocytomas.

Alterations in the spontaneous release of dopamine and the density of the DA D2 receptor mRNA after chronic postnatal exposure to cocaine.

The influence of cocaine administration on dopamine (DA) release and D2 dopamine receptor mRNA levels was examined in developing rat brain. In the rat pup, cocaine (25 mg/ kg SC) was administered daily from postnatal days 1-9 and extracellular DA measured 24 h after the last injection of cocaine, using in vivo micro dialysis. Twenty-four hours after discontinuing cocaine administration, a decrease in the extracellular concentration of DA of more than 100% was found in treated pups compared to control pups. Pups were tested on postnatal days 10-12, 20-21, or 35-36. After 1 month, basal release of DA returned to control levels. To examine the structural basis of the alteration in basal release of DA, in situ hybridization studies were performed to access the effect of chronic administration of cocaine on the mRNA encoding the D2 DA receptor. These preliminary studies, on postnatal day 10, indicate that drug treatment alters the developmental pattern of D2 mRNA. The changes in D2 mRNA expression were accompanied by delayed disaggregation of neostriatal cells and diminished growth of neostriatal neurons. These structural changes may lead to functional impairment in the development of dopamine target cells, thus altering the balance of synaptic and trophic effects of DA.

Golli-MBP proteins mark the earliest stages of fiber extension and terminal arboration in the mouse peripheral nervous system.

The Golli-myelin basic protein (MBP) transcription unit gives rise to two sets of products. One set (i.e., the MBPs) is expressed exclusively in myelin forming cells and the other set (i.e., the golli isoforms) is expressed in both oligodendrocytes and neurons in the CNS. The two major golli proteins, generated from RNAs transcribed from the most upstream promoter of the gene, contain MBP peptide sequences in their C-terminal halves and are, therefore, structurally and immunologically related to the MBPs. We have examined the distribution and localization of golli proteins in the mouse peripheral nervous system (PNS) using immunocytochemistry with a golli-specific antibody. Golli immunoreactivity was first observed in sensory and motor fibers of the mouse at E11 during fiber tract extension, but prior to the maturation of terminal connections. Once neuromuscular junctions had formed, golli immunoreactivity appeared in motor endplates and persisted to the latest age examined, P60. Golli immunoreactivity was also observed in the cell bodies and processes of the dorsal root ganglia throughout development. Strong staining in the PNS of the dysmyelinating mutant shiverer suggested that the major golli protein in peripheral fibers was the BG21 isoform. Interestingly, golli immunoreactivity was also found in adrenal chromaffin cells, which share a common neural crest derivation with other postganglionic neurons that express golli protein. These results suggest that in addition to its role in early forming neuronal systems of the CNS, golli protein also plays a role in the early development and maintenance of neurons in the PNS.

Expression of the myelin basic protein gene locus in neurons and oligodendrocytes in the human fetal central nervous system.

The myelin basic protein (MBP) gene locus is composed of two overlapping transcription units that share all of the MBP exons. One of these transcription units expresses the MBPs and the other expresses a family of proteins structurally related to the MBPs. This second transcription unit is called the Golli gene, and the entire complex is called the Golli-mbp gene. In this study, the expression of the Golli gene was examined in the human fetal central nervous system (CNS). By using reverse transcriptase-polymerase chain reaction cloning we have identified eight new members of the Golli gene family of transcripts expressed in the human CNS. Golli gene expression was examined by in situ hybridization and immunohistochemistry, and surprisingly, Golli products were found to be expressed in neurons as well as oligodendrocytes. Furthermore, the subcellular distribution of Golli immunoreactivity in fetal spinal cord interneurons shifted between the various laminae. Golli protein was localized within the nuclei of interneurons in the posterior horn, but was found in the cell bodies and processes of interneurons in the anterior horn. Within oligodendrocytes, Golli protein was detected in the cell bodies and processes, including processes which were wrapping axonal segments. Golli mRNA expression was also observed in neurons within the cerebral cortex between 18 and 20 weeks postconception, prior to myelination of this brain region. During this period, there was a striking developmental increase in the numbers and in the locations of neurons expressing Golli mRNAs within the cortical plate. The diverse distribution of Golli proteins within neurons and oligodendrocytes indicates that their function is quite different from that of the MBPs to which they are closely related.

Myelin basic protein gene expression in neurons: developmental and regional changes in protein targeting within neuronal nuclei, cell bodies, and processes.

The myelin basic protein (MBP) gene is part of the golli-mbp gene complex. In mouse, the golli-mbp gene produces two families of mRNAs from different transcription start sites that generate either MBPs or golli proteins (which contain MBP sequences in addition to unique peptide sequences). In situ hybridization and immunocytochemical analyses indicate that golli products are expressed in selected neuronal populations in postnatal mouse brain, in addition to oligodendrocytes, as shown earlier. The principal subcellular location of golli proteins in neurons was in axonal and dendritic processes. In a small subset of neurons, golli proteins were located in nuclei. With development and neuronal maturation, golli-mbp expression decreased and/or there was a striking shift in subcellular localization from nuclei and cell soma to the cell processes in specific neuronal populations. Golli protein was localize in neurites of migrating cerebellar granule cells, but it shifted to a nuclear localization when the cells took up residence in the internal granule cell layer. In some regions, (e.g., olfactory bulb and cerebellum) golli proteins were expressed over the entire postnatal period examined (birth to 75 d). The unique patterns of developmental expression within individual populations of neurons, and the unusual shift in subcellular localization of golli proteins with neuronal migration and maturation, suggest a complex regulation of this gene at both the transcriptional and posttranslational levels. The data also suggest that the cellular function(s) of the golli proteins is very different from the structurally related MBPs.

Cellular influences on RNA sorting in neurons and glia: an in situ hybridization histochemical study.

The unique structures of process-bearing cells in the central nervous system (CNS) present an ideal model with which to study the differential distribution of mRNA. We conducted a side-by-side examination of the intracellular distribution of nine neural mRNAs by in situ hybridization histochemistry in mammalian brain and observed four general types of mRNA distributions. (1) Some mRNA species were confined to cell somas and included those encoding the glial proteins, myelin proteolipid protein and 2'3'-cyclic nucleotide-3'-phosphodiesterase and the neuronal enzymes, neuron-specific enolase and glutamate decarboxylase-67. (2) Some mRNAs were found abundantly within the cell soma and were also located throughout cellular processes. These included myelin basic protein (MBP) mRNA, which was localized to the cell soma and myelin sheaths of oligodendrocytes, and glial fibrillary acidic protein (GFAP) mRNA, which was localized to the cell soma and processes of reactive and some non-reactive astrocytes in the adult brain and radial glia in embryonic brain. (3) Some mRNAs were found primarily in perinuclear cytoplasm but in some cells were also observed in cell processes. These included mRNAs encoding the protein kinase C/calmodulin-binding substrates, RC3 (neurogranin) and GAP-43, which were identified in the somas as well as within the proximal dendritic branches of specific forebrain neurons. (4) Some mRNAs were localized primarily within cell processes. These included MAP2 mRNA, which was identified by deep staining within dendritic fields but by only light staining within neuronal cell bodies. The data also indicated that the stage of cellular development and the regional location of a cell within the CNS had a profound influence on translocation events. MAP2 mRNA was found in the dendritic processes of most neurons but was confined to the soma of neurons in specific brainstem nuclei. MBP mRNA was confined to the perinuclear cytoplasm of immature oligodendrocytes and was then transported into the myelin sheath at a developmental stage corresponding to myelination. The distribution patterns of these mRNAs are likely to reflect the mechanism by which the protein products of these molecules are targeted within neurons and glia. In addition, mRNA movement may be influenced by cellular and regional factors not encoded solely within the structure of the translocated mRNA.

Structure and developmental regulation of Golli-mbp, a 105-kilobase gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain.

We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.

Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization.

The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10.

Effect of a discrete dorsal forebrain lesion in the rat on the expression of neuronal and glial-specific genes: induction of calmodulin, NF-L, SC1, and GFAP mRNA.

The influence of a localized tissue injury on the expression of genes encoding specific neuronal and glial proteins was examined using in situ hybridization. A pronounced induction of neuron-specific calmodulin (CaM) mRNA was evident within cells proximal to the wound site by 2 hours following a cortical lesion in rats. By 12 hours postlesion, intense signal corresponding to CaM mRNA was found to extend 1 mm from the wound site. Changes in the expression of mRNA encoding two additional neuronal proteins, the 68 kilodalton neurofilament protein and the extracellular matrix protein, SC1, were also evident at 12 hours following the cortical injury. Of the two glial proteins examined, a dramatic elevation in levels of mRNA for glial fibrillary acidic protein (GFAP) was observed at the wound site by 12 hours postlesion. This intense labeling corresponding to GFAP mRNA was evident in the ipsilateral glial limitans and hippocampus as well as in the contralateral glial limitans. In contrast, the pattern of labeling for the beta-subunit of the S100 protein did not differ from that of control animals at either of the postlesion intervals examined. This study identifies four genes, CaM, GFAP, SC1, and NF-L, which are induced following a localized cortical injury and which encode mRNA species enriched in specific cell-types in the central nervous system.

Heterogeneity in expression of S100 beta mRNA in human melanoma and pleomorphic adenoma demonstrated by in situ hybridization.

S100 protein is a widely used immunohistochemical marker for identification of a number of tumors including malignant melanoma and pleomorphic adenoma of the salivary gland. To extend the detection techniques for S100 protein to the level of its mRNA, sections of malignant melanoma and pleomorphic adenoma were hybridized in situ with a 35S-labeled anti-sense RNA probe complementary to the mRNA for the beta subunit of human S100 protein. Both tumors were labeled with the anti-sense RNA probe but not with a sense RNA probe. In addition, sections of normal and tumor tissues which were known not to express S100 protein on the basis of immunohistochemical studies were not labeled with the anti-sense RNA probe. These results established the specificity of the in situ hybridization technique for the detection of S100 protein mRNA. Although most of the tumor cells in both malignant melanoma and pleomorphic adenoma were labeled with the anti-sense RNA probe, unlabeled tumor cells were also present in their vicinity, suggesting there was a heterogeneity among the cells in both tumor types with respect to S100 protein mRNA expression.

Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization.

Glial fibrillary acidic protein (GFAP) accumulates in astrocytes during development. We have characterized the increase in GFAP mRNA during development of the rat brain by using Northern blotting and in situ hybridization histochemistry and have found a caudal to rostral gradient of expression, consistent with overall brain maturation. GFAP mRNA was first observed at embryonic day 16 (E16) in the glial limitans of the ventral hindbrain. During brain development message levels increased rostrally and by postnatal day 5 (P5) the entire glial limitans showed a positive signal which persisted into adulthood. GFAP mRNA was also found to accumulate in a caudal to rostral direction within the Purkinje cell layer of cerebellum beginning shortly after birth. By P5 the entire layer was positive and signal in this region could be localized to Bergmann glia by P15. A transient elevation in GFAP mRNA was apparent during the second postnatal week in cerebellum and cerebrum. Using in situ hybridization, a peak in message levels was observed at P15 and could be localized primarily to the deep white matter of cerebellum, to the corpus callosum, and to certain hippocampal fiber tracts. The pattern of GFAP expression in these regions is consistent with the differentiation of interfascicular glia and the appearance of type-2 astrocytes during the initial events of myelination. GFAP mRNA levels in white matter were greatly reduced in the adult. The pronounced regional differences in GFAP mRNA expression during development may reflect the differentiation of subpopulations of astrocytes.

Expression of the beta-S100 gene in brain and craniofacial cartilage of the embryonic rat.

The distribution of beta-S100 mRNA within the brain and skull of the 14-day rat embryo was determined using in situ hybridization. In nonnervous tissue, signal corresponding to beta-S100 mRNA was prominent within cartilaginous regions. Signal appeared to be confined to chondrocytes and was not observed within undifferentiated cells of the perichondrium. Within the skull, expression of beta-S100 mRNA was prominent in the nasal region and maxillary process as well as within Meckel's cartilage. Signal was also observed within the cranial base of the embryonic skull. In the embryonic brain, beta-S100 mRNA was prominent within the ventricular region of the myelencephalon and could be localized to a layer of cells lining the ventricle. Although beta-S100 mRNA has been shown to accumulate postnatally in the central nervous system, the present results clearly identify high levels of beta-S100 mRNA within specific regions of the embryonic brain and skull.

Expression of the gene encoding the beta-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization.

To investigate patterns of expression of the gene encoding the beta-subunit of S-100 protein during development of the rat brain we have used Northern blotting and in situ hybridization histochemistry. During late prenatal development beta-S-100 mRNA was observed first in the germinal zone lining the 4th ventricle. In the postnatal cerebellum this mRNA accumulated primarily in Bergmann glia and astrocytes of the deep white matter. In the hindbrain, expression of S-100 mRNA increased steadily in specific regions during the first postnatal week while levels remained low in more anterior brain regions. By the end of the second postnatal week, a dense punctate signal was distributed throughout the midbrain and hindbrain. Expression in forebrain, first observed at E18, was confined to cells lining the ventricle until the second postnatal week when accumulation of mRNA was observed in specific regions of the hippocampus, neocortex and olfactory bulb. The adult brain pattern of beta-S-100 mRNA distribution is attained during the third postnatal week. These results demonstrate a caudal-rostral gradient in expression of the beta-S-100 gene during rat brain development, as well as pronounced regional differences which may reflect the differentiation of subpopulations of astrocytes.