mRNA Binding Sites Research Articles

Localization of mRNA and Receptor Binding Sites for the alpha sub 1a-Adrenoceptor Subtype in the Rat, Monkey and Human Urinary Bladder and Prostate

Localization of mRNA and Receptor Binding Sites for the alpha sub 1a-Adrenoceptor Subtype in the Rat, Monkey and Human Urinary Bladder and Prostate

5-HT1A and 5-HT2A receptor mRNAs and binding site densities are differentially altered in schizophrenia

We have investigated 5-HT1A (serotonin1A) and 5-HT2A (serotonin2A) receptor mRNA abundance and binding site densities in various neocortical and hippocampal regions of schizophrenics and control subjects. Age, agonal state (brain pH), and post mortem interval were included where necessary as covariates in our analyses. In schizophrenics, 5-HT1A binding site densities, determined autoradiographically by [3H]8-hydroxy-2,3-(dipropylamino)-tetralin ([3H]8-OH-DPAT), were significantly increased (+23%) in the dorsolateral prefrontal cortex, with a similar trend in anterior cingulate gyrus. These increases were not accompanied by any change in 5-HT1A receptor mRNA. No differences between the groups in [3H]8-OH-DPAT binding or 5-HT1A receptor mRNA were seen in superior temporal gyrus, striate cortex, or hippocampus. 5-HT2A binding sites, determined by [3H]ketanserin, were decreased in the dorsolateral prefrontal cortex (-27%) and parahippocampal gyrus (-38%) of schizophrenics, with a similar trend in cingulate gyrus, but not in superior temporal gyrus or striate cortex. 5-HT2A receptor mRNA abundance was reduced in schizophrenics in the dorsolateral prefrontal (-49%), superior temporal (-48%), anterior cingulate (-63%) and striate (-63%) cortices, but not in parahippocampal gyrus. Parallel analyses of rat brain tissue showed no changes in 5-HT1A or 5-HT2A receptor mRNAs or binding site densities after chronic administration of haloperidol. These data show that schizophrenia is associated with alterations in the expression of central 5-HT1A and 5-HT2A receptors. They confirm reports of increased 5-HT1A and decreased 5-HT2A binding site densities in prefrontal cortex, and reveal more extensive decreases in 5-HT2A receptor gene expression at the mRNA level. The resulting imbalance in the 5-HT1A to 5-HT2A receptor ratio, when considered in terms of the chemoarchitectural distribution of these receptors, may contribute to an impairment of corticocortical association pathways. The apparent dissociation of the normal relationships between the abundance of each 5-HT receptor and its mRNA in schizophrenia introduces a separate complexity to the data, which may give clues to the underlying molecular mechanisms.

Localization of alpha 7 nicotinic receptor subunit mRNA and alpha-bungarotoxin binding sites in developing mouse somatosensory thalamocortical system.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.

Chronic infusion of a CRH 1 receptor antisense oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-related behavior in socially defeated rats

We studied the role of central amygdala CRH receptors in behavioral responses to an anxiogenic stimulus. An antisense oligodeoxynucleotide corresponding to the rat CRH 1 receptor mRNA was infused chronically into the central amygdaloid nucleus of male rats via osmotic minipumps (0.25 μg/0.5 μl/h). Control groups received infusions of either a scrambled sequence oligodeoxynucleotide or vehicle. On the 4th day of treatment, rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Antisense oligodeoxynucleotide-treated rats spent significantly more time exploring the open arms of the plus-maze than scrambled sequence- and vehicle-treated animals, both of which did not differ from each other. The social discrimination test, on the other hand, revealed no difference in juvenile recognition abilities among the treatment groups. Using in situ hybridization and receptor autoradiography, we were not able to detect clear signals of CRH 1 receptor mRNA and CRH binding sites in the central amygdaloid nucleus of either group, confirming the reportedly low expression and density of CRH receptors in this brain area. The present data support the view that CRH receptors in the central nucleus of the amygdala are involved in the mediation and expression of anxiety-related behavior, but simultaneously raise questions as to the mechanisms of antisense oligodeoxynucleotide action.

Distribution of Cross-links between mRNA Analogues and 16 S rRNA in Escherichia coli 70 S Ribosomes Made under Equilibrium Conditions and Their Response to tRNA Binding

The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical cross-linking using mRNA analogues that contain 4-thiouridine (s4U) or s4U modified with azidophenylacyl bromide (APAB), either two nucleotides upstream or eight nucleotides downstream from the nucleotide sequence ACC, the codon for tRNA(Thr). The sequences of the mRNA analogues were described earlier (Stade, K., Rinke-Appel, J., and Brimacombe, R. (1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe, R. (1991) EMBO J. 10, 2195-2202). Under equilibrium conditions, both of these mRNA analogues bind and cross-link to 70 S ribosomes without the presence of tRNA(Thr); however, there are significant increases both in binding and particularly in cross-linking in the presence of the tRNA(Thr). Four regions contain cross-linking sites that increase in the presence of tRNA, C1395, A532, A1196 (and minor sites around these three positions), and C1533/U1532. Three other cross-linking sites, U723, A845, and U1381, show very little change in extent of cross-linking when tRNA is present. A conformational change in the 30 S subunit allowing additional accessibility to the 16 S rRNA by the mRNA analogues upon tRNA binding best explains the behavior of the tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.

Repeated ECS differentially affects rat brain 5-HT1A and 5-HT2A receptor expression.

We investigated the effect of electroconvulsive shock (ECS), administered five times over 10 days, on 5-HT1A and 5-HT2A receptor mRNA and binding site densities in the rat brain using in situ hybridization histochemistry and quantitative autoradiography. ECS treatment increased 5-HT1A receptor mRNA abundance and binding site densities in the dentate gyrus, but decreased these parameters in the CA3c layer of the hippocampus. No changes in 5-HT1A receptor mRNA and binding sites occurred in other hippocampal subfields, neocortex or raphe nuclei. Repeated ECS was also found to increase 5-HT2A receptor binding site densities in the neocortex and this was accompanied by a non-significant increase in cortical 5-HT2A receptor mRNA abundance. Our study demonstrates that in the rat, repeated ECS produces anatomically and molecularly discrete effects on 5-HT1A and 5-HT2A receptor gene expression. These changes may be relevant to the therapeutic effect of repeated ECS in depression.

Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.

Universal barrier to lateral spread of specific genes among microorganisms.

A genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed. It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3' end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera. Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis. While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non-immune recipients results in killing of such recipients. This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude. In combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels. This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms.

Detection and quantitation of 5-HT 1A and 5-HT 2A receptor mRNAs in human hippocampus using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique and their correlation with binding site densities and age

The presence and abundance of 5-HT 1A and 5-HT 2A receptor mRNAs in post mortem human hippocampus was investigated using a novel quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique using cyclophilin mRNA as an internal standard. 5-HT 1A and 5-HT 2A receptor mRNAs were each co-amplified with varying dilutions of cyclophilin primers, and their abundance expressed as a ratio of cyclophilin mRNA. Using this technique in combination with quantitative autoradiography we have investigated the effect of aging on hippocampal 5-HT 1A and 5-HT 2A receptor mRNA abundance and binding site densities. There was a significant negative correlation between hippocampal 5-HT 1A receptor binding site densities and age and a similar trend for 5-HT 1A receptor mRNA abundance. Neither 5-HT 2A receptor binding site densities nor mRNA abundance were affected by age. Both 5-HT 1A and 5-HT 2A receptor binding site densities in individual subjects correlated significantly with abundance of their encoding mRNA. This study demonstrates that 5-HT 1A and 5-HT 2A receptor mRNAs can be measured in small samples of human brain. Combining studies of mRNA with those directed at binding sites will help reveal mechanisms underlying changes in expression of these receptors in various neuropsychiatric disorders.

Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.

Tianeptine decreases both serotonin transporter mRNA and binding sites in rat brain

In situ hybridization histochemistry and quantitative autoradiography were used to evaluate the effects of repeated administration of the serotonin reuptake enhancer, tianeptine. Tianeptine (10 mg/kg twice daily, for 14 days) significantly reduced both the expression of serotonin transporter mRNA and serotonin transporter binding sites labeled by [ 3H]paraxetine in rat dorsal raphe nucleus. In median raphe nucleus, tianeptine did not change either expression of serotonin transporter mRNA or binding. This effect, which is similar to a reported effect for serotonin reuptake inhibitors, may help to explain the antidepressant effect of tianeptine.

An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.

An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.

μ-Opioid receptor mRNA expression in the rat CNS: comparison to μ-receptor binding

The distribution of cells expressing μ-receptor mRNA and μ-receptor binding sites were compared in brain and spinal cord tissue sections using a combination of in situ hybridization and receptor autoradiographic techniques. μ-Receptor mRNA was visualized with a 35S-labeled cRNA probe directed to transmembrane III–VI of the rat μ-receptor, while μ-receptor binding sites were labeled with the μ-selective ligand [ 3H]DAMGO. A high correspondence between the μ-receptor mRNA and receptor binding distributions was observed in the nucleus of the accessory olfactory bulb, anterior olfactory nuclei, striatal patches of the nucleus accumbens and caudate-putamen, endopiriform nucleus, claustrum, diagonal band of Broca, globus pallidus, ventral pallidum, bed nucleus of stria terminalis, most thalamic nuclei, medial and posteriocortical medial amygdala, lateral, dorsomedial, posterior and mammillary nuclei of the hypothalamus, presubiculum, subiculum, rostral interpeduncular nucleus, median raphe, inferior colliculus, parabrachial nucleus, locus coeruleus, central grey, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis, nucleus cuneatus, and the dorsal motor nucleus of vagus. Differences in μ-receptor mRNA and receptor binding distributions were observed in several regions, including the olfactory bulb, cortex, hippocampus, superior colliculus, spinal trigeminal nucleus, cochlear nucleus and spinal cord, and may be due to μ-receptor transport to presynaptic terminals.

The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction.

Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions.

Ovariectomy increases the level of estrogen receptor mRNA and estrogen receptor binding sites in female rat adipose tissue.

The roles of estrogen in the changes in estrogen receptor (ER) mRNA and ER binding sites in rat adipose tissue were studied in female rats. To elucidate the mechanism(s) behind ovariectomy (OVX)-induced obesity, the levels of ER mRNA and ER binding sites in adipose tissue were analyzed three weeks after OVX using Northern blot analysis of ER mRNA and the [3H]E2 binding assay, respectively. OVX induced an increase in body weight, and replacement of estradiol (E2) prevented that increase. Significant increases in the amounts of ER mRNA and in [3H]E2-specific binding were observed after OVX, and E2 replacement reduced both of those increases. These results suggest that E2 may regulate rat obesity directly through ER in adipose tissues.

Distribution of nicotinic receptors in cynomolgus monkey brain and ganglia: Localization of α3 subunit mRNA, α-bungarotoxin and nicotine binding sites

The distribution of nicotinic receptors in the brain and ganglia of the Cynomolgus monkey was studied by in situ hybridization and receptor autoradiography. A 35S-labeled antisense riboprobe for the mRNA of the α3 subunit of the human nicotinic receptor, [ 3H] l-nicotine and [ 125]α-bungarotoxin were used as markers. The highest levels of α3-mRNA were observed in the hippocampus, the medial habenula, the lateral geniculate, the granular layer of the cerebellum, as well as in the pineal gland; moderate levels were found in other nuclei of the thalamus and in the deeper layers of the cerebral cortex. High-affinity binding sites for [ 3H] l-nicotine were observed mainly in the thalamus. The distribution of [ 125I]α-bungarotoxin binding sites was different from that observed for α3-mRNA and [ 3H] l-nicotine; they were most abundant in a few specific thalamic nuclei, in the medial habenula and in lamina I of the cerebral cortex. The localization of these three markers was also investigated in the sympathetic, parasympathetic and sensory ganglia of the monkey. Intense labeling was observed for α3-mRNA and for [ 125I]α-bungarotoxin in the sympathetic and parasympathetic ganglia, whereas no positive signal was seen in the ganglion of Gasser. [ 3H] l-nicotine binding was not detected in any of the ganglia examined. High levels of mRNA for the α3 subunit of the nicotinic receptor were also detected in human sympathetic ganglia. Comparison between α3-mRNA distribution and [ 3H] l-nicotine binding suggests that in the Cynomolgus monkey brain, the α3 subunit may participate in the formation of more than one nicotinic receptor subtype: a high-affinity binding site for [ 3H] l-nicotine in the thalamus and other sites with low affinity for nicotine in the medial habenula and cerebral cortex. Both the α3-mRNA and the [ 125I]α-bungarotoxin are highly expressed in the sympathetic ganglia; however, since no information is presently available on the intraneuronal cellular localization, it cannot be established whether or not they are both present at synaptic sites.

Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation

To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA∗30S subunit∗tRNA f Mei), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5′-end region of the cro gene of λ bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5′-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5′-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5′-end side of the initiation AUG condon in the P site. According to the second approach, 5-azidouridine (5N 3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N 3UTP and UTP. A message that had U residues at either −4, −3, −1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N 3U transcripts were essentially ‘zero-length’, the 5′-derivatized transcripts were covalently attached to ribosomal components about 14 Å from the 5'-end. We found the proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site. The 5′-end of the start codon might be within 14 Å from protein S7, while the 5′-end of the Shine-Dalgarno sequence probably has no definite binding site. Finally we discuss a possible way of mRNA positioning on the 30S ribosomal subunit.

Ontogeny of gene expression of adenosine A2 receptor in the striatum: early localization in the patch compartment.

The ontogeny of adenosine A2 receptor mRNA and adenosine A2 binding sites distributions was studied by in situ hybridization histochemistry and receptor autoradiography in pre- and post-natal rat striatum, postnatal dog striatum, and a human fetus striatum and compared to that of dopamine D1 and mu opiate receptors. The early postnatal striatum demonstrated heterogeneous distributions of adenosine A2 receptor mRNA and adenosine A2 binding sites with patches of dense labeling corresponding to dopamine D1 and mu opiate receptors enriched zones. This patchy pattern evolved to the homogeneous distribution observed in the adult. The higher intensity of adenosine A2 receptor mRNA enriched patches correspond at the microscopical level to a higher density of labeled neurons in the patches areas and also to a higher level of expression per labeled patches neuron than in the matrix ones. This demonstrates for the first time that differences in patch/matrix receptor density is at least partly linked to different levels of receptor gene expression.

Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [ 125I]estrogen autoradiography and non-isotopic in situ hybridization histochemistry

The spatial distribution of cells in the adult rodent forebrain which express estrogen receptor mRNA, as shown by in situ hybridization histochemistry with isotopically-labeled probes, has been reported to overlap with regions that are known targets of estrogen and which bind estrogen. The extent to which detection of estrogen receptor mRNA within developing forebrain neurons of the postnatal day 10–12 female rat is accompanied by translation into estrogen binding sites was investigated by combining [ 125I]estrogen autoradiography with non-isotopic (digoxigenin) in situ hybridization, using a 48-base oligodeoxyribonucleotide probe encoding a sequence of the estrogen-binding domain of rat uterine estrogen receptor cDNA. Estrogen receptor mRNA and estrogen binding sites appeared to be restricted to neurons. No mRNA or binding was seen in ependymal cells. Cells expressing estrogen receptor mRNA were widely distributed in the developing rat forebrain and were found in brain regions generally corresponding to those previously shown in the adult, with the addition of some regions not previously described, such as the medial habenula and dorsal endopiriform nucleus. Although there was widespread overlapping of estrogen receptor mRNA expression with known estrogen binding sites, there were regional and cellular variations in the extent of receptor mRNA translation. This pattern was true for developing forebrain regions previously defined as estrogen receptor-containing (hypothalamus, preoptic area, medial and lateral septum, vertical and horizontal nuclei of the diagonal band, cerebral cortex, hippocampus and amygdala) as well as for regions heretofore not considered estrogen targets (the thalamus, dorsal endopiriform nucleus, claustrum, ventral pallidum/substantia innominata and the basal nucleus of Meynert) or characterized as estrogen-responsive in the adult without previously documented estrogen binding [caudate-putamen (striatum)]. While estrogen binding and receptor mRNA expression always co-localized, neurons expressing estrogen receptor mRNA did not always exhibit ligand binding and there was no clear-cut relationship between the intensity of the hybridization signal and estrogen binding. Little, however, is known about translational control of estrogen receptor expression in the brain. Localization of estrogen binding sites to regions not generally considered targets of estrogen would appear to reflect the greater sensitivity of the iodinated ligand than the tritiated estrogens more commonly used for autoradiography. Non-isotopic in situ hybridization histochemistry combined with [ 125I]estrogen autoradiography represents a very powerful tool with which to study regulation of estrogen receptor gene expression at the single cell level with an exceptional degree of cellular and anatomical resolution. These findings suggest that the extent of estrogen receptor mRNA translation in the developing brain has been underestimated. Differential patterns of translation may be related to regional differences in the ontogeny of the estrogen receptor and to developmentally-regulated differences in post-translational processing as well as to the autoradiographic method itself, particularly with respect to regional differences in the length of exposure required.