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Current understanding of IGF-I-mediated neuroprotection implies the activation of phosphatidylinositol-3-kinase (PI-3K), which leads to the activation of Akt/Protein Kinase B. In non-neuronal cells, Akt phosphorylates and activates the transcription factor CREB, implicated in the transcription of the anti-apoptotic bcl-2 gene. This paper further analyses the anti-apoptotic IGF-I action in neurons. We show that IGF-I protects cortical neurons against ceramide-induced apoptosis. Ceramide decreases Akt phosphorylation during apoptotic process whereas a simultaneous treatment with IGF-I increases Akt phosphorylation. Analysis of the signal transduction pathways revealed that IGF-I induces CREB phosphorylation via PI-3K and ERK, whereas simultaneous ceramide and IGF-I treatment decreases CREB phosphorylation. Although an overexpression of Bcl-2 protects cortical neurons against ceramide-induced apoptosis, our data indicate that the Bcl-2 protein level is not modulated during IGF-I, ceramide and/or LY294002 treatment. In consequence, we demonstrated that IGF protects neurons against ceramide-induced apoptosis and that IGF-I protection involves the PI-3K/Akt and ERK pathways; this protection may be independent of CREB and Bcl-2.

The frog (Xenopus laevis) retina has been an important model for the analysis of retinal circadian rhythms. In this paper, several isoforms of X. laevis casein kinase I (CKI) were analyzed to address whether they are involved in the phosphorylation and degradation of period protein (PER), as they are in the circadian oscillators of other species. cDNAs encoding two splice variants of CKI(delta) (a full-length form and deletion isoform, which is missing an exon that encodes a putative nuclear localization signal and two evolutionarily conserved protein kinase domains) were isolated and analyzed, together with a previously isolated CKI(epsilon) isoform. Both CKI(delta) and CKI(epsilon) were shown to be constitutively expressed in the photoreceptors of the retina, where a circadian clock has been localized. Both the full-length CKI(delta) and CKI(epsilon) were shown to have kinase activity in vitro, and the full-length CKI(delta) phosphorylated and degraded Drosophila PER when expressed in Drosophila S2 cells. The expression and biochemical characteristics of these CKIs are consistent with an evolutionarily conserved role for CKI in the Xenopus retinal clock. The CKI(delta) deletion isoform did not exhibit kinase activity and did not trigger degradation of PER. Subcellular localization of both CKI(delta) isoforms was cytoplasmic in several cell culture lines, but the full-length CKI(delta) , and not the deletion CKI(delta) isoform, was localized to both the nucleus and the cytoplasm in Drosophila S2 cells. These results indicate that the sequences missing in the deletion CKI(delta) isoform are important for the nuclear localization and kinase activity of the full-length isoform and that one or both of these features are necessary for degradation of Drosophila PER.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi is an important system that allows sequence-specific gene silencing through targeted degradation of mRNA by cognate double-stranded RNA (dsRNA). RNAi plays a role in endogenous cellular processes, such as developmental control and heterochromatin formation, and serves as an antiviral defense mechanism. Recent findings suggest that RNAi and related pathways are involved in protecting the genome against instabilities caused by transposons and repetitive sequences. Several rapidly developing RNAi methodologies provide a new approach for elucidation of gene functions. RNAi technology is also currently being evaluated as a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics. In this paper, we review the use of RNAi in neuroscience research and as a possible therapeutic tool for treatment of neurological diseases.

Recent studies indicated that junctophilins (JPs) contribute to the formation of junctional membrane structures in excitable cells by interacting with the plasma membrane and spanning the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane. In the brain, functional crosstalk between cell-surface and intracellular channels is proposed in the "subsurface cistern" as the junctional membrane complex observed in neurons. So far, three JPs have been identified as tissue-specific subtypes derived from different genes; JP-1 is specifically expressed in skeletal muscle, JP-2 is detected throughout muscle cell types, and JP-3 is predominantly expressed in the brain. In this paper, we report a novel JP subtype, JP-4, encoded in the human (chromosome 14q11.1) and mouse (chromosome 14C1-2) genomes. Cloning the cDNA showed that JP-4 shares characteristic structural features with other JP subtypes, and Northern and Western blot analyses demonstrated its brain-specific expression. In situ hybridization analysis revealed that both JP-3 and JP-4 mRNAs are expressed in discrete neuronal sites, and their overall regional distribution patterns were similar in the brain. Furthermore, both the JP mRNAs and subsurface cistern showed somatodendritic localization in hippocampal pyramidal neurons. The results obtained suggest the collaborative contribution of JP-3 and JP-4 to the subsurface cistern formation in neurons.

Cloning and molecular characterization of an homologous gene to proenkephalin in a teleost, the zebrafish Danio rerio is presented in this paper. The new zebrafish proenkephalin (zfPENK) encodes a 249 amino acid polypeptide that displays an identity of 40% to mammalian PENKs and which contains the consensus sequences for four Met-enkephalins, one Leu-enkephalin and one Met-enkephalin-Gly-Tyr, described for the first time in teleosts. Expression studies indicate that zfPENK is selectively expressed in zebrafish brain. Our findings support the concept that PENK genes might have been highly conserved throughout evolution and that its origins might be placed more than 400 million years ago. Moreover, we present evidence that the heptapeptide Met-enkephalin-Gly-Tyr present in fish might be anterior in evolution to the heptapeptide Met-enkephalin-Arg-Phe present in tetrapods. Also another homologous sequence to proenkephalin in zebrafish genome is presented. This sequence might stand for the third exon of a possible duplicate gene of zfPENK. Our findings not only present new data in relation to the evolution of opioid peptides in vertebrates, but also we present a new heptapeptide with putative differential activity than the other peptides derived from the mammalian proenkephalins. Future research will define the functional role of this new heptapeptide in the mechanism that describes opioid activity.

The antioxidant enzyme Cu,Zn superoxide dismutase has so far been considered costitutively expressed and exclusively localized into cytosol. In this paper we investigated Cu,Zn superoxide dismutase export in neuroblastoma SK-N-BE cells by flow cytometry analysis, confocal immunofluorescence analysis and enzyme-linked immunosorbed assay. Immunofluorescence analysis shows that the enzyme is exported by microvesicular granules; moreover the treatment of cells with brefeldin A and with 2-deoxy-D-glucose and sodium azide strongly decreases the amount of CuZn superoxide dismutase detected in the medium. Therefore the involvement of ATP-dependent mechanisms, likely including BFA-sensitive intracytoplasmic vesicles in Cu,Zn SOD export from SK-N-BE cells, has to be hypothesized. Microvesicular-mediated Cu,Zn SOD export in neurons could represent a relevant phenomenon able to influence cell excitability that is affected by reactive oxygen species.

This paper reports on the initial analysis of protein expression in the mouse cerebellum with the proteomics approach. Proteins from cerebellar tissue homogenates were separated by two-dimensional gel electrophoresis, and the proteins were stained with colloidal Coomassie Blue to produce a high-resolution map of the cerebellum proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray quadrupole ion trap mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. To date, 30 prominent proteins with various functional characteristics were identified. These data will be used for future studies of differential protein expression in mouse models of neurological disorders.

Nitric oxide synthases (NOS) are heme-containing enzymes which catalyse the oxidation of L-arginine to nitric oxide and L-citrulline. Some nitrogenated compounds have been reported to coordinate with the iron atom from the heme group, thus inhibiting NOS. 1,4-Benzodiazepines are nitrogenated compounds which have many physiological effects such as antianxiety, antiepileptic, hypnotic, and muscle relaxation properties. The aim of this paper was to measure the effect of different benzodiazepines on NOS activity in pig brain extracts. Medazepam, pinazepam, diazepam, oxazepam and alprazolam competitively inhibited NOS with IC(50) in the micromolar range. Other benzodiazepines showed no effect at concentrations as high as 200 microM. Due to the structural similarity of the benzodiazepine ring nucleus with L-arginine, we propose a benzodiazepine-enzyme interaction to explain the competitive inhibitions. By comparing benzodiazepine effects and their structures, the inhibitory effect of benzodiazepines on NOS is related to the absence of substituents on N4 and to the absence of a halogen substituent on C5 phenyl group. Although benzodiazepine's inhibitions observed in this study are not in the physiological range in normal cases, these inhibitions could be significant in drug abuse situations and should be taken into account for the rational design of drugs which specifically inhibit NOS.

To clarify the possible mechanisms by which the recreational drug, methamphetamine (METH), induces apoptosis, we investigated its effects on the expression of Myc apoptotic genes. This paper presents the characterization of c-myc and L-myc gene transcription in the striatum and the cortex. In addition, the expression of the corresponding proteins was also evaluated. Our observations reveal that c-myc and L-myc were up-regulated by METH at both the mRNA and protein levels. Thus, myc transcription factors might be responsible for some aspects of METH-induced apoptotic processes.

Proteins of the caspase family are involved in the signalling pathway that ultimately leads to programmed cell death (apoptosis), which has been reported to occur in some experimental models of stroke. In a previous paper we used quantitative reverse transcription and polymerase chain reaction (RT-PCR) to characterise changes in the mRNA expression of one member of this family, caspase-3, in a rat model of permanent focal ischemia. Here we have used this technique to study the expression of a further three caspases which are involved in different aspects of caspase signalling. Caspase-8, involved in Fas-mediated apoptosis, was upregulated in the cortex of ischemic rats. Caspase-11, which leads to the synthesis of the functional form of the cytokine interleukin-1 beta, also showed increased expression, but with a different temporal profile from caspase-8. In contrast, caspase-9, which forms part of the pathway signalling through the mitochondria, showed a decrease in expression. The expression of a further four caspases (1, 2, 6 and 7) has also been characterised in a simpler experiment. These caspases all showed distinctive patterns of expression following the induction of ischemia. These data lead us to conclude that caspase expression as a whole is under very strict transcriptional control in this model. Certain elements of caspase signalling, such as the Fas-induced pathway and the events upstream of IL-1 beta processing, are upregulated, while others are not. This may be due to some form of genetic program activated in response to ischemia in the brain and may highlight which biological pathways are modulated.

The hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of alpha and beta subunits, plays an important role in the cellular response to O(2) deprivation. In this paper, Drosophila HIF-1beta (dHIF-1beta) homolog is cloned and characterized. Further, Northern analyses showed that dHIF-1alpha and dHIF-1beta expressed their highest level at an embryonic stage. From the pupal stage on, their expression was sharply reduced and maintained at a steady level. Anoxia treatment up-regulated the expression of the both alpha and beta subunits. Over-expression of dHIF-1alpha in transgenic embryos resulted in embryonic lethality, while over-expression of dHIF-1beta significantly prolonged fly recovery time from a 5-min anoxic stupor. The cloning and characterization dHIF-1beta reported in this paper provide a framework for further genetic dissection of the HIF-1 complex in its role in the cellular or tissue response to O(2) deprivation.

The present results show that androgens are able to modulate the Po gene expression in different models. In particular, we have shown that: (1) the messenger for the androgen receptor (AR) is present in the rat sciatic nerve but not in cultured Schwann cells; (2) castration induces a decrease of Po mRNA levels in the sciatic nerve of male rats, which is counteract by the subsequent treatment with dihydrotestosterone (DHT), the 5alpha-reduced metabolite of testosterone; (3) castration is also able to significantly decrease in the sciatic nerve the activity of the enzyme 5alpha-reductase (which converts testosterone into DHT); and (4) DHT is able to stimulate Po gene expression in cultured Schwann cells. These observations seem to indicate that androgens may exert their effect on Po gene expression via indirect mechanisms; modulation of neuronal influences reaching the Schwann cells through the binding of the androgen to the AR present in neurons may be postulated. However, alternative mechanisms may also be taken in consideration. The data presented suggest indeed that androgens might act on Schwann cells via the progesterone receptor (PR) rather than the AR. It has been observed that: (1) the messenger for PR is present in Schwann cells; (2) DHT may activate the transcriptional activity of a PR-responsive gene by binding to the PR; and (3) putative steroid responsive elements have been described in this paper to be present in the Po promoter region., (Copyright 1999 Elsevier Science B.V.)

Recently, sortilin a non G-protein-coupled receptor has been identified as the 100-kDa neurotensin receptor. In this paper we describe the expression of its gene during mouse embryonal development. We show that the nervous system is the main location of sortilin gene expression and that with ongoing development the forebrain exhibits the highest accumulation of transcripts., (Copyright 1999 Elsevier Science B.V.)

A novel class of immediate early genes that encode enzymes of the MAP kinase phosphatase family has recently been described. These enzymes are dual-specificity protein phosphatases and some show tissue-specific distribution, like the hVH-5 gene (homologue of vaccinia virus H1 phosphatase gene clone 5), which is expressed predominantly in the adult brain. In this paper, we investigated whether the hVH-5 gene is induced by psychostimulants in rat brain, as has been demonstrated for immediate early genes encoding transcription factors. Using in situ hybridization, we found that i.p. injection of cocaine, amphetamine and caffeine induced hVH-5 mRNA expression within 40 min in the nucleus accumbens (NAc), caudate putamen, frontal cortex and hippocampus, with a maximal effect in the NAc. The cocaine-induced hVH-5 gene induction involves the serotonergic system, since it was abolished in the NAc by lesioning serotonergic raphé projections with 5,7-dihydroxytryptamine. Moreover, the effect of cocaine was fully mimicked by the selective serotonin uptake inhibitor fluoxetine. In contrast to what has been described for c-fos and egr-1 immediate early genes, we found that hVH-5 mRNA expression in the NAc and hippocampus was as significant after repeated cocaine injections for 10 days as after a single injection. The considerable and prolonged induction of the MAP kinase phosphatase hVH-5 gene by psychostimulant drugs in postmitotic brain cells, particularly in the NAc, could indicate that MAP kinase substrates are involved in the reinforcing properties of drugs of abuse., (Copyright 1998 Elsevier Science B.V.)

Parasympathetic innervation of rat submandibular and parotid glands regulates saliva volume, its rate of secretion and its composition. It also has a regulatory role in hypertrophy and hyperplasia of salivary glands, and in the expression of specific sets of genes. Rat cystatin S is a member of family 2 of the cysteine proteinase inhibitor superfamily. Cystatin S gene expression is tissue- and cell type-specific, temporally regulated during postnatal development, and not observed in adult animals. Isoproterenol (IPR), a beta-adrenergic agonist, induces hypertrophic and hyperplastic enlargement of rat salivary glands and expression of a number of genes including cystatin S. Sympathectomy reduces, but does not completely block IPR-induced expression of the cystatin S gene in the submandibular glands of adult female rats, indicating the participation of sympathetic factor(s) in this regulation. Since both sympathetic and parasympathetic branches of the autonomic nervous system act in parallel in the submandibular gland, it is possible that parasympathetic nerve terminals also provide factor(s) that play a role in regulation of cystatin S gene expression. Experiments described in this paper were designed to test the hypothesis that the parasympathetic nervous system participates in IPR-induced cystatin S gene expression. Bilateral parasympathectomy reduced IPR-induced cystatin S gene expression, suggesting a role of the parasympathetic nervous system in its regulation. Unilateral parasympathectomy in contrast, had no effect on IPR-induced cystatin S gene expression, suggesting that the presence of an intact parasympathetic innervation in the contralateral side permits the 'normal' IPR-induced expression of the cystatin S gene in the parasympathectomized gland., (Copyright 1998 Elsevier Science B.V.)

The 150-kDa oxygen-regulated protein (ORP150) first was described with reference to the central nervous system in cultured astrocytes subjected to dense hypoxia. Subsequently its transcript was found in macrophages within human aortic atherosclerotic plaques, suggesting a role in protecting cells under hypoxic stress. In a mouse model of permanent focal brain ischemia, we aimed to elucidate the constitutive cellular localization in vivo of ORP150 in the central nervous system as well as the sequential alteration in its mRNA and protein expression during this severe ischemic insult. Immunohistochemical study demonstrated that ORP150 protein normally is present predominantly in neurons. The 78-kDa glucose-regulated protein, which is another well-known stress protein retained in the endoplasmic reticulum, also was stained in neurons. During the first 3 h after ischemia, ORP150 antigenicity was markedly enhanced in severely damaged neurons, while the amount of the glucose-regulated protein was decreased. Preceding this change, orp150 mRNA was selectively induced in neurons undergoing postischemic cytoskeletal proteolysis, as early as 1 h after middle cerebral artery occlusion. These results indicated that ORP150 might be regulated by transcriptional level as for many stress proteins, but unlike previously described other stress proteins it was translated in the center of ischemic lesions despite nearly complete energy depletion. In this paper, the biological potentials of ORP150 protein in the setting of brain ischemia in vivo will also be discussed., (Copyright 1998 Elsevier Science B.V.)

The involvement of nerve growth factor (NGF) in neuroendocrine regulation is supported by several lines of evidence. In this paper, we investigated the NGF content and expression in the pituitary gland and other endocrine organs during dysendocrine states (thyroidectomized, adrenalectomized and gonadectomized male rats). We found an increase of NGF-IR in the pituitary gland and testis of hypothyroid rats whereas no differences were found in the adrenal gland and blood. Also, NGF mRNA expression had increased in the anterior pituitary of hypothyroid rats whereas it had not changed after adrenalectomy and gonadectomy. Moreover, other neurotrophins and neurotrophin high-affinity receptors were unchanged in the anterior pituitary of hypothyroid rats. These data indicate that pituitary NGF is selectively modulated by thyroid status of the animal, further supporting a close link between NGF and thyroid hormone.

Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that Ad-LacZ, a prototype first generation adenoviral vector containing an expression cassette for the reporter gene LacZ, directs transgene expression to mature and immature olfactory neurons and to sustentacular cells. The technique to apply the vector to the nasal cavity and the amount of viral vector per mouse are important variables that determine the success of viral vector-mediated gene transfer to the mouse olfactory neuroepithelium. Slow infusion of the viral vector solution in fully anaesthetized mice yields the best result in terms of the number of epithelial cells transduced. Infection of the olfactory neuroepithelium with a moderate amount of viral vector (10(9) plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 8-12 days, followed by a decline in transduced cells at 25 days postinstillation of the virus This decrement in transgene expression is consistent with the natural turnover process that occurs in the epithelium throughout adulthood. At high viral loads (1.3 x 10(10) PFU) extinction of transgene expression occurs as early as 8 days postinjection and is accompanied by epithelial degeneration indicating that the vector dose used should be carefully chosen. Taken together, the current observations demonstrate that adenoviral vectors are effective tools to genetically modify the adult mouse olfactory neuroepithelium in vivo.

The myelin sheath in higher vertebrates consists predominantly of proteolipid protein (PLP) and its smaller isoform DM20. Mutations in the PLP gene produces several neurological disorders such as Pelizaeus-Merzbacher disease and the rumpshaker phenotype in mice. This paper describes the cloning and expression of DM20 from the brain of Rainbow trout. We have isolated a nearly full-length cDNA clone containing 1835 bp that codes for a protein of 258 amino acids. Trout DM20 shows extensive homology with DM20 from higher vertebrates and includes the four hydrophobic regions that are believed to span the myelin membrane. The DM20 transcript is expressed throughout the central nervous system of the trout but appears at its highest levels in the spinal cord and medulla oblongata. The transcript is expressed at very low levels on hatching day but increases 179-fold by the 5th week. Contrary to higher vertebrates, there is no switch to the PLP transcript in maturing trout. Moreover, the rsh mutation (186 Thr to Ile) that produces the rumpshaking neurological disorder in mice has no effect in trout.

Considerable evidence points to an involvement of natural polyamines (putrescine, spermidine and spermine) in trophic regulation of brain tissue. Spermidine/spermine N1-acetyltransferase is the key enzyme in the interconversion pathway which leads to the formation of spermidine and putrescine from spermine and spermidine, respectively. In the present paper we have studied using in situ hybridization histochemistry the levels of spermidine/spermine N1-acetyltransferase mRNA in the rat central nervous system after transient forebrain ischemia. In the first hours after the insult, a modest increase in spermidine/spermine N1-acetyltransferase mRNA levels was observed in ependymal cells and other non-neuronal cells of all telencephalic and diencephalic regions. In addition, major increases in spermidine/spermine N1-acetyltransferase mRNA levels were observed in regions selectively vulnerable to the ischemic insult, such as striatum, hippocampus and cerebral cortex, during the first day post-reperfusion. The time course and extent of labelling increase were subregion- and cell-specific. At the cellular level, the labelling appeared markedly increased in neurons (8-10 fold in ventromedial striatum and CA1 region) and, to a lesser extent, in non-neuronal cells. The increase in SSAT mRNA levels was not directly related to cell degeneration, as it was detected in both some vulnerable and some resistant cell populations. However, the peak increase of SSAT labelling was precocious in resistant neurons (such as those of ventromedial striatum and dentate gyrus granular layer) and delayed or very limited in vulnerable neurons (such as those of CA1 pyramidal layer and dorsolateral striatum). The increase in spermidine/spermine N1-acetyltransferase may contribute to the increase in putrescine and decrease in spermidine levels observed after ischemia and gives further support to the notion that polyamine metabolism in the early phase after lesion is oriented towards putrescine production. This phenomenon could be relevant in determining the prevalence of neurotrophic vs. neurotoxic effects of polyamines.

We have previously reported that limbic seizures regulate the gene expression of fibroblast growth factor-2 (basic, FGF-2) according to a specific spatio-temporal pattern. In the present paper we have investigated the role of adrenal hormones on seizure-induced elevation of fibroblast growth factor-1 (acidic, FGF-1) and FGF-2 gene expression. Adrenalectomy reduces FGF-2 mRNA expression in specific brain regions, such as frontal cortex, hippocampus and striatum, whereas FGF-1 mRNA levels were decreased only in the frontal cortex. The injection of kainic acid in adrenalectomized rats produced a widespread increase of FGF-2 mRNA with a pattern similar to sham animals as indicated by in situ hybridization. In contrast, although kainate-induced elevation of FGF-1 mRNA in the hippocampus was not influenced by adrenalectomy, its induction in frontal cortex was prevented by this surgery procedure. Taken together, these data indicate that adrenal hormones play a role in the regulation of the gene expression for fibroblast growth factors, but different mechanisms are operative in their induction following seizure activity.

Angiotensin II (Ang) injected intracerebroventricularly stimulates neurohypophyseal vasopressin (AVP) release into the peripheral circulation. As we have shown previously, central actions of Ang II in the rat forebrain are mediated by the AT1A receptor subtype. In the present paper, we attempted to clarify the cellular localization of the AT1A receptor mRNA in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, in order to reappraise the conflicting data on the nature of the angiotensin II receptor involved in Ang induced vasopressin release. For this purpose, double in situ hybridization was performed using a radioactive AT1A receptor riboprobe and a digoxygenin labeled AVP oligoprobe, and immunohistochemical localization of the glial marker glial fibrillary acidic protein (GFAP) on the same brain slice. The results show neuronal expression of AT1A receptor mRNA mainly in dorsal and medial parvocellular parts of the PVN, its localization in some magnocellular PVN neurons and the absence of its expression in AVP producing neurons either in the PVN or in the SON. Thus, while indirect evidence indicates the involvement of the AT1A receptor subtype in the regulation of CRH and oxytocin release, the stimulation of vasopressinergic neurons is likely due to indirect mechanisms, or to a yet unknown type of angiotensin receptor.

The fibroblast growth factor (FGF) receptor-4 mRNA in the adult rat brain is expressed preferentially in the medial habenular nucleus. In this paper, we examined the expression of FGFR-4 mRNA in the brain during postnatal development. Interestingly, in addition to the persistent expression of FGFR-4 mRNA in the medial habenular nucleus, FGFR-4 mRNA was transiently expressed in the proliferative zone of the external granule layer of the developing cerebellum. The localization and transient expression of FGFR-4 mRNA in the developing cerebellum suggest that FGFR-4 mRNA was expressed by proliferative granule cells. The present findings indicate that FGFR-4 in the brain has an important role in the postnatal development of the cerebellar cortex.

A role of thyroid hormone in the regulation of neuropeptide synthesis has been demonstrated in different tissues. In this paper we investigated the vasoactive intestinal peptide (VIP) mRNA expression by means of in situ hybridization in several brain areas of hypo- and hyperthyroid adult rats. Neither hypo- nor hyperthyroidism modified the VIP mRNA levels in the thalamus and in the hypothalamic suprachiasmatic nucleus. In contrast, in the anterior cingulate and frontoparietal motor cortex of hypothyroid rats there was a marked increase in the signal for VIP mRNA per cell, but the number of VIP expressing neurons did not change. These data indicate that also central VIP synthesis can be influenced by the levels of circulating thyroid hormone, but that this effect is confined to specific areas and cell populations of the brain.

Different NMDA receptor subunits have been recently cloned. The present paper describes the developmental profile of expression of the NR-1 subunit and three NR-2 subunits (A, B, C) in the rat central nervous system. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of these receptor subunits. We found low levels of NR-1 mRNA (comprising all different splicing isoforms) in newborn rats with a progressive increase of its expression in the following 2-3 weeks. NR-2 subunits can be regarded as 'modulatory' since their expression can produce differences in the properties of NMDA receptors. More than one NR-2 subunits can be expressed in the same brain region. NR-2A and NR-2C are concomitantly expressed in the cerebellum and during development their mRNAs increase with a similar profile from low levels in P-8 rats to maximal expression in P-21 animals. NR-2A and NR-2B are concomitantly expressed in several brain regions with a different ontogenetic profile. In the hippocampus NR-2B mRNA increases rapidly during the first week of life as compared to the NR-2A subunits which at this time is expressed to low levels indicating that NR-2B will probably be dominant in determining the NMDA properties during the first period of life. Our data can provide a molecular correlate with properties of NMDA receptors such as voltage dependent Mg2+ block and deactivation kinetics which undergo significant changes during development and have been shown to depend upon the NR-2 subunit co-expressed with the common NR-1 subunit in various brain regions.

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.

In this paper, we tested whether physiological activators of the cAMP second messenger pathway in primary cultures of neurons from rat cerebral cortex directly induce c-fos and other immediate early gene (IEG) transcription factors. We have found that brief (30 s to 2 min) stimulation of neurons with vasoactive intestinal peptide (VIP) and SKF-38393, a D1-dopaminergic receptor agonist, potently increased mRNA levels for the IEGs c-fos, jun-B, and NGFI-A, with weaker increases for c-jun. This action was mimicked by forskolin and dibutyryl cAMP. IEG induction by VIP and dibutyryl cAMP was not blocked by excitatory amino acid receptor antagonists or by blockers of dihydropyridine-sensitive calcium channels. Moreover, calcium-free medium did not modify IEG induction by dibutyryl cAMP, suggesting that cAMP can directly regulate IEG expression in differentiated neurons independently of calcium.

Sulphated glycoprotein-2, thought to be involved in programmed cell death in peripheral organs, has been detected at increased levels in brain degenerative states. In this paper we have investigated the regional and cellular expression of this protein during development of brain lesion. With this aim sulphated glycoprotein-2 mRNA levels were studied in models of ischemic (transient forebrain ischemia) or mechanical (partial mesodiencephalic hemitransection) brain injuries using in situ hybridization histochemistry. Marked increases in sulphated glycoprotein-2 mRNA were observed in lesioned brains in both models. In addition, we report a shift in the regional distribution of positive cells in both lesion models 1-7 days post-lesion. After transient forebrain ischemia, sulphated glycoprotein-2 mRNA increases were always localized in selectively vulnerable regions (caudate-putamen, hippocampal formation), showing a temporal change in the pattern of intraregional distribution from less to more lesioned parts. In the case of mechanical lesion at 1 day, increased labelling had a widespread distribution on the lesioned side and was also observed on the intact side near the midline. In contrast, at 7 days increased labelling was restricted to regions directly lesioned (either areas whose input and/or output connections were severed by the transection or areas which were directly affected by the mechanical lesion). Analysis at the cellular level revealed that at both time intervals and in both lesion models most cell bodies overlain by dense clusters of specific grains were non-neuronal cells. The distribution patterns and their change over time suggest that at least some of these cells are inflammatory and phagocytic cells. The majority of degenerating neuronal cells after ischemia did not show increased levels of sulphated glycoprotein-2 mRNA. However, seven days after hemitransection and at all time intervals after transient ischemia, some cells clearly identifiable as neurons exhibited increased sulphated glycoprotein-2 mRNA levels.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) catalyzes the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeric gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631-1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5'-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.

Our earlier work demonstrated that certain populations of brain neurons which do not synthesize catecholamine (CA) neurotransmitters in vivo, will, when grown in culture with muscle-derived differentiation factor (MDF), unexpectedly express the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH). In this paper, we sought to determine whether MDF could also regulate TH expression in those neurons which normally synthesize CA neurotransmitters. Incubation of cultured dopamine neurons from the ventral midbrain with MDF elevated the levels of TH mRNA and TH enzyme activity 5- to 40-fold higher than that measured in control cultures. Sympathetic neurons were unaffected by a similar MDF treatment. Unlike the 2-day critical period for MDF-responsivity in non-CA neurons. CA neurons remained susceptible to MDF's influence over an extended developmental interval (E14-18), suggesting that MDF may be important for TH gene regulation in brain CA neurons even differentiation is complete. Because of these unique properties, MDF may provide a unique opportunity to explore ways in which the TH gene might be directly manipulated in these cell populations in order to correct the CA imbalances that occur in certain neurological diseases and disorders.

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes.

Expression of the mammalian prepro-gastrin-releasing peptide (preproGRP) gene has been shown to be restricted to neural and neuroendocrine cell types. In this paper, the structure and nucleotide sequence of the rat preproGRP gene coding regions and promoter is described and analyzed. The gene is divided into 3 exons, encoding a signal sequence, the 29 amino acid rat GRP, and a 92 amino acid extension peptide. While the overall prohormone structure is similar to that predicted from the sequence of the human gene, differences in transcription are apparent. Several forms of the rat preproGRP mRNA are found in brain: a 1.1 kb form which initiates in both brain and gut primarily from a TATAA-directed promoter, and less abundant forms of about 1.5 kb, whose initiation sites are heterogeneous, located 300-400 base pairs upstream of the 1.1 kb initiation site, and found only in spinal cord and a subset of brain nuclei expressing preproGRP mRNA. Comparison of the human and rat promoter region sequences identifies regions of high similarity upstream from both the 1.1 kb and 1.5 kb mRNA initiation sites, which may be important in the cell type-specific regulation of the preproGRP gene.

In this paper we validate a methodology, ligand autoradiographic receptor screening (LARS), for detecting expression of full length receptor cDNAs in COS cells. The method involves transfection of COS cells with receptor cDNAs by spheroplast fusion, production of filter replicas of the cell fragments, ligand binding to the receptors expressed in the membranes, and autoradiographic detection of bound ligand. A beta-adrenergic receptor cDNA cloned into a eukaryotic expression vector reliably induces high levels of beta-adrenergic receptor expression in 2-12% of COS cell colonies transfected with this plasmid after experimental conditions are optimized. Receptor expression is monitored by autoradiographic detection of 125iodocyanopindolol binding to COS cell fragments immobilized on polyester filter replicas. Binding displays appropriate pharmacological properties. The number of high-density binding spots per filter depends on the fraction of the spheroplasts in the fusion mixture that contain the beta-adrenergic receptor cDNA. A 100-plate LARS experiment can assess receptor expression in more than 10(4) transfected colonies. Thus detection of receptor-encoding sequences present in libraries in proportions as low as 1 in 10(4) should be possible. This technique may therefore be useful in detecting expression of other receptor cDNAs for which selective radioligands are available.

We have begun studies on regulatory mechanisms of muscarinic acetylcholine receptors (mAChRs) in slices of rat cerebral cortex. This paper, the first of two, deals with the viability of the cells in the slices (a prerequisite for studying receptor regulation) and provides a characterization of binding sites for [3H]N-methyl scopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in this preparation. Trypan blue exclusion tests in 400-microns-thick cortical slices showed a number of dead cells in a 100 microns zone from each cut edge, for a total of about 15-30% of all cells in the slice. In agreement with previous reports, electron microscopy revealed healthy tissue in the middle of the slice, but after incubation for several hours, swollen cells and dendrites were seen without cytoplasmic organelles. Axon terminals, however, were still seen to synapse upon these processes. Electrophysiological single unit recordings showed spontaneous action potentials in the slices. For receptor binding experiments, slices were incubated with either [3H]NMS, a hydrophilic mAChR ligand which does not penetrate the cell membrane, or the lipophilic ligand [3H]QNB which readily enters cells. For both ligands, equilibrium binding was reached after 8 h at 4 degrees C, and after 3 h at 30 degrees C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a Bmax of 2187 fmol/mg protein for [3H]QNB (reflecting all mAChRs) and 1335 fmol/mg protein for [3H]NMS (only mAChRs on the cell surface) at 30 degrees C. Kd values were 8.2 and 5.2 nM for [3H]QNB and [3H]NMS, respectively. These values are high compared with values obtained from homogenates, frozen sections or dissociated cells, and presumably reflect the use of intact, living tissue. These data are probably a better reflection of the actual, in vivo mAChR number and affinity than those obtained from dead tissue. This slice preparation suggests itself as a simple but effective method with which to study the regulation of mAChRs in living brain tissue.

In order to investigate the mechanism underlying ovarian steroid action on gene expression of hypothalamic luteinizing hormone releasing hormone (LHRH), changes in LHRH mRNA level were determined by RNA-blot hybridization assay. Twenty-eight-day-old female rats were ovariectomized (OVX) and implanted with Silastic capsule containing either 17 beta-estradiol (E) or vehicle (V). Two days later (day 30), OVX + E-primed rats were given s.c. progesterone (P, 1 mg) 6 h prior to decapitation. Four experimental groups were studied: (1) intact, (2) OVX + V, (3) OVX + E, and (4) OVX + E + P-treated rats. Poly(A) RNA fractions from hypothalami (40-50/group) were isolated, blotted onto nitrocellulose paper and hybridized with 32P-end-labeled LHRH oligonucleotides (29 mer) which are complementary to rat LHRH mRNA. The hypothalamic LHRH mRNA signal markedly attenuated 2 days following ovariectomy. E replacement to OVX rats slightly increased LHRH mRNA level, which is lower than that of the intact group. However, a single injection of P to OVX + E-treated rats notably augmented the LHRH mRNA level over that observed in the intact group. In addition, LHRH content and release in vitro were examined to correlate with changes in LHRH gene expression. Ovariectomy and the replacement of E and/or P resulted in a similar fashion of changes in LHRH release and content as compared to alteration of LHRH mRNA level. This study clearly demonstrates that P increases LHRH mRNA level in the hypothalamus of OVX + E-primed immature rats.(ABSTRACT TRUNCATED AT 250 WORDS)