Laminin γ1-dependent basement membranes are instrumental to ensure proper olfactory placode shape, position and boundary with the brain, as well as olfactory axon development.

Despite recent progress, the complex roles played by the extracellular matrix in development and disease are still far from being fully understood. Here, we took advantage of the zebrafish sly mutation which affects Laminin γ1, a major component of basement membranes, to explore its role in the development of the olfactory system. Following a detailed characterisation of Laminin distribution in the developing olfactory circuit, we analysed basement membrane integrity, olfactory placode and brain morphogenesis, and olfactory axon development in sly mutants, using a combination of immunochemistry, electron microscopy and quantitative live imaging of cell movements and axon behaviours. Our results point to an original and dual contribution of Laminin γ1-dependent basement membranes in organising the border between the olfactory placode and the adjacent brain: they maintain placode shape and position in the face of major brain morphogenetic movements, they establish a robust physical barrier between the two tissues while at the same time allowing the local entry of the sensory axons into the brain and their navigation towards the olfactory bulb. This work thus identifies key roles of Laminin γ1-dependent basement membranes in neuronal tissue morphogenesis and axon development in vivo .

The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction.

The function of the beta-amyloid protein precursor (betaAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood. We recently reported the presence of a fraction of betaAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction. To investigate whether betaAPP actually interferes with cell signaling, we reexamined the interaction between betaAPP and Go GTPase. In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22C11, an antibody that recognizes an N-terminal betaAPP epitope, reduces high-affinity Go GTPase activity. This inhibition is specific of Galphao and is reproduced, in the absence of 22C11, by the addition of the betaAPP C-terminal domain but not by two distinct mutated betaAPP C-terminal domains that do not bind Galphao. This inhibition of Galphao GTPase activity by either 22C11 or wild-type betaAPP cytoplasmic domain suggests that intracellular interactions between betaAPP and Galphao could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Galphao and betaAPP in CSEM. We show that inhibition of basal Galphao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Galphao and betaAPP. The regulation of Galphao GTPase activity by betaAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of betaAPP.

Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein.

BACKGROUND: Engrailed-1 and Engrailed-2 are homeoproteins--transcription factors implicated in the morphogenesis of discrete structures. Engrailed proteins have a role in patterning the midbrain-hindbrain region and are expressed in the nuclei of rat embryo midbrain-hindbrain cells. We have previously found that both endogenous and exogenously expressed Engrailed proteins also associate with membrane regions implicated in signal transduction and secretion. Within total membrane fractions, a small proportion of Engrailed--about 5%--is protected against proteinase K proteolysis, suggesting that Engrailed has access to a luminal compartment. Together with our finding that homeodomains and homeoproteins can be internalized by live cells, these observations suggest that Engrailed might act as a polypeptidic messenger. In order to investigate this possibility, we looked to see if Engrailed could be secreted. RESULTS: Engrailed expressed in COS cells can be recovered in abutting primary neurons and this is dependent on a short sequence in its homeodomain distinct from 'classical' secretion signals. This sequence, which overlaps with the sequence necessary for Engrailed internalization and which is highly conserved among homeoproteins, is the first example of an 'unconventional' sequence necessary for secretion. Less than 50% of total intracellular Engrailed is secreted and there is a correlation between secretion and access to the membrane compartment where the protein is protected against proteinase K. CONCLUSIONS: Our results lend weight to the proposal that Engrailed, and possibly other homeoproteins, might act as intercellular polypeptidic messengers.

Subcellular localization of tyrosine hydroxylase (TH) gene transcripts: new insights into the pattern of TH gene expression in the locus coeruleus under pharmacological stimulation.

To document the subcellular compartmentalization of the tyrosine hydroxylase (TH) primary (heterogeneous nuclear, hnRNA) and processed mRNA transcripts in vivo, we have studied the expression of the TH gene in the locus coeruleus of the rat brain in basal conditions and under pharmacological stimulation by reserpine. Using exon-specific probes and electron microscopic in situ hybridization in reserpine-treated animals, we have demonstrated that the TH mRNA is localized both in the perikaryal cytoplasm (in association with domains of the endoplasmic reticulum) and in the nucleus (in large and smaller aggregates). By comparing hybridization patterns with intron- and exon-specific probes, we have shown that the two main foci of nuclear TH RNA labeling correspond to the primary (hnRNA) transcript, while accessory secondary tracks or dots represent mature transcript devoid of introns. Finally, our data indicate that the pattern of expression of the TH gene is heterogeneous within the locus coeruleus neuron population. Under basal conditions, many neurons exhibit no detectable intron signal, although all locus coeruleus neurones express the TH gene. Other neurons display high intron labeling, and obviously express the gene at a much higher level. Upon reserpine stimulation, the number of neurons displaying detectable intron signals, as well as the intensity of the intron signal per neuron progressively increases during the first 24 h, suggesting that TH expressing neurons are progressively recruited for higher expression of this gene as the stimulation progresses.

Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice.

The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the beta-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHbeta gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.

Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization.

In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed. In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides. The sense and antisense probes are then yielded by the action of the lambda bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated. We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target.

Clusterin/ApoJ expression is associated with neuronal apoptosis in the olfactory mucosa of the adult mouse.

The molecular events orchestrating neuronal degeneration and regeneration remain poorly understood. Attempts at identifying genes specifically expressed during these processes, have constantly led to the (re)isolation of the clusterin/ApoJ gene, whose expression is highly reactive to injury in a wide variety of tissues. To get insight into the function of clusterin in neuron loss, we have assessed in detail the clusterin gene expression in an experimental model of neurodegeneration, using the peripheral olfactory system of adult mouse. The sensory neurons of olfactory nasal mucosa can be massively induced to degenerate in vivo, by surgical removal of their only synaptic target: the olfactory bulb. We have previously shown that this neuron loss results from a near-synchronized induction of apoptosis genetic programs. We present here evidence that clusterin gene expression is tightly correlated to the onset of neuronal apoptoses in lesioned olfactory mucosae. The simultaneous preparation of DNA and RNA from the same tissue samples reveals that a strong clusterin mRNA accumulation coincides with the wave of nucleosome-sized DNA fragmentation. However, double detection of apoptotic nuclei by the TUNEL method and of clusterin messengers by in situ hybridization revealed that the clusterin gene expression is not induced in dying neurons, but in the glial sheath surrounding the axon bundles of degenerating olfactory neurons. Clusterin immunocytochemistry reveals that the clusterin protein accumulates not only in these producing cells, but also in the olfactory epithelium, suggesting the possibility of clusterin internalization by cells located at a distance from the synthesis loci. In view of this localization and of the activities of the clusterin protein reported so far, possible functions of clusterin in nervous plasticity are discussed.

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties.

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

Pharmacological model of catecholamine depletion in the hypothalamus of fetal and neonatal rats and its application.

1. The present study aimed to develop a pharmacological model of catecholamine (CA) depletion in the hypothalamus during the period of its morphofunctional development, i.e. in fetal and neonatal rats of both sexes. 2. In the first series of experiments, pregnant females and, hence, fetuses were systemically treated daily from the embryonic day (E) 13 to E20 with the inhibitor of the CA synthesis alpha-methyl-m-tyrosine. The CA concentrations were subsequently measured in the fetal hypothalamus at E21 by high performance liquid chromatography with electrochemical detection (HPLC-ED). In the second series of experiments, neonatal rats were injected with neurotoxin, 6-hydroxydopamine and/or alpha-methyl-m-tyrosine daily from the 2nd postnatal day (P2) to P10. 3. The HPLC-ED assay of hypothalamic catecholamines (CA's) at E21 and P11 showed that both in fetuses and neonates, alpha-methyl-m-tyrosine caused more than 50% depletion of hypothalamic noradrenaline and adrenaline, while the dopamine (DA) level remained unchanged. The combined treatment of neonatal rats with alpha-methyl-m-tyrosine and 6-hydroxydopamine resulted additionally in a 25% decreased level of DA. 4. The influence of CA deficiency on the developing hypothalamic CA system was further evaluated by measuring [3H]DA uptake by nervous tissue in vitro. 5. The CA deficiency caused a 50% drop of [3H]DA uptake by the hypothalamic tissue in treated fetuses suggesting a stimulating effect of CA's on the early development of the CA system. In pharmacologically treated neonatal rats [3H]DA uptake remained at the control level showing no influence of the CA deficiency on the developing CA system after birth. 6. The usefulness of the proposed pharmacological model for studying of CA influence on differentiating hypothalamic target neurons is discussed.

Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent.

We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A.(1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10 degrees C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37 degrees C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the alpha-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A.(1995) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L.(1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors.

Differential compartmentalization of vasopressin messenger RNA and neuropeptide within the rat hypothalamo-neurohypophysial axonal tracts: light and electron microscopic evidence.

Arginine vasopressin messenger RNA is axonally transported in the rat hypothalamo-neurohypophysial system [for review see Mohr et al. (1993) In Vasopressin (eds Gross P., Richter D. and Robertson C. L.), pp. 119-129, John Libbey Eurotext]. Upon chronic dehydration (2% saline-feeding for seven days), vasopressin messenger RNA within this axonal compartment is dramatically increased and appears aggregated in a selected subset of axonal swellings confined to the median eminence and posterior pituitary. In this study, we analysed the axonal distribution of the vasopressin messenger RNA within the hypothalamo-neurohypophysial tracts of control and saline-fed animals, and compared this distribution to that of the vasopressin peptide. Our data further support a selective aggregation of the vasopressin messenger RNA in a subset of distal axonal swellings and/or terminals of the median eminence and posterior pituitary. The selective aggregation is observed not only in saline-fed animals, but also in control animals. Although the osmotic stimulus dramatically enhances the axonal transport of vasopressin messenger RNA, the consequent general distribution pattern of the messenger RNA in the hypothalamo-neurohypophysial system is not changed. However, the physiological perturbation does increase the number of vasopressin messenger RNA-containing swellings within the median eminence and the posterior pituitary. In both saline-fed and control animals, the level of messenger RNA label within individual swellings appeared roughly similar to that found in the perikaryal cytoplasm of extra-hypothalamic vasopressinergic neurons. A detailed comparison of the axonal compartmentalization of vasopressin messenger RNA and vasopressin peptide demonstrates that the axonal distribution of vasopressin messenger RNA does not precisely overlap that of vasopressin peptide along the hypothalamo-neurohypophysial tract. In seven-day saline-fed animals, the majority of the messenger RNA-containing swellings of the median eminence also contain detectable vasopressin peptide; however in the same animals, nearly all the messenger RNA-containing swellings of the posterior pituitary appear devoid of vasopressin peptide. Therefore, our work strongly suggests that at least in the posterior pituitary, the vasopressin messenger RNA might be selectively targeted and aggregated in a selected subset of axonal swellings containing little if any vasopressin, and hence very few neurosecretory granules. Given this evidence that vasopressin messenger RNA and neuropeptide are differentially compartmentalized in axons of magnocellular neurons, we propose that vasopressin messenger RNA and peptide probably rely on different intracellular transport systems with respect to packaging, transport and/or aggregation within these selected axonal locations.

Spatial segregation of G alpha s mRNA and vasopressin mRNA to distinct domains of the rough endoplasmic reticulum within secretory neurons of the rat hypothalamus.

The comparative subcellular localization of the mRNAs encoding the stimulatory subunit of heterotrimeric G-proteins (G alpha s) and the vasopressin-secreted peptide was performed at the light and electron microscopic level. We find that although the G alpha s membrane protein is devoid of signal peptide sequence at its N-terminal extremity, its mRNA is aggregated on defined domains of the rough endoplasmic reticulum (RER). This suggests that the G alpha s protein is probably synthesized close to the RER, and that, on the pathway to the plasma membrane, this protein might be primarily associated with RER membranes. We further find that the mRNAs encoding the G alpha s membrane-attached protein and the secreted peptide vasopressin have different patterns of distribution within the neuronal perikarya. Overall, our results show that these two mRNAs are segregated to distinct domains of the RER. We speculate that the RER might be organized in specialized domains involved in distinct functions with respect to mRNA translation and/or protein postranslational modifications.

BC1 RNA and vasopressin mRNA in rat neurohypophysis: axonal compartmentalization and differential regulation during dehydration and rehydration.

Brain cytoplasmic 1 (BC1) RNA is a small non-translated RNA polymerase III transcript. Because this RNA can be detected in the rat posterior pituitary with 35S in situ hybridization autoradiography, it has been hypothesized that this RNA might be transported in the axons of hypothalamo-neurohypophyseal neurons. In the present study, we aimed to determine the cellular localization of BC1 more precisely by using non-radioactive in situ hybridization of BC1 RNA at both the light and electron microscopic levels. Our studies revealed that BC1 RNA was indeed located intra-axonally. Furthermore, BC1 RNA was abundant within a subset of axonal swellings and/or terminals, and was also found in discrete cytoplasmic domains of undilated axonal segments. Using a semiquantitative in situ hybridization approach, we have measured and compared the changes in BC1 RNA and arginine vasopressin (AVP) mRNA during dehydration (chronic salt-loading) and rehydration. Chronic salt-loading significantly increased both BC1 RNA and AVP mRNA. The increase in BC1 RNA labelling (2.5-fold), however, was modest and somewhat less enduring than the increase in AVP mRNA labelling (13-fold). Upon rehydration, both the BC1 and vasopressin transcripts in the posterior pituitary rapidly returned to control values. In conclusion, like vasopressin mRNA, BC1 RNA is transported in axons of the hypothalamo-neurohypophyseal system where it aggregates in a subset of axonal swellings, and its axonal transport is similarly regulated. Therefore, we propose that BC1 RNA might be involved in the axonal targeting, docking and/or transport of AVP or other axonal mRNAs.

Isolation and characterization of a chicken homolog of the E2F-1 transcription factor.

In higher eukaryotes, the E2F-1 transcription factor is an essential and limiting component of cell cycle progression in late G1. E2F-1 heterodimerizes with members of the DP gene family and the resulting heterodimer regulates the expression of several proto-oncogenes and the genetic machinery of DNA replication. Cell cycle regulation of E2F activity is mediated through its association with the tumor suppressor Rb gene product. To examine the evolutionary conservation of the E2F-1 protein sequence and its developmental expression pattern we have isolated and sequenced the chick E2F-1 gene (chE2F-1) cDNA. The chicken protein is 34 amino acids (a.a) shorter than its human counterpart (403/437 a.a.) but has extremely well conserved bHLH and pRb binding domains, with respectively 94% and 83% identity. The position of the leucine zipper is also strictly conserved thereby accounting for ability of E2F-1 to form heterodimers with human and chicken DP-1. E2F-1 expression was analysed in synchronized cells as well as in embryonic or newborn chick tissues and appears to be closely correlated to the cell proliferation rate. In situ hybridization studies have shown very high expression levels in the neuroretina during the early stages of embryonic development when active neuroblast division occurs. In contrast, a sharp down-regulation is observed when cells become postmitotic. Overexpression of the chE2F-1 protein leads to oncogenic transformation only when a truncated version of the transgene lacking the pRb binding domain is used; the full length protein either has no effect or may be deleterious for cell survival.

Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes.

Although oligonucleotide probes are useful for in situ hybridization, their low sensitivity compared to riboprobes and cDNA remains a problem. We have systematically examined the protocols to provide a general procedure that increases the sensitivity of oligoprobes for light and electron in situ hybridizations by using mixtures of multiple non-overlapping oligonucleotides (multi-oligoprobes). The protocol achieves these improvements with both radioactive and non-radioactive oligoprobes. With 33P-labeled probes in a semiquantitative assay, we found that mixtures of up to six vasopressin-directed multi-oligoprobes, each employed at saturating concentration, led to an additive signal with no significant increase of the background. Using this approach with non-radioactive oligoprobes, we were able to detect in the hypothalamus several low or moderately abundant mRNAs, such as vasopressin heterogeneous nuclear RNA and the galanin, dynorphin, and tyrosine hydroxylase mRNAs. Moreover, we showed that multi-oligoprobes used in a pre-embedding procedure were suitable for studying the ultrastructural compartmentalization of moderately abundant mRNAs. Finally, with the same basic approach we demonstrated that two sets of multi-oligoprobes can be combined for simultaneous detection of two different mRNAs using fluorescent dyes, making this approach suitable for high-resolution confocal analyses. Overall, our data demonstrate that multi-oligoprobes provide a sensitive tool of choice for various applications in which both well-preserved morphology and high sensitivity are needed. In particular, these probes appear ideal for study of the comparative subcellular localization of mRNAs at both the light and the electron microscopic level.

A Bcl-2-related gene is activated in avian cells transformed by the Rous sarcoma virus.

The oncoprotein p60v-src encoded by the Rous sarcoma virus (RSV) genome is the prototype of non-receptor tyrosine kinases. More than 50 targets of p60v-src have been described to date. However, the precise mechanisms of RSV transformation remain to be elucidated. Here, we present the study of a new v-src-activated gene, NR-13, which encodes a protein identified as a new member of the Bcl-2 family. This protein is localized in the membrane with a pattern already observed with Bcl-2. In quail embryos, this gene is mainly expressed in neural and muscular tissues. Its expression is dramatically down-regulated after embryonic day 7 (E7) in the optic tectum. To evaluate a possible role for NR-13 in the control of apoptotic processes in this particular brain area, in situ hybridization and DNA ladder fractionation studies were performed to correlate NR-13 expression with typical situations of apoptosis during brain development. Our results support the idea that RSV could activate anti-apoptotic functions of the host cell resulting in an increase of their lifespan, which could be particularly relevant to tumour formation.

Vasopressin and oxytocin gene expression in intact rats and under catecholamine deficiency during ontogenesis.

The development of the hypothalamic vasopressin (VP) and oxytocin (OT) systems has been studied in rats from the 16th embryonic day (E16) until the 11th postnatal day (P11). The VP and OT mRNA-producing neurons were identified on cryostat sections by in situ hybridization using oligonucleotide probes labeled by [35S], [3H] or digoxigenin. Moreover, VP and OT gene expressions were evaluated either at E21 or at P11 following chronic depletion of catecholamines (CA). For this purpose, pregnant rats were daily injected with alpha-methyl-m(p)-tyrosine from gestational day 13 to 20 while neonates were daily injected with alpha-methyl-m(p)-tyrosine and neurotoxin 6-hydroxydopamine from postnatal day 2 to 10. No VP mRNA- or OT mRNA-expressing cells were observed in the hypothalamus of intact fetuses at E16, while 2 days later rather numerous VP and OT neurons occupied the anterior hypothalamus. One major bilateral group of VP and OT neurons was located in the supraoptic nucleus (SON). Less numerous labeled cells were found in the developing paraventricular nucleus (PVN). Some VP and OT neurons were also spread along the ventrolateral surface of the hypothalamus from the level of the median eminence, caudally, to the level of the optic nerves, rostrally. From E18 until birth, the OT neurons were localized in the dorsal portion of the SON, while its ventral portion was occupied by the VP neurons. The VP mRNA- and OT mRNA-expressing cells seemed to increase both in size and in number over the perinatal period. Frequent relatively long neuronal processes contained VP and OT mRNAs in fetuses and in newborns. When performed during the second half of the fetal life, the chronic depletion of CA did not cause any change in the VP and OT mRNA concentrations in the SON and PVN of fetuses. By contrast, similar treatment of neonates resulted in a significant increase of both mRNA levels in the SON. These data suggest that at least in the SON VP and OT gene expression might be under the inhibitory control of CA during the neonatal period.

Detection and regulation of tyrosine hydroxylase mRNA in catecholaminergic terminal fields: possible axonal compartmentalization.

Reverse transcriptase coupled with nested polymerase chain reaction amplification (RT/nested-PCR) was used to detect mRNA encoding tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in adult rat cerebellum, striatum, and pituitary neurointermediate lobe (NIL). These regions receive catecholaminergic innervation from the locus coeruleus, substantia nigra, and arcuate and periventricular nuclei of the hypothalamus, respectively, but are devoid of catecholamine-synthesizing cells. The RT/nested-PCR products, which were generated using primers located on different exons of the tyrosine hydroxylase gene, indicate that the tyrosine hydroxylase mRNA detected is devoid of introns and, hence, is processed. These findings raise the possibility that tyrosine hydroxylase mRNA may be axonally transported. Using the same RT/nested-PCR protocol, we were unable to detect mRNA encoding dopamine beta-hydroxylase, a different catecholaminergic biosynthetic enzyme, in either cerebellum, striatum, or NIL pituitary tissue. Thus, the detection of tyrosine hydroxylase mRNA in catecholamine terminal regions is biochemically specific. We were unable to detect tyrosine hydroxylase mRNA in optic nerve, indicating some degree of anatomical specificity as well. Expression of tyrosine hydroxylase mRNA in the cerebellum was markedly increased by subcutaneous administration of the catecholamine-depleting agent, reserpine, suggesting that tyrosine hydroxylase mRNA in catecholamine terminal regions may be functionally important. This finding also indirectly supports the hypothesis that tyrosine hydroxylase mRNA can be axonally transported since the ability of reserpine to induce expression of this transcript in conventional catecholamine cell groups is considered secondary to catecholamine depletion, and cerebellar cells do not synthesize catecholamines. Finally, lesions of the nigrostriatal pathway significantly decreased levels of tyrosine hydroxylase mRNA in the striatum, providing strong additional support for this hypothesis.

Altered vasoactive intestinal polypeptide gene expression in the fetal rat suprachiasmatic nucleus following prenatal serotonin deficiency.

This study has evaluated the possible role of serotonin, a potential morphogen, in the regulation of vasoactive intestinal polypeptide (VIP) gene expression in the target neurons of the suprachiasmatic nucleus (SCN) before and after the onset of the serotonin neurotransmitter function. VIP gene expression was quantified by in situ hybridization of the corresponding mRNA on cryostat sections with subsequent film autoradiography and densitometry. The content of VIP mRNA was measured in the SCN in fetuses at the 21st embryonic day (E21) and in postnatal rats at day 11 (P11) following chronic depletion of serotonin by p-chlorophenylalanine, an inhibitor of serotonin synthesis. This inhibitor was daily injected to pregnant rats for E13-20 or to postnatal animals for P2-10. Results of this study indicate that prenatal serotonin depletion caused a significant increase in VIP mRNA content in the SCN compared to control fetuses. On the contrary, the same treatment performed postnatally did not change VIP mRNA levels in the SCN. These data suggest that the VIP gene expression in differentiating target neurons of the SCN might be under serotonin inhibitory control during prenatal neurogenesis, prior to the onset of the serotoninergic neurotransmission.