The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Atmospheric-Pressure Plasma-Enhanced Spatial ALD of SiO2 Studied by Gas-Phase Infrared and Optical Emission Spectroscopy.

An atmospheric-pressure plasma-enhanced spatial atomic layer deposition (PE-s-ALD) process for SiO2 using bisdiethylaminosilane (BDEAS, SiH2[NEt2]2) and O2 plasma is reported along with an investigation of its underlying growth mechanism. Within the temperature range of 100-250 °C, the process demonstrates self-limiting growth with a growth per cycle (GPC) between 0.12 and 0.14 nm and SiO2 films exhibiting material properties on par with those reported for low-pressure PEALD. Gas-phase infrared spectroscopy on the reactant exhaust gases and optical emission spectroscopy (OES) on the plasma region are used to identify the species that are involved in the ALD process. Based on the identified species, we propose a reaction mechanism where BDEAS molecules adsorb on -OH surface sites through the exchange of one of the amine ligands upon desorption of diethylamine (DEA). The remaining amine ligand is removed through combustion reactions activated by the O2 plasma species leading to the release of H2O, CO2, and CO in addition to products such as N2O, NO2, and CH-containing species. These volatile species can undergo further gas-phase reactions in the plasma as indicated by the observation of OH*, CN*, and NH* excited fragments in OES. Furthermore, the infrared analysis of the precursor exhaust gas indicated the release of CO2 during precursor adsorption. Moreover, this analysis has allowed the quantification of the precursor depletion yielding values between 10 and 50% depending on the processing parameters. Besides providing insights into the chemistry of atmospheric-pressure PE-s-ALD of SiO2, our results demonstrate that infrared spectroscopy performed on exhaust gases is a valuable approach to quantify relevant process parameters, which can ultimately help evaluate and improve process performance.

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation.

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.

Targeting oncogene expression to endothelial cells induces proliferation of the myelo-erythroid lineage by repressing the Notch pathway.

Human oncogenes involved in the development of hematological malignancies have been widely used to model experimental leukemia. However, models of myeloid leukemia rarely reproduce the human disease in full, due to genetic complexity or to difficulties in targeting leukemia initiating cells. Here, we used a zebrafish genetic model to induce the expression of oncogenic RAS in endothelial cells, including the hemogenic endothelium of the dorsal aorta that generates hematopoietic cells, and observed the development of a myelo-erythroid proliferative disorder. In larvae, the phenotype is characterized by disruption of the vascular system and prominent expansion of the caudal hematopoietic tissue. In few surviving juveniles, increased number of immature hematopoietic cells and arrest of myeloid maturation was found in kidney marrow. Peripheral blood showed increased erythroblasts and myeloid progenitors. We found that the abnormal phenotype is associated with a downregulation of the Notch pathway, whereas overexpressing an activated form of Notch together with the oncogene prevents the expansion of the myelo-erythroid compartment. This study identifies the downregulation of the Notch pathway following an oncogenic event in the hemogenic endothelium as an important step in the pathogenesis of myelo-erythroid disorders and describes a number of potential effectors of this transformation.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Overlapping expression of zebrafish T-brain-1 and eomesodermin during forebrain development.

T-box transcription factors are important determinants of embryonic cell fate and behaviour. Two T-box genes are expressed in the developing telencephalon of several vertebrate species, including amphibia, birds and mammals. Here we report the cloning of zebrafish T-brain-1 (tbr1) and eomesodermin (eom). As a prelude to genetic studies of neuro-ectodermal fate determination we studied their expression pattern during embryogenesis and early larval development. Eom is expressed in the presumptive telencephalon from around the 4-5 somite stage in bilaterally symmetric groups of cells; the number of positive cells increases dramatically with time and encompasses the entire dorsal telencephalon by the 22 somite stage. Tbr1 is expressed from the 18 somite stage in a subset of eom-expressing cells. By 24 hpf eom and tbr1 are expressed in largely overlapping domains in the dorsal telencephalon, tbr1 is expressed in postmitotic cells whereas eomes is also expressed in proliferative ventricular zone cells. Both genes are also found in a small domain of the diencephalon bordering the telencephalon. A detailed analysis of the expression of tbr1 and eom in the brain of 4 day old larvae shows that the two T-box genes are differentially expressed in various cell populations of the developing brain.

DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.

The homeobox genes in the Dlx family are required for differentiation of basal forebrain neurons and craniofacial morphogenesis. Herein, we studied the expression of Dlx-1, Dlx-2, and Dlx-5 RNA and protein in the mouse forebrain from embryonic day 10.5 (E10.5) to E12.5. We provide evidence that Dlx-2 is expressed before Dlx-1, which is expressed before Dlx-5. We also demonstrate that these genes are expressed in the same cells, which may explain why single mutants of the Dlx genes have mild phenotypes. The DLX proteins are localized primarily to the nucleus, although DLX-5 also can be found in the cytoplasm. During development, the fraction of Dlx-positive cells increases in the ventricular zone. Analysis of the distribution of DLX-1 and DLX-2 in M-phase cells suggests that these proteins are distributed symmetrically to daughter cells during mitosis. We propose that DLX-negative cells in the ventricular zone are specified progressively to become DLX-2-expressing cells during neurogenesis; as these cells differentiate, they go on to express DLX-1, DLX-5, and DLX-6. This process appears to be largely the same in all regions of the forebrain that express the Dlx genes. In the basal telencephalon, these DLX-positive cells differentiate into projection neurons of the striatum and pallidum as well as interneurons, some of which migrate to the cerebral cortex and the olfactory bulb.

Differential origins of neocortical projection and local circuit neurons: role of Dlx genes in neocortical interneuronogenesis.

Herein we review the evidence that neocortical projection neurons and interneurons are derived from distinct regions within the telencephalon. While neocortical projection neurons are derived from the ventricular zone of the neocortex, neocortical interneurons appear to be derived from the germinal zone of the basal ganglia. These interneurons follow a tangential migratory pathway from the ganglionic eminences to the cortex. Interneurons of the olfactory bulb follow a distinct tangential migration from the basal ganglia. The Dlx homeobox genes, which are essential for basal ganglia differentiation, are also required for the development of neocortical and olfactory bulb interneurons. Furthermore, evidence is presented that retroviral-mediated expression of DLX2 in neocortical cells can induce GABAergic interneuron differentiation.

Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters.

Basic fibroblast growth factor (bFGF) has been shown to influence the survival, proliferation and differentiation of a variety of cell types in the nervous system. In this investigation we have examined the action of bFGF on: (i) the rate of proliferation; (ii) cell cycle parameters; (iii) the maintenance of cell division; (iv) the recruitment of quiescent cells; and (v) the degree of differentiation of cortical progenitor cells in cultures prepared from E16 rat embryos. The proliferation rate (labelling index) of cortical progenitor cells doubled in the presence of bFGF over 48 h. However, the lengths of the cell cycle phases were unchanged. Clones marked with a recombinant retrovirus on the first day in vitro (DIV) grew significantly larger in the presence of bFGF. Furthermore, many of the clones examined in control cultures had ceased to divide after a maximum of four cell cycles, whereas almost all clonally related cells were still dividing in the presence of bFGF 4 days later, i.e. for at least six cell cycles. Basic FGF also stimulated the division of quiescent progenitor cells, which otherwise would have differentiated or undergone cell death. The degree of neuronal and glial differentiation was studied after 5 DIV using MAP-2 and GFAP immunocytochemistry. In the presence of bFGF, the percentage of MAP-2-labelled cells was less than half that of control cultures, whereas the number of cells immunoreactive for nestin (a marker of progenitor cells) remained very high. Cells immunoreactive for GFAP were present in bFGF-treated cultures, yet were extremely rare in control conditions. These experiments show that bFGF, a potent mitogen for cortical progenitor cells, has no effects on the parameters of their cell cycle but extends their proliferative capability, promotes their survival and delays their differentiation into neurons.

Cell fate specification and symmetrical/asymmetrical divisions in the developing cerebral cortex.

Two different modes of cell division are adopted by progenitor cells to generate the neurons and glia of the cerebral cortex: they either divide symmetrically to generate other progenitors or a pair of postmitotic cells or divide asymmetrically to generate both a progenitor and a postmitotic cell. In this study we used a lineage marker, the BAG retrovirus, in embryonic day 16 rats in combination with bromodeoxyuridine (BrdU) to identify patterns of cell generation in the cerebral cortex, and investigated the relationship between the phenotype of cells and the history of their lineages. The location, phenotype and birth order of clonally related cells were studied in the subsequent 3 weeks. Only pyramidal neurons and/or astrocytes formed discrete clusters in which several generations of family members were present, whereas nonpyramidal neurons were found exclusively in pairs or as single cells. Analysis of BrdU levels in these cells showed that nonpyramidal neurons were originally part of larger clones and were found dispersed in the neocortex because of tangential migration of their progenitors, dispersion of postmitotic cells, or death of clonal relatives. These results suggest that both symmetrical and asymmetrical division can be adopted by progenitor cells to generate cortical neurons and glial cells and that cell extrinsic events contribute to the isolation of nonpyramidal neurons.

Apoptosis and its relation to the cell cycle in the developing cerebral cortex.

Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre- and early postnatal development of the rat cerebral cortex using in situ end labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [3H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.

Neuronal clones in the cerebral cortex show morphological and neurotransmitter heterogeneity during development.

The mammalian cerebral cortex, although a structure of great complexity, is characterized by a high degree of organization where the proportions, spatial relationships, and properties of the various cell types are rigidly controlled. The mechanisms responsible for the creation of such a rigid distribution of cell types are not known. Lineage studies in adult rats have suggested that each of the cortical progenitor cells lining the telencephalic ventricles during embryonic development gives rise to progeny of the same phenotype (homogeneous clones). However, the possibility that homogeneous clones are the result of complex processes affecting both the final number and the phenotype of clonally related cells during development has not been investigated. In the present study, we followed the development of cortical cell lineages labeled with retroviral injections at embryonic day (E) 16 in rats of 7, 14, or 21 d of age using electron microscopy and immunocytochemistry for the neurotransmitters glutamate and GABA. We found that a significant number of cortical clones at postnatal day (P) 7 and P14, and fewer at P21, showed mixed pyramidal/nonpyramidal cell composition. We sometimes observed that "mixed" neuronal clones contained cells immunoreactive for both glutamate and GABA. In the general population of cortical cells, "bireactive" neurons represented 3.7% of all neurons at P7, 18% at P14, and 0.6% in adult rats. Although the change in the composition of neuronal clones between the third week of postnatal life and adulthood may be due to changes in the phenotype of some developing neurons, we would like to suggest that it is probably due to selective neuronal cell death.

The cell lineage of neuronal subtypes in the mammalian cerebral cortex.

We have studied the lineage relationships of pyramidal and nonpyramidal neurons, the principal neuronal types in the cerebral cortex, using a recombinant retrovirus that carries the gene encoding Escherichia coli beta-galactosidase as a lineage marker. The phenotype of every cell of clones of beta-galactosidase-labelled neurons generated by intraventricular injection of recombinant retrovirus in rat embryos at different stages of cortical neurogenesis was identified using light and electron microscopy as well as immunohistochemistry for known markers of neuronal subtypes. We found that clonally related neurons in adult rats showed the same morphological and neurotransmitter phenotypes, suggesting that lineages of pyramidal and nonpyramidal neurons are specified as early as E14, the time of onset of neurogenesis. However, when we followed the development of cortical cell lineages, we noted that a significant number of neuronal clones showed a mixed pyramidal/nonpyramidal cell composition during the first three weeks of life. We suggest that the change in the composition of neuronal clones between the third week of postnatal life and adulthood may either be due to changes in the phenotype of some developing neurons or, more likely, to selective cell death.

Lineage analysis reveals neurotransmitter (GABA or glutamate) but not calcium-binding protein homogeneity in clonally related cortical neurons.

Studies of cell lineage in the rat cerebral cortex have provided new insights into the mechanisms of neuronal and glial determination. They have shown that clonally related cells, marked with retrovirus injection at embryonic day 16 (E16), express the same glial or neuronal phenotype, suggesting that separate progenitors for each of these cell phenotypes exist in the ventricular zone at that stage of corticogenesis. However, it is not known if such committed progenitors are present in the ventricular zone before E16. Another important question concerns which neurochemical features are shared by clonally related cells of the adult cerebral cortex. In this study we have addressed the first question by injecting a retroviral vector expressing beta-galactosidase into the telencephalic ventricles of rat embryos at different stages (E14-E19). In order to classify clonally related neurons in the cerebral cortex of these rats, we have used postembedding immunohistochemistry for the amino acid neurotransmitters glutamate, aspartate, and GABA. Glutamate and GABA immunoreactivity marked nonoverlapping populations of cells that corresponded to the pyramidal and nonpyramidal neuron types of the rat cerebral cortex. Clonally related neurons, marked by retrovirus injection at any day between E14 and E19, homogeneously expressed one or other phenotype and accordingly displayed glutamate or GABA immunoreactivity. This finding indicates that committed progenitor cells for pyramidal and nonpyramidal neurons are present in the ventricular zone before E16. To investigate whether lineage dictates other features in clonally related neurons, we performed an immunohistochemical analysis for the calcium-binding proteins calbindin, parvalbumin, and calretinin in clusters of clonally related nonpyramidal neurons. The same calcium-binding protein was rarely found in members of the same cluster, suggesting that lineage does not control the expression of calcium-binding proteins in cortical nonpyramidal neurons. As a result of examining a large number of clonally related neurons from brains injected at different ages, we observed remarkable differences in number and laminar distribution of pyramidal and nonpyramidal neurons marked with retrovirus. Clusters of nonpyramidal neurons were usually composed of two or three cells, and resided in the cortical layers that were just being generated at the time of injection. Clusters of pyramidal neurons were larger and dispersed in several layers in the earlier injections; their size and laminar distribution were progressively reduced for later injections. These observations suggest the existence of different mechanisms that generate the pyramidal and nonpyramidal neurons of the cerebral cortex.

Uptake of 5-hydroxydopamine into non-sympathetic nerves of guinea-pig uterine artery in late pregnancy.

Perivascular nerve fibres of the uterine artery of virgin and late pregnant guinea-pigs were examined under the electron microscope following loading with 5-hydroxydopamine, a marker for catecholamine uptake, and immunohistochemistry for dopamine beta hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Varicosities, loaded with 5-hydroxydopamine labelled vesicles, and immunoreactive axons were counted in whole transverse sections of uterine arteries. Localization of the immunoreactivities in 5-hydroxydopamine-labelled vesicles was also studied. Colocalization of substance P and dopamine beta hydroxylase immunoreactivities was investigated at the light microscopic level. Both total and relative number of varicosities with 5-hydroxydopamine-labelled vesicles in a whole section of the artery increased in late pregnancy (61.2 +/- 10.2 versus 24.5 +/- 3.2 in virgin, representing 35% and 27% respectively, of all varicosities). Also the number of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons increased, but their relative proportion remained unchanged. In virgin guinea-pigs only calcitonin gene-related peptide and neuropeptide Y immunoreactivities were associated with varicosities loaded with small dense-cored vesicles, while in late pregnancy 5-hydroxydopamine-labelled vesicles were also seen in a number of vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons. Double immunolabelling for dopamine beta hydroxylase and substance P immunoreactivity showed that substance P immunoreactivity was not present in dopamine beta hydroxylase-immunoreactive axons of the uterine artery, of neither virgin nor late pregnant guinea-pigs. It is concluded that vascular hypertrophy of the uterine artery in late pregnancy is associated with an increase in the number of perivascular nerve fibres, that involves many, if not all of the subpopulations of neurons supplying the uterine artery. Also 5-hydroxydopamine-labelled varicosities were increased, but the results of the present study indicate that some of the nerve fibres that are able to take up 5-hydroxydopamine in late pregnancy are not sympathetic (i.e. are sensory and/or parasympathetic in origin). The relevance of these findings in pregnancy is discussed.

Plasticity in expression of calcitonin gene-related peptide and substance P immunoreactivity in ganglia and fibres following guanethidine and/or capsaicin denervation.

This study was designed to investigate the effects of multiple denervation procedures on calcitonin gene-related peptide- and substance P-immunoreactive neurons in sympathetic and sensory cranial ganglia and in selected targets. Sympathectomy by long-term guanethidine treatment induced a pronounced increase in calcitonin gene-related peptide-immunoreactive and substance P-immunoreactive nerve fibres in all the tissues investigated, in contrast to a significant reduction of immunoreactive cell bodies. Neonatal capsaicin treatment abolished substance P immunoreactivity in many targets and caused a dramatic reduction of substance P-immunoreactive sensory nerve cell bodies; calcitonin gene-related peptide-immunoreactive nerve density was decreased, but the number of immunoreactive nerve cell bodies was unchanged. Guanethidine treatment of capsaicin-injected rats reversed the loss of calcitonin gene-related peptide-immunoreactive nerves, but not that of substance P-immunoreactive neurons. In the iris, capsaicin treatment had little effect on calcitonin gene-related peptide- and substance P-immunoreactive nerves, suggesting that in rats the majority of these fibres originate from capsaicin-insensitive neurons. The results suggest that the denervation procedures used in this study alter the synthesis and transport of neuropeptides in sensory neurons in conjunction with changes in the number of nerve fibres.

Nerve fibres in the uterine artery increase in number in pregnant guinea-pigs.

Perivascular nerves of the uterine artery were studied in four-month-old pregnant and non-pregnant guinea-pigs. In pregnancy, the uterine artery hypertrophies (more than two-fold growth in diameter and length and 50% increase in wall thickness), but its density of innervation remains high, because of the growth of many new axons. The axons in a complete transverse section of the vessel increase by 40%. The nerve bundles grow by 100%. Small nerve bundles increase more than large bundles: nearly 70% of the nerve bundles contain 10 or fewer axons in pregnant guinea-pigs (37% in non-pregnant animals). The increase in axon number is accompanied by formation of new varicosities, hence presumably of new neuromuscular junctions.

Increase of dopamine beta-hydroxylase immunoreactivity in non-noradrenergic nerves of rat cerebral arteries following long-term sympathectomy.

The expression of dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) immunoreactivity (IR) after short-term (2 days) and long-term (3 weeks) sympathectomy was investigated in rat cerebral vessels, dura mater and pterygopalatine ganglion neurones (which are known to project to cerebral arteries) by immunohistochemistry at both the light and electron microscopical levels. TH-IR, like glyoxylic acid-induced fluorescence, was completely abolished by sympathectomy. By contrast, DBH-IR was localized in nerve fibres, lacking 5-hydroxydopamine (5-OHDA)-labelled vesicles, along cerebral vessels of long-term sympathectomized rats, but not in the dura mater, and in pterygopalatine ganglia, where the number of DBH-IR neurons increased from 27.87% to 54.11%. Since virtually all the pterygopalatine neurons displayed choline acetyltransferase (ChAT)-IR, both in control and sympathectomized rats, it is concluded that long-term sympathectomy caused an increase of the expression of DBH-IR in cholinergic neurones of the pterygopalatine ganglion, without these neurons producing or storing noradrenaline.

Pregnancy reduces noradrenaline but not neuropeptide levels in the uterine artery of the guinea-pig.

Using histochemical, immunohistochemical and biochemical techniques, noradrenaline-, neuropeptide Y-, vasoactive intestinal polypeptide-, substance P- and calcitonin gene-related peptide-containing nerve fibres were studied in the uterine artery of virgin, progesterone-treated and pregnant guinea-pigs. Morphological changes following hormone treatment or in pregnancy were also evaluated in a quantitative study on semithin sections of the uterine artery. In late pregnancy, the number of noradrenaline-containing nerve fibres, which formed the densest plexus in virgin animals, was significantly decreased, a finding supported by a significant reduction in noradrenaline levels. This reduction was not mimicked by systemic progesterone treatment. In contrast, the innervation of the uterine artery by neuropeptide Y-containing nerve fibres was increased in pregnancy, while the other peptidergic nerves and peptide levels were unchanged after progesterone treatment and in pregnancy. These changes led to a predominance of innervation by neuropeptide Y- rather than noradrenaline-containing nerve fibres in late pregnancy. No morphological changes were detected following progesterone treatment, but pregnancy led to a marked increase in the cross-sectional area of the vessel accompanied by an increase in the thickness of the media.