Biomedical subjects

A Beaudet

Publications and source records attributed to A Beaudet.

At least 19 recordsLinked to original sources

PubMed2006

Morphine priming in rats with chronic inflammation reveals a dichotomy between antihyperalgesic and antinociceptive properties of deltorphin.

We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (deltaOR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of deltaOR, and thereby the analgesic properties of the deltaOR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Freund's adjuvant (CFA) into the hind paw resulted in a bilateral increase in the binding and internalization of fluorescent deltorphin in neurons of the lumbar spinal cord as did prolonged morphine treatment [Morinville A, Cahill CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, Stroh T, Sadikot AF, O'Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord. J Neurosci 24:5549-5559]. This effect was accompanied by an increase in the antinociceptive efficacy of intrathecal deltorphin as measured using the tail-flick test. Treatment of CFA-injected rats with morphine decreased the cell surface availability of deltaOR in neurons of the dorsal horn of the lumbar spinal cord as compared with treatment with CFA alone. Behaviorally, it significantly enhanced the antihyperalgesic effects of deltorphin (plantar test; % maximum possible antihyperalgesic effect (MPAHE)=113.5%+/-32.4% versus 26.1%+/-11.6% in rats injected with CFA alone) but strongly reduced the antinociceptive efficacy of the drug (tail-flick test; % maximum possible antinociceptive effect (MPE)=29.6%+/-3.6% versus 66.6%+/-6.3% in rats injected with CFA alone) suggesting that the latter, but not the former, is linked to the deltaOR trafficking events observed neuroanatomically. These results demonstrate that in chronic inflammation, the antihyperalgesic effects of deltaOR agonists may be enhanced by morphine pre-treatment. They also reveal a dichotomy between mechanisms underlying antihyperalgesic and antinociceptive effects of deltaOR agonists.

Analgesics↗

PubMed2005

PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile.

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.

Adenoviridae↗

PubMed2004

Role of neuronal nicotinic receptors in the effects of nicotine and ethanol on contextual fear conditioning.

Nicotine can enhance contextual learning while ethanol impairs some forms of learning. Nicotine can overcome some of the impairing effects of ethanol when the two drugs are co-administered. The specific brain nicotinic acetylcholine receptors (nAChRs) that mediate nicotine's effects on contextual learning and whether any of ethanol's actions are mediated by nAChRs are unknown. The potential roles of nAChRs in contextual and cued fear conditioning as well as the effects of nicotine, ethanol, or co-administration of nicotine and ethanol were examined in wild type and homozygous null mutant mice from alpha7, beta2, beta3, and beta4 mouse lines at 24 h after training. Nicotine was given prior to training and testing, whereas ethanol was given only before training. Nicotine enhanced contextual learning in both alpha7 wild types and mutants when mice were trained at 0.17 mA, but not 0.35 mA. Mutants lacking the alpha7 subunit were less sensitive to the memory impairing effects of ethanol trained at 0.35 mA. beta2 Null mutants receiving saline showed a small, but significant, impairment in contextual learning compared with wild type littermates when the shock stimulus was 0.35 mA. Beta2 Null mutant mice also did not respond to the cognitive enhancing effects of nicotine alone, or after ethanol administration. beta3 and beta4 null mutants did not differ from wild types either after saline or any of drug treatments. These results show that beta2-containing nAChRs, but not beta3- or beta4-containing receptors, mediate the enhancing effects of nicotine on contextual learning and confirm previous studies implicating beta2 in other forms of learning. A new role for alpha7 nAChRs in regulating sensitivity to the cognitive disrupting effects of ethanol is proposed.

Animals↗

PubMed2003

Up-regulation and trafficking of delta opioid receptor in a model of chronic inflammation: implications for pain control.

Pharmacological and physiological evidence supports a role for delta (delta) opioid receptors in the nociceptive mechanisms of inflammation. However, few data exist regarding delta opioid receptor expression and localization in such conditions. In this study, we have assessed the distribution and function of delta opioid receptors in the rat spinal cord following induction of chronic inflammation by intraplantar injection of complete Freund's adjuvant (CFA). Intrathecal administration of the selective delta opioid receptor agonist, D-[Ala(2), Glu(4)] deltorphin, dose-dependently reversed thermal hyperalgesia induced by CFA. In situ hybridization and Western blotting experiments revealed an increase in delta opioid receptor mRNA and protein levels, respectively, in the dorsal lumbar spinal cord ipsilateral to the CFA injection site compared to the contralateral side and sham-injected controls. By electron microscopy, immunopositive delta opioid receptors were evident in neuronal perikarya, dendrites, unmyelinated axons and axon terminals. Quantification of immunopositive signal in dendrites revealed a twofold increase in the number of immunogold particles in the ipsilateral dorsal spinal cord of CFA-injected rats compared to the contralateral side and to sham-injected rats. Moreover, the relative frequency of immunogold particles associated with or in close proximity to the plasma membrane was increased in the ipsilateral dorsal spinal cord, indicating a more efficient targeting of delta opioid receptors to neuronal plasma membranes. These data demonstrate that CFA induces an up-regulation and increased membrane targeting of delta opioid receptors in the dorsal spinal cord which may account for the enhanced antinociceptive effects of delta opioid receptor agonists in chronic inflammatory pain models.

Animals↗

PubMed2001

Immunohistochemical distribution of delta opioid receptors in the rat central nervous system: evidence for somatodendritic labeling and antigen-specific cellular compartmentalization.

Many studies have reported on the distribution of delta opioid receptors (delta OR) in the mammalian central nervous system (CNS) by using a variety of techniques. However, no general consensus has emerged with regards to the localization of this receptor due to inconsistencies in the immunohistochemical literature. In the present study, we analyzed the cellular and subcellular distribution of immunoreactive delta OR in the rat CNS using two different antibodies (directed against a sequence in the C-terminus or N-terminus of the rat delta OR). By using Western blotting, these two antibodies recognized similar forms of the delta OR in COS-7 cells transfected with this receptor, but distinct forms in membranes from the rat spinal cord. By using light microscopic immunohistochemistry, both antibodies recognized identical populations of nerve cell bodies throughout the CNS; the distribution of these cell bodies conformed to that of delta OR mRNA-expressing cells detected by in situ hybridization. However, whereas the C-terminus-directed antibody recognized predominantly perikarya and proximal dendrites, the N-terminus-directed antibody also labeled extensively dendritic and terminal arbors. Furthermore, by using electron microscopy, the two antibodies were found not only to label differentially somatodendritic versus axonal compartments, but also plasma membrane versus cytoplasmic ones, suggesting that distinct immunological forms of the receptor are being targeted preferentially to different cellular and subcellular domains.

Animals↗

PubMed2001

Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain.

Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.

Animals↗

PubMed2001

Prolonged morphine treatment targets delta opioid receptors to neuronal plasma membranes and enhances delta-mediated antinociception.

Opioid receptors are known to undergo complex regulatory changes in response to ligand exposure. In the present study, we examined the effect of morphine on the in vitro and in vivo density and trafficking of delta opioid receptors (deltaORs). Prolonged exposure (48 hr) of cortical neurons in culture to morphine (10 microm) resulted in a robust increase in the internalization of Fluo-deltorphin, a highly selective fluorescent deltaOR agonist. This effect was mu-mediated because it was entirely blocked by the selective mu opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) and was reproduced using the selective mu agonist fentanyl citrate. Immunogold electron microscopy revealed a marked increase in the cell surface density of deltaORs in neurons exposed to morphine, indicating that the increase in Fluo-deltorphin internalization was caused by increased receptor availability. Prolonged morphine exposure had no effect on deltaOR protein levels, as assessed by immunocytochemistry and Western blotting, suggesting that the increase in bioavailable deltaORs was caused by recruitment of reserve receptors from intracellular stores and not from receptor neosynthesis. Complementary in vivo studies demonstrated that chronic treatment of adult rats with morphine (5-15 mg/kg, s.c., every 12 hr) similarly augmented targeting of deltaORs to neuronal plasma membranes in the dorsal horn of the spinal cord. Furthermore, this treatment markedly potentiated intrathecal d-[Ala(2)]deltorphin II-induced antinociception. Taken together, these results demonstrate that prolonged stimulation of neurons with morphine markedly increases recruitment of intracellular deltaORs to the cell surface, both in vitro and in vivo. We propose that this type of receptor subtype cross-mobilization may widen the transduction repertoire of G-protein-coupled receptors and offer new therapeutic strategies.

Analgesics↗

PubMed2001

Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.

BACKGROUND: Familial hypercholesterolemia (FH) that results from LDL receptor (LDLR) deficiency affects approximately 1 in 500 persons in the heterozygous state and approximately 1 in 1 million persons in the homozygous state. We tested a novel gene therapy strategy for the treatment of FH in a mouse model. METHODS AND RESULTS: We delivered the VLDL receptor (VLDLR) to the liver of LDLR-deficient mice and compared the effect of a helper-dependent adenoviral vector with all viral coding sequences deleted (HD-Ad-mVLDLR) with a first-generation vector (FG-Ad-mVLDLR), an HD-Ad (HD-Ad-0) that contained no expression cassette, and dialysis buffer (DB). A single intravenous injection of HD-Ad-mVLDLR led to a lowering of plasma cholesterol that lasted >/=6 months. Acute liver toxicity (as measured with liver enzyme elevation) occurred after FG-Ad-mVLDLR but not after HD-Ad-mVLDLR, HD-Ad-0, or DB treatment. At 6 months, VLDLR was detected in the liver with Western blotting and with immunofluorescence staining only in HD-Ad-mVLDLR-treated mice. Aortic atherosclerosis was almost completely prevented in these animals. CONCLUSIONS: HD-Ad-mediated intravenous delivery of VLDLR to hepatocytes is well tolerated. It produces long-term lowering of plasma cholesterol and prevents atherosclerosis development in LDLR-deficient mice. These data provide support for the feasibility and safety of this approach for therapy of human subjects.

Adenoviridae↗

PubMed2001

Homologous upregulation of sst2 somatostatin receptor expression in the rat arcuate nucleus in vivo.

In vitro studies using various cell systems have provided conflicting results regarding homologous regulation of somatostatin (SRIH) receptors, and information on whether SRIH regulates the expression of its own receptors in vivo is lacking. In the present study we examined, by in situ hybridization, the effects of pretreatment with the sst2-preferring SRIH analog, octreotide, in vivo, on mRNA levels of two SRIH receptor subtypes, sst1 and sst2, in rat brain and pituitary. (125)I-[DTrp(8)]-SRIH binding was also measured in these regions. Three hours after the iv injection of 50 microg octreotide to conscious adult male rats, there was a 46% increase (p < 0.01) in the labeling density of sst2 mRNA-expressing cells in the hypothalamic arcuate nucleus compared to normal saline-pretreated controls, but not in any of the other brain regions examined. Computer-assisted image analysis revealed that 3 h exposure to octreotide significantly (p < 0.01) augmented both the number and labeling density of sst2 mRNA-expressing cells in the arcuate nucleus, compared to those in saline-treated controls. By contrast, within the anterior pituitary gland, in vivo exposure to octreotide did not affect the expression of sst2 mRNA. No changes in sst1 mRNA-expressing cells were observed after octreotide treatment in any of the regions measured, indicating that the observed effects were homologous, i.e. specific of the receptor subtype stimulated. Octreotide pretreatment was also without effect on the density of (125)I-[DTrp(8)]-SRIH binding in either the arcuate nucleus or pituitary. These results demonstrate, for the first time, that SRIH preexposure in vivo upregulates the expression of a subtype of its own receptors, sst2, within the central nervous system. They further suggest that pretreatment with SRIH in vivo does not cause sst2 receptor desensitization in arcuate nucleus and pituitary. Such homologous regulatory mechanisms may play an important role in the neuroendocrine control of growth hormone (GH) secretion by the arcuate nucleus.

Animals↗

PubMed2000

Neurotensin-induced bursting of cholinergic basal forebrain neurons promotes gamma and theta cortical activity together with waking and paradoxical sleep.

Cholinergic basal forebrain neurons have long been thought to play an important role in cortical activation and behavioral state, yet the precise way in which they influence these processes has yet to be fully understood. Here, we have examined the effects on the electroencephalogram (EEG) and sleep-wake state of basal forebrain administration of neurotensin (NT), a neuropeptide that has been shown in vitro to potently and selectively modulate the cholinergic cells. Microinjection of (0.1-3.0 mm) NT into the basal forebrain of freely moving, naturally waking-sleeping rats produced a dose-dependent decrease in delta ( approximately 1-4 Hz) and increase in both theta ( approximately 4-9 Hz) and high-frequency gamma activity (30-60 Hz) across cortical, areas with no increase in the electromyogram. These EEG changes were accompanied by concomitant decreases in slow wave sleep (SWS) and transitional SWS (tSWS), increases in wake, and most remarkably, increases in paradoxical sleep (PS) and transitional PS (tPS), despite the virtual absence of SWS. The effects were attributed to direct action on cholinergic neurons as evidenced by selective internalization of a fluorescent ligand, Fluo-NT, in choline acetyltransferase (ChAT)-immunoreactive cells and stimulation by NT of bursting discharge in juxtacellularly recorded, Neurobiotin-labeled, ChAT-immunoreactive neurons. We conclude that NT-induced rhythmic bursting of cholinergic basal forebrain neurons stimulates rhythmic theta oscillations and gamma across the cerebral cortex. With the selective action of NT on the cholinergic cells, their bursting discharge promotes theta and gamma independent of motor activity and thereby also stimulates and enhances PS.

Animals↗

PubMed2000

Immunohistochemical evidence for the implication of PC1 in the processing of proneurotensin in rat brain.

Biochemical studies have shown that prohormone convertases PC1, PC2 and PC5A all have the capacity to process, with different specificities, the neurotensin/neuromedin N precursor, pro-NT/NN. A previous study from our laboratory has demonstrated that in rat brain, both PC2 and PC5A may be co-expressed with NT, lending support to a physiological implication of these two enzymes in the endoproteolytic maturation of pro-NT/NN. In the present study, we sought to determine whether PC1 might also be involved in this process by comparing the immunohistochemical distribution of the enzyme with that of NT in both singly labeled and dually labeled serial brain sections. PC1 was found to co-localize extensively with NT throughout the rat neuraxis. However, there were important regional variations in the proportion of NT neurons co-expressing PC1. Furthermore, this proportion was negatively correlated with that of NT neurons expressing PC5, suggesting that PC1 may serve as an alternative to PC5A for processing pro-NT/NN in mammalian brain.

Animals↗

PubMed2000

Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina.

Before phototransduction, spontaneous activity in the developing mammalian retina is required for the appropriate patterning of retinothalamic connections, and there is growing evidence that this activity influences the development of circuits within the retina itself. We demonstrate here that the neural substrate that generates waves in the mouse retina develops through three distinct stages. First, between embryonic day 16 and birth [postnatal day 0 (P0)], we observed both large, propagating waves inhibited by nicotinic acetylcholine receptor (nAChR) antagonists and small clusters of cells displaying nonpropagating, correlated calcium increases that were independent of nAChR activation. Second, between P0 and P11, we observed only larger propagating waves that were abolished by toxins specific to alpha3 and beta2 subunit-containing nAChRs. Third, between P11 and P14 (eye opening) we observed propagating activity that was abolished by ionotropic glutamate receptor antagonists. The time course of this developmental shift was dramatically altered in retinas from mice lacking the beta2 nAChR subunit or the beta2 and beta4 subunits. These retinas exhibited a novel circuit at P0, no spontaneous correlated activity between P1 and P8, and the premature induction at P8 of an ionotropic glutamate receptor-based circuit. Retinas from postnatal mice lacking the alpha3 nAChR subunit exhibited spontaneous, correlated activity patterns that were similar to those observed in embryonic wild-type mice. In alpha3-/- and beta2-/- mice, the development and distribution of cholinergic neurons and processes and the density of retinal ganglion cells (RGCs) and the gross segregation of their dendrites into ON and OFF sublaminae were normal. However, the refinement of individual RGC dendrites is delayed. These results indicate that retinal waves mediated by nAChRs are involved in, but not required for, the development of neural circuits that define the ON and OFF sublamina of the inner plexiform layer.

Action Potentials↗

PubMed2000

Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain.

Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry.

Animals↗

PubMed2000

Immunohistochemical evidence for the involvement of protein convertases 5A and 2 in the processing of pro-neurotensin in rat brain.

The neuropeptides/neurotransmitters neurotensin (NT) and neuromedin (NN) are synthesized by endoproteolytic cleavage of a common inactive precursor, pro-NT/NN. In vitro studies have suggested that the prohormone convertases PC5A and PC2 might both be involved in this process. In the present study, we used dual immunohistochemical techniques to determine whether either one or both of these two convertases were co-localized with pro-NT/NN maturation products and could therefore be involved in the physiological processing of this propeptide in rat brain. PC2-immunoreactive neurons were present in all regions immunopositive for NT. All but three regions expressing NT were also immunopositive for PC5A. Dual localization of NT with either convertase revealed that NT was extensively co-localized with both PC5A and PC2, albeit with regional differences. These results strongly suggest that PC5A and PC2 may play a key role in the maturation of pro-NT/NN in mammalian brain. The regional variability in NT/PC co-localization patterns may account for the region-specific maturation profiles previously reported for pro-NT/NN. The high degree of overlap between PC5A and PC2 in most NT-rich areas further suggests that these two convertases may act jointly to process pro-NT/NN. At the subcellular level, PC5A was largely co-localized with the mid-cisternae Golgi marker MG-160. By contrast, PC2 was almost completely excluded from MG-160-immunoreactive compartments. These results suggest that PC5A, which is particularly efficient at cleaving the two C-terminal-most dibasics of pro-NT/NN, may be acting as early as in the Golgi apparatus to release NT, whereas PC2, which is considerably more active than PC5A in cleaving the third C-terminal doublet, may be predominantly involved further distally along the secretory pathway to release NN.

Animals↗

PubMed2000

Somatostatin-induced regulation of SST(2A) receptor expression and cellsurface availability in central neurons: role of receptor internalization.

To investigate the effects of somatostatin (somatotropin release-inhibiting factor, SRIF) on the regulation of SST(2A) receptors in mammalian brain, we examined how blockade of SRIF release or stimulation by the SRIF analog [d-Trp(8)]-SRIF would affect the expression and cell surface availability of SST(2A) receptors in rat brain slices. First, we measured the intensity of SST(2A) immunoreactivity, using quantitative light microscopic immunocytochemistry, and levels of SST(2A) mRNA, using semiquantitative RT-PCR, under conditions of acute SRIF release blockade. Incubation of slices from the claustrum or basolateral amygdala, two regions previously shown to contain high concentrations of SST(2A) receptors, in Ca(2+)-free Ringer's for 40 min induced a decrease in the intensity of SST(2A) receptor immunoreactivity and concentration of SST(2A) mRNA as compared with control values obtained in Ca(2+)-supplemented Ringer's. These effects were counteracted in a dose-dependent manner by the addition of 10-100 nm [d-Trp(8)]-SRIF to the Ca(2+)-free medium. Furthermore, both of these effects were abolished in the presence of the endocytosis inhibitors phenylarsine oxide or hyperosmolar sucrose, suggesting that they were dependent on receptor internalization. Electron microscopic immunogold labeling confirmed the existence of an agonist-induced internalization of SST(2A) receptors in central neurons. At a high (10 microm), but not at a low (10 nm), concentration of agonist this internalization resulted in a significant decrease in cell surface receptor density, irrespective of the presence of Ca(2+) in the medium. Taken together, these results suggest that ligand-induced endocytosis is responsible for rapid transcriptional (increase in SST(2A) expression) and trafficking (loss of cell surface receptors) events involved in the control of the somatostatinergic signal.

Amygdala↗

PubMed2000

Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture.

Luteinizing hormone-releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression of immortalized hypothalamic LHRH (GT(1-1)) neurons in vitro. Culturing GT(1-1) cells in the presence of young glia for different time intervals caused a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT(1-1) cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of processes/cell and greater surface area of growth cones than GT(1-1) cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT(1-1) neurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT(1-1) neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT(1-1) neurons cocultured with young glia exhibited a 50% lower incorporation of [(3)H]thymidine than GT(1-1) neurons cultured without glia. By contrast, in the presence of old glia [(3)H]thymidine incorporation was significantly higher in cells cocultured with glia than in GT(1-1) neurons cultured alone. Localization of the proliferating cells by dual immunohistochemical staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted to nuclei of GT(1-1) neurons when these were cocultured with young glia, but associated with both neurons and astrocytes in the presence of old glia. At the functional level, coculture of GT(1-1) neurons with young glia increased the spontaneous release of LHRH as compared to GT(1-1) neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium of young glia (ACM-Y) induced significant neurotrophic and functional effects on GT(1-1) cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM-Y totally abolished its neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release-promoting effects. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged bFGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth-supporting state when treated with bFGF and this functional shift involves a diffusible molecule with potent neurotrophic and functional effects on immortalized LHRH neurons. (c) 2000 Wiley-Liss, Inc.

Animals↗

PubMed2000

Receptor-mediated internalization of somatostatin in rat cortical and hippocampal neurons.

Binding of neuropeptides to their receptors usually results in internalization of receptor-ligand complexes. This process serves a crucial role in receptor downregulation, resensitization, and transmembrane signaling. It has mainly been investigated in cells ectopically expressing recombinant receptors. In the present study, we investigated whether rat central neurons and astrocytes naturally expressing somatostatin (SRIF) receptors internalized this neuropeptide. We demonstrated that 29% of cortical and 45% of hippocampal neurons in culture expressed the SRIF receptor sst(2A) and that 40-50% of the neurons internalized fluorescent SRIF. Similarly, an important proportion of astrocytes expressed sst(2A) (up to 60% in cortical cultures) and internalized fluo-SRIF. Competition experiments using the sst(2)/sst(5)-preferring agonist SMS 201-995 (octreotide) showed that a subpopulation of neurons internalized fluo-SRIF via sst(2) and/or sst(5) receptors, but that others also did so via other subtypes. Fluo-SRIF labeling was barely competed for by the sst(1)-selective agonist CH-275, indicating that sst(1) was unlikely to be mediating SRIF internalization in hippocampal and cortical neurons. Given the paucity of sst(5) receptors in cerebral cortex and hippocampus and the poor yield of sst(4) internalization in transfected cells, we conclude that sst(2) and sst(3) subtypes are the most likely to be responsible for SRIF internalization in our culture systems.

Animals↗

PubMed2000

Basic fibroblast growth factor priming increases the responsiveness of immortalized hypothalamic luteinizing hormone releasing hormone neurones to neurotrophic factors.

The participation of growth factors (GFs) in the regulation of luteinizing hormone releasing hormone (LHRH) neuronal function has recently been proposed, but little is known about the role played by GFs during early LHRH neurone differentiation. In the present study, we have used combined biochemical and morphological approaches to study the ability of a number of GFs normally expressed during brain development, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I) to induce survival, differentiation, proliferation, and phenotypic expression of immortalized (GT1-1) LHRH neurones in vitro, at early (3-days in vitro, 3-DIV) and late (8-DIV) stages of neuronal differentiation. Comparison of GF-treated vs untreated neurones grown in serum-deprived (SD) medium demonstrated bFGF to be the most potent, and insulin the least active in promoting neuronal differentiation. Thus, at both 3-DIV and 8-DIV, but especially at 8-DIV, bFGF induced the greatest increase in the total length and number of LHRH processes/cell and in growth cone surface area. bFGF was also the most active at 3-DIV, and IGF-I at 8-DIV, in counteracting SD-induced cell death, whereas EGF was the most potent in increasing [3H]thymidine incorporation. All GFs studied decreased the spontaneous release of LHRH from GT1-1 cells when applied at 3-DIV or 8-DIV, except for insulin which was inactive at both time-points and bFGF which was inactive at 8-DIV. Pre-treatment of GT1-1 cells with a suboptimal ('priming') dose of bFGF for 12 h followed by application of the different GFs induced a sharp potentiation of the neurotrophic and proliferative effects of the latter and particularly of those of IGF-I. Moreover, bFGF priming counteracted EGF-induced decrease in LHRH release and significantly stimulated LHRH secretion following IGF-I or insulin application, suggesting that bFGF may sensitize LHRH neurones to differentiating effects of specific GFs during development.

Cell Division↗