Membranes from pituitary intermediate lobe cells enhance differentiation of fetal hypothalamic dopaminergic neurons in primary culture

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.     

Polysialylated Neural Cell Adhesion is Involved in Target-induced Morphological Differentiation of Arcuate Dopaminergic Neurons

We have previously shown that the morphological and biochemical maturation of developing rat hypothalamic dopaminergic neurons is accelerated when they are cocultivated with pituitary intermediate lobe cells, one of their targets. Only two subsets of hypothalamic dopaminergic neurons (arcuate, A12, and periventricular, A14, nuclei) may project to the pars intermedia. In order to determine whether the two populations are equally responsive to coculture conditions, we microdissected the hypothalamus of 17-day-old rat fetuses in two fragments containing cell bodies from the A12 and from the A14 regions, prepared neuronal cultures from both portions and incubated them separately with intermediate lobe cells. The presence of intermediate lobe cells increased tyrosine hydroxylase levels in both dopaminergic neuron subsets, but morphological differentiation was accelerated in dopaminergic neurons originating in the arcuate nucleus only. We then investigated whether physical contact between developing arcuate neurons and their target cells was a prerequisite of the morphological effect by interposing a semipermeable membrane between cultivated neurons and intermediate lobe cells in transwell culture dishes. The morphological effect was no longer observed under transwell coculture conditions, pointing to the involvement of membrane-bound molecules. Accordingly, the stimulating effect of coculture on arcuate dopaminergic neurons was completely abolished by the removal of polysialic acid on neural cell adhesion molecules by endoneuraminidase N treatment. Thus, maturation of A12 and A14 dopaminergic neurons exhibits differential susceptibility to intermediate lobe target cells, and polysialylated-NCAM is required for the contact-dependent effect.     

Distinct populations of hypothalamic dopaminergic neurons exhibit differential responses to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3)

We have previously demonstrated that differentiation of hypothalamic dopaminergic (DA) neurons can be induced in culture by their pituitary intermediate lobe target cells, through both membrane and diffusible factors. We also showed that subpopulations of DA neurons from the arcuate nucleus only, not the periventricular area, can respond to the target. Here we investigated the possibility that both neuronal subsets could also respond differentially to brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3). Addition of NT3, but not BDNF, enhanced growth and branching of neurites, tyrosine hydroxylase (TH) as well as increasing levels of cultured arcuate DA neurons. Conversely, BDNF, but not NT3, affected the same parameters in cultured periventricular DA neurons. The neurotrophins thus affect DA neurons in a structure and neuronal type-selective manner, since general neuronal markers were not affected by either neurotrophin. Neurotrophin effects were reversed by addition of specific antibodies directed against them or their respective receptors, TrkB or TrkC. By themselves, the antibodies inhibited development of DA neurons below that of control cultures, suggesting involvement of endogenous neurotrophins. BDNF and NT3 were indeed found in both arcuate and periventricular neurons and in the intermediate lobe. BDNF was always present as the mature peptide. The mature form of NT3 was only detected in the periventricular area; a precursor-like heavier form was present in all tissues studied. The present data suggest that NT3, but not BDNF, could participate in the differentiating action of intermediate lobe cells on arcuate DA neurons.     

The involvement of nitric oxide in the secretion of β-endorphin from the pituitary intermediate lobe of the rat

Nitric oxide (NO) generated by the enzyme nitric oxide synthase (NOS) has been implicated in the regulation of a variety of endocrine functions. A number of biochemical and anatomical studies have demonstrated the presence of neuronal NOS (nNOS) in the neuroendocrine axis and have shown significant effects of NO on the release of hypothalamic and pituitary hormones. Using a C-terminal directed peptide antibody that is specific for nNOS we have found a predominance of nNOS in the neural lobe of the pituitary and in a single layer of epithelial cells, possibly a remnant of Rathke's pouch that form a border between the intermediate lobe and the anterior lobe. Furthermore, we have examined the effect of sodium nitroprusside (SNP), a donor of NO on the secretion of β-endorphin (β-EP) from the isolated neuro-intermediate lobe (NIL) of the rat and cultured rat melanotrophs. It was shown that in explant cultures of intact neuro-intermediate lobes, SNP (100 μM) was able to cause an inhibition of β-EP secretion. In the presence of sulpiride (10 μM), a dopamine D₂-receptor antagonist, there was a partial reversal of the SNP effect. On the other hand SNP did not affect β-EP secretion in primary cultures of melanotrophs that no longer possessed any innervation. Taken together these data suggest that NO has an indirect inhibitory effect on the secretion of β-EP by the intermediate lobe via the release of dopamine.     

Target-dependent synaptogenesis: Inductive effect of pituitary melanotrophs on their central innervation

The glandular activity of the vertebrate pituitary intermediate lobe (IL) is regulated by direct cellular innervation, in contrast with the purely humoral regulation of adjacent pituitary anterior lobe (AL). Thus in the rat IL, melanotrophs receive a dopaminergic and GABAergic innervation from the basal hypothalamus, which tonically inhibit their glandular activity. We studied this model of neuron–target interactions in cocultures in defined medium of fetal hypothalamic neurons with neonate pituitary glandular cells. In the cocultures with IL cells, neuroglandular contacts occurred after 4 days in vitro (DIV) but required another 8 DIV to exhibit ultrastructural and immunocytochemical features of fully differentiated functional synapses; by contrast, neuroneuronal synapses developed much faster and could already be detected after 4 DIV. In the cocultures with AL cells, neuroglandular contacts never mature in differentiated synapses. Confocal microscope observation revealed that dopaminergic neurons, which represented less than 1% of total neurons in the cocultures, established 50% of the synapses detected on the melanotrophs. These cells are thus able, contrary to the AL cells, to promote the establishment of functional synapses and, to some extent, to select their specific innervation. Synapse 27:267–277, 1997. © 1997 Wiley-Liss, Inc.     

Diffusible factors from rat arcuate nucleus and Broca's diagonal band nucleus increase size and neurite outgrowth, respectively, of cultured melanin-concentrating hormone containing neurons

Using a co-culture model, we showed that diffusible factors from arcuate nucleus (AN) specifically increased the number and the size of hypothalamic neurons producing melanin-concentrating hormone (MCH). In this model neurite outgrowth and contacts between MCH neurons and dopaminergic neurons were also prominently increased, as compared to control lateral areas of the posterior hypothalamus (LH) primary cultures. These effects were mediated in part by AN glia but also by neurons of both fetal and adult AN. AN glia produced diffusible factor(s) mainly responsible for an important MCH neurite outgrowth and expressed inhibiting factors, preventing the adhesion of LH cells on AN glial cells. Furthermore, we report here a nerve growth factor-like effect from Broca's diagonal band on MCH hypothalamic neurons.     

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

Striatal target-induced axonal branching of dopaminergic mesencephalic neurons in culture via diffusible factors

The effects of striatal target cells on the morphological development of dopaminergic neurons were studied in dissociated cultures of embryonic rat mesencephalon. Mesencephalic neurons were cultured for four days in presence of target striatal cells or non target cerebellar ones. The outgrowth of dopaminergic neurons, visualized after tyrosine hydroxylase immunohistochemistry, was examined by quantitative morphometry. In cocultures, the increased complexity of dopaminergic neurites (branching) was the most striking pattern. It was dependent on the presence of target striatal cells as compared to non target ones. Cultures raised in presence or absence of serum lead to suggest the implicaton of striatal neurons rather than glia. Using MAP2 and phosphorylated neurofilaments immunohistochemistry in combination with tyrosine hydroxylase immunolabelling, it could be shown that the target-induced branching effect concerned only axonal and not dendritic processes. To further define whether diffusible factors from the striatal target would participate in the axonal branching effect, mesencephalic cells were cultured in conditioned medium from striatal neurons. Striatal conditioned medium enhanced dopamine uptake and dopamine neuron branching to the same extent as that observed in striatal cocultures. These findings demonstrate that soluble factors secreted by striatal neurons themselves selectively influence the branching of dopaminergic axons in vitro. J. Neurosci. Res. 48:358–371, 1997. © 1997 Wiley-Liss, Inc.     

Selective injury to dopaminergic neurons up-regulates GDNF in substantia nigra postnatal cell cultures: role of neuron-glia crosstalk

The effect of selective injury to dopaminergic neurons on the expression of glial cell line-derived neurotrophic factor (GDNF) was examined in substantia nigra cell cultures. H(2)O(2), mimicking increased oxidative stress, or l-DOPA, the main symptomatic treatment for Parkinson's disease, increased GDNF mRNA and protein levels in a time-dependent mode in neuron-glia mixed cultures. The concentration dependence indicated that mild, but not extensive, injury induced GDNF up-regulation. GDNF neutralization with an antibody decreased dopaminergic cell viability in H(2)O(2)-treated cultures, showing that up-regulation of GDNF was protecting dopaminergic neurons. Neither H(2)O(2) nor l-DOPA directly affected GDNF expression in astrocyte cultures, but conditioned media from challenged mixed cultures increased GDNF mRNA and protein levels in astrocyte cultures, indicating that GDNF up-regulation was mediated by neuronal factors. Since pretreatment with 6-OHDA completely abolished H(2)O(2)-induced GDNF up-regulation, we propose that GDNF up-regulation is triggered by failing dopaminergic neurons that signal astrocytes to increase GDNF expression.     

Tenascin and laminin function in target recognition and central synaptic differentiation

The influence of the target cell-issued extracellular molecules tenascin-C and laminin on synaptogenesis was studied in mixed primary cultures of pituitary melanotrophs and hypothalamic neurons. We could demonstrate in this neuron-target co-culture system a new role for tenascin-C, which appeared to be expressed as an early and transitory signal of target recognition for selective afferent fibers. Tenascin-C expression disappeared from the melanotrophs soon after the establishment of neural contacts. Concomitantly, the melanotrophs became immunoreactive for laminins, and more specifically for the synaptic isoform beta2 chain-containing laminin. The laminin signal appeared to be involved in the induction of synaptic differentiation, selectively with fibers containing both dopamine and GABA, like those innervating the melanotrophs in situ.     

Estradiol Modulates Insulin-Like Growth Factor I Receptors and Binding Proteins in Neurons from the Hypothalamus

Trophic effects of 17β-estradiol (βE²) on in vitro developing hypothalamic cells have been reported. Insulin-like growth factor I (IGF-I) is also a potent trophic factor for cultured hypothalamic cells. An interaction between sexual steroids and insulin-like growth factors (IGFs) in modulating growth of hypothalamic cells has been suggested. Thus, we tested whether βE² modulates the levels of IGF-I, its membrane receptor and its binding proteins in rat hypothalamic culturs. Using both neuron- and glial-enriched cultures obtained from fetal rat hypothalami we found that addition of βE² elicited a significant increase in IGF-I receptor levels in neurons, without affecting its affinity. On the other hand, the three different IGF-binding proteins (IGFBPs) found in the conditioned medium of the cultures were differentially modulated by βE² in the two types of cells studied. Overall, neuronal cultures produced greater amounts of IGFBPs after treatment with βE², with IGFBP2 reaching significantly higher levels. On the contrary, treatment with βE² did not significantly alter the amounts of IGFBPs produced by glial cells. Finally, the levels of immunoreactive IGF-I found either in the medium or in cellular extracts in both neuronal and glial cultures were not modified by treatment with βE². These results strongly support previous observations of a trophic synergistic interaction between IGFs and βE² on hypothalamic cells. Thus, an increase in IGF-I receptors and/or IGFBPs after exposure to βE² may result in an enhanced response of hypothalamic neurons to IGF-I. Further, the present findings strengthen our recent observation that the effects of βE² on hypothalamic glial cells are neuronally mediated, since IGF-I receptors and IGFBPs are modulated by this sex hormone in neurons, but not in glial cells.     

Pro-opiomelanocortin neuronal systems

Proopiomelanocortin (POMC) is a glycoprotein which serves as a multihormonal precursor for corticotropin (ACTH), lipotropins (beta and gamma-LPH), melanotropins (alpha, beta- and gamma-MSH) and endorphins (alpha-, beta- and gamma-endorphins). This precursor protein is primarily synthesized in corticotrophs of the anterior lobe and in melanotrophs of the intermediate lobe of the pituitary, as well as in other organs or tissues such as the genitourinary tract, the gastrointestinal tract and leukocytes. POMC is also present in the central nervous system (CNS) and numerous studies have been conducted to determine the localization, biosynthesis and functions of POMC-derived peptides. The identification of POMC-neuronal systems has been achieved by combining immuno histochemical studies, biochemical analysis, bioassays and radioimmunoassays. Three groups of perikarya containing various POMC-related peptides have been identified. One of these is located in the arcuate nucleus in the basal hypothalamus and projects towards the septum, thalamus and telencephalon. Some fibers originating from the arcuate nucleus terminate in the nucleus of the solitary tract in the brainstem where a second group of POMC-containing nerve cells are located. The latter innervates both the mesencephalon, the brainstem and the spinal cord. A third group of neurons, which contain alpha-MSH but not other POMC-derivates, has been identified in the zona incerta in the dorso-lateral hypothalamus. Processing of POMC in the cell bodies of the arcuate nucleus follows a similar pattern as in the pituitary intermediate lobe. Endopeptidases called "acid-thiol-arginyl-proteases" cleave the prohormone at paired basic amino acids. The basic residues remaining on the resulting peptides are subsequently eliminated by the joint action of the less specific B-type carboxypeptidases and B-type aminopeptidases. alpha-MSH and beta-endorphin are among the major end products. Enzymatic modifications including N-alpha-acetylation by opiomelanotropin-acetyltransferase (OMAT) and/or C-terminal amidation by peptidyl-glycine alpha-amidating monooxygenase (PAM) occur after proteolytic processing. However, the rate of acetylation observed in hypothalamic POMC neurons is much lower than in the melanotrophs of the pars intermedia. Acetylation of MSH and endorphin is crucial in determining the biological potency of these peptides. Desacetyl alpha-MSH is far less active than alpha-MSH (monoacetyl alpha-MSH), whereas acetylated beta-endorphin has no opiate activity. The mechanisms regulating the activity of POMC-containing neurons are still unknown.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nuclear translocation of STAT5 and increased expression of Fos related antigens (FRAs) in hypothalamic dopaminergic neurons after prolactin administration

Ample evidence indicates feedback relationships between pituitary prolactin and hypothalamic dopaminergic neurons. Since the presence of prolactin receptors was earlier demonstrated in hypothalamic dopaminergic neurons, our working hypothesis was that prolactin induced activation of prolactin receptor coupled signaling leads to increased neuronal activity in these neurons. The aim of this study was to correlate prolactin receptor mediated signaling and prolactin induced activation in hypothalamic dopaminergic neurons. We used nuclear translocation of STAT5 as a marker of prolactin receptor induced signaling and expression of Fos related antigens (FRAs) as an indicator of neuronal activation. We performed double label immunocytochemical studies to determine the time course of the presence of FRAs and STAT5 in the nuclei of hypothalamic dopaminergic neurons after ovine prolactin treatment. Exogenous ovine prolactin treatment of ovariectomized rats resulted in an increase in serum ovine prolactin levels and a decrease in endogenous serum prolactin levels, indicating that ovine prolactin activated mechanisms inhibited pituitary prolactin secretion. Indeed, ovine prolactin activated the prolactin receptors in most subpopulations of hypothalamic dopaminergic neurons, resulting in nuclear translocation of STAT5. Also, increased neuronal activity, indicated by expression of FRAs, was observed in the same neuron populations after ovine prolactin treatment. These results suggest that signal transduction mechanisms coupled to prolactin receptors in hypothalamic dopaminergic neurons resemble those observed in other tissues; and nuclear translocation of STAT5 can be used as a marker of prolactin receptor activation in hypothalamic dopaminergic neurons.     

Soluble striatal extracts enhance development of mesencephalic dopaminergic neurons in vitro

Recent studies have suggested that diffusible factors released by neural target tissues enhance survival, growth, and differentiation of neurons within the central, as well as the peripheral, nervous system. In this report, we use catecholamine cytofluorescence to demonstrate that a soluble factor from the striatum produces a 4-fold increase in number of catecholamine cytofluorescent-positive dopaminergic neurons in dissociated mesencephalon cultures prepared from embryonic 14-day-old rats. The same soluble extract enhances the number of neurites per cell and the length of neurites, and also produces a greater than 3.5-fold stimulation of high affinity dopamine uptake into neurons. Such stimulation is significantly reduced following trypsin treatment. The trophic effects on dopaminergic neurons are maximal in extracts of the striatum, but are also found in extracts of the hippocampus-entorhinal cortex-amygdaloid nucleus and the cerebral cortex, although they are less in extracts of the cerebellum, negligible in the olfactory bulb, and absent in the liver. With molecular sieving chromatography, the soluble factors stimulating high affinity dopamine uptake are partially separable from the factors stimulating neuronal high affinity GABA uptake. The approximate molecular weight of the factors influencing dopaminergic neurons is 1500-2200 Da.     

Disruption of dopamine homeostasis underlies selective neurodegeneration mediated by alpha-synuclein

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.     

Identification of MSH release-inhibiting elements in the neurointermediate lobe of the rat

Neurointermediate lobes of rats comprise elements which, when excited in vitro, bring about an inhibition of the release of melanocyte stimulating hormone (MSH). Superfusion of neurointermediate lobes of intact donor rats with medium containing 45 mM K+ induced a stimulation of the release of oxytocin, arginine-vasopressin and dopamine (DA) and inhibited the release of MSH. Fluorescence histochemical observations and the results of release studies indicate that electrothermic lesions in the mediobasal hypothalamus induced a more rapid degeneration of dopaminergic than of peptidergic terminals in the neurointermediate lobe. Dopaminergic nerve terminals and the stimulated release of DA had vanished completely on the second day after these lesions, which coincided with the disappearance of K+-induced inhibition of MSH release. Frontal hypothalamic deafferentations resulted in disappearance of peptidergic nerve terminals as evidenced by the development of diabetes insipidus and the strong decline of depolarization-induced release of oxytocin and vasopressin from neurointermediate lobes in vitro. In contrast, the dopaminergic plexus was left intact, as was the K+-induced stimulation of DA release and inhibition of MSH release. We conclude that the K+-induced inhibition of MSH release is mediated by DA rather than by neuropeptides from terminals in the neurointermediate lobe. The results are in agreement with the proposed MSH release-inhibiting role of the dopaminergic tuberoinfundibular neurones.     

Expression of tetrodotoxin-sensitive and resistant sodium channels by rat melanotrophs

Rat melanotrophs fire Na+ and Ca2(+)-dependent action potentials. Whereas the molecular identity of Ca2+ channels expressed by these cells is well documented, less is known about Na channels. We characterize the expression of seven sodium channel alpha-subunit and the beta1- and beta2-subunit mRNAs. The tetrodotoxin-resistant Nav1.8 and Nav1.9 alpha subunit mRNAs are detected in the newborn intermediate lobe and in cultured melanotrophs. Electrophysiological recordings further demonstrate the expression of both tetrodotoxin-sensitive and tetrodotoxin-resistant currents by dissociated melanotrophs. Moreover, activated sodium channels are able to elicit intracellular calcium waves, both in the absence or in the presence of tetrodotoxin. This work shows that rat melanotrophs express functional tetrodotoxin-resistant sodium channels, whose activation can lead to the generation of intracellular calcium waves.     

Activation of tuberoinfundibular and tuberohypophysial dopamine neurons following intracerebroventricular administration of bombesin.

The effect of bombesin on the activity of dopamine (DA) neurons comprising the nigrostriatal, mesolimbic, tuberoinfundibular and tuberohypophysial systems in the male rat was determined by measuring: (1) the accumulation of 3,4-dihydroxyphenylalanine (DOPA) after administration of a decarboxylase inhibitor, and (2) the concentration of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in brain (striatum, nucleus accumbens, median eminence) and pituitary regions (intermediate and neural lobes) containing terminals of these neurons. Intracerebroventricular (i.c.v.) injection of bombesin caused a dose- and time-related increase in the activity of DA neurons projecting to the median eminence and intermediate lobe of the pituitary, and a corresponding decrease in the concentrations of prolactin and alpha-melanocyte-stimulating hormone (alpha MSH) in the plasma. In contrast, doses of bombesin up to 10 ng i.c.v. failed to alter the activity of DA neurons terminating in the striatum, nucleus accumbens or neural lobe of the pituitary gland. Equimolar doses of bombesin and gastrin-releasing peptide (GRP), a bombesin-like peptide, increased the concentrations of DOPAC in the median eminence and intermediate lobe of the pituitary, suggesting that GRP-preferring receptors may be responsible for the stimulatory effects of bombesin on DA neuronal activity in these regions. The results of these studies suggest that bombesin increases the activity of tuberoinfundibular and tuberohypophysial DA neurons projecting to the median eminence and intermediate lobe of the pituitary, respectively, and thereby inhibits the secretion of prolactin and alpha MSH.     

Atrial natriuretic peptide-induced suppression of basal and dehydration-induced vasopressin secretion is not mediated by hypothalamic tuberohypophysial or tuberoinfundibular dopaminergic neurons

The purpose of this study was to examine the effects of atrial natriuretic peptide (ANP) on the secretion of vasopressin and the activities of hypothalamic tuberohypophysial and tuberoinfundibular dopaminergic neurons in normal and dehydrated male rats. Neuronal activity was estimated by measuring the concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and dopamine (DA) in brain and posterior pituitary regions containing terminals of tuberohypophysial (neural lobe; intermediate lobe) and tuberoinfundibular (median eminence) DA neurons. Intracerebroventricular (icv) administration of 20 micrograms ANP decreased basal arginine vasopressin concentrations in the plasma, but had no effect on the concentrations of DOPAC or DA in any region examined. Water deprivation caused a time-dependent increase in plasma arginine vasopressin concentrations, with maximal levels measured 2 days after removal of water bottles. Water deprivation had no effect on DOPAC concentrations in the neural lobe, intermediate lobe or median eminence, but increased DA concentrations in the neural lobe. ANP (20 micrograms/rat; icv) decreased arginine vasopressin concentrations in the plasma of water-deprived rats without altering DOPAC or DA concentrations in the neural lobe, intermediate lobe or median eminence. These results indicate that ANP-induced suppression of basal and dehydration-induced vasopressin secretion is not mediated by tuberohypophysial or tuberoinfundibular DA neurons.     

Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia.

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.