Dario Sonetti

Restraint stress alters the secretory activity of neurons co-expressing urocortin-1, cocaine- and amphetamine-regulated transcript peptide and nesfatin-1 in the mouse Edinger–Westphal nucleus

Central stress regulatory pathways utilize various neuropeptides, such as urocortin-1 (Ucn1) and cocaine- and amphetamine-regulated transcript peptide (CART). Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn1, CART and nesfatin-1 are highly expressed in neurons of the npEW, but the way these three neuropeptides act together in response to acute stress is not known. We hypothesized that Ucn1, CART and nesfatin-1 are colocalized in npEW neurons and that these neurons are recruited by acute stress. Using quantitative immunocytochemistry and the reverse transcriptase polymerase chain reaction (RT-PCR), we support this hypothesis, by showing in B6C3F1/Crl mice that Ucn1, CART and nesfatin-1 occur in the same neurons of the npEW nucleus. More specifically, Ucn1 and CART revealed a complete colocalization in the same perikarya, while 90% of these neurons are also nesfatin-1-immunoreactive. Furthermore, acute (restraint) stress stimulates the general secretory activity of these npEW neurons (increased presence of Fos) and the production of Ucn1, CART and nesfatin-1: Ucn1, CART and nesfatin-1(NUCB2) mRNAs have been increased compared to controls by x1.8, x2.0 and x2.6, respectively (p<0.01). We conclude that Ucn1, CART and nesfatin-1/NUCB2 are specifically involved in the response of npEW neurons to acute stress in the mouse.

Ascaris suum, an Intestinal Parasite, Produces Morphine

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 ± 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals’ nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal μ receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.

Endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma.

The aim of this study was to demonstrate by biochemical and immunocytochemical methods the presence of endogenous morphine in nervous and immune tissues of the freshwater snail, Planorbarius corneus. High performance liquid chromatography (HPLC) coupled to electrochemical detection performed on tissues from control snails, revealed that the CNS contains 6.20+/-2.0 pmol/g of the alkaloid, the foot tissue contains a much lower level, 0.30+/-0.03 pmol/g, whilst morphine is not detected in the hemolymph and hepatopancreas. In specimens that were traumatized, we detected a significant rise of the CNS morphine level 24 h later (43.7+/-5.2 pmol/g) and an initial decrease after 48 h (19.3+/-4.6 pmol/g). At the same times, we found the appearance of the opiate in the hemolymph (0.38+/-0.04 pmol/ml and 0.12+/-0.03 pmol/ml) but not in the hepatopancreas. Using indirect immunocytochemistry, a morphine-like molecule was localized to a number of neurons and a type of glial cell in the CNS, to some immunocytes in the hemolymph and to amoebocytes in the foot, as well as to fibers in the aorta wall. Simultaneously to the rise of morphine biochemical level following trauma, morphine-like immunoreactivity (MIR) increased in both intensity and the number of structures responding positively, i.e., neurons and fiber terminals. In another mollusc, the mussel Mytilus galloprovincialis, the same pattern of enhanced MIR was found after trauma. Taken together, the data suggest the presence of a morphinergic signaling in invertebrate neural and immune processes resembling those of classical messenger systems and an involvement in trauma response.

Serotonin and Retzius cell depress the hyperpolarization following impulses of leech touch cell

Intracellular recordings from T mechanosensory cells of Hirudo medicinalis showed, as previously demonstrated, that repetitive firing is followed by a long-lasting hyperpolarization. Serotonin application at two concentrations (1 microM and 50 microM) depressed this hyperpolarization by up to 2/3; the effect was dose-dependent, long-lasting and reversible. Intracellular stimulation of giant serotonergic neurons (Retzius cells, Rz) mimicked serotonin perfusion: the effect was proportional to the number of spikes fired by Retzius cells. The combined use of intracellular iontophoretic injection of horseradish peroxidase and lucifer yellow indicated the possible sites of contact between Rz and T cells. The effect of serotonin, released by Rz cells, is discussed with respect to its possible physiological significance.

Endogenous morphine modulates acute thermonociception in mice

The endogenous synthesis of morphine has been clearly demonstrated throughout the phylogenesis of the nervous system of mammals and lower animals. Endogenous morphine, serving as either a neurotransmitter or neurohormone, has been demonstrated in the nervous system of both verteb‐rates and invertebrates. As one of the effects of exogenous morphine is the modulation of pain perception, we investigated the effects that the depletion of endogenous morphine had on nociceptive transmission. The immunoneutralization of endogenous morphine from brain extracellular spaces was obtained through the intracerebroventricular administration of affinity purified anti‐morphine IgG to mice, which then underwent the hot plate test. Endogenous morphine immunoneutralization decreased thermal response latency and attenuated the anti‐nociceptive effect of the mu selective agonist DAMGO in hot plate test suggesting that endogenous morphine is involved in pain modulation.

Antagonizing effect of morphine on the mobility and phagocytic activity of invertebrate immunocytes

In the present study we have demonstrated that lipopolysaccharide (LPS) acts as an activator in the immunocytes of molluscs Planorbarius corneus, Mytilus edulis, and the insect Leucophaea maderae. This stimulatory effect, demonstrated by cellular conformational changes, is concentration- and time-dependent, and is antagonized by morphine. The inhibitory effect of morphine can be counteracted by naloxone. Morphine inhibitory action on immunocyte activity is also demonstrated by a decrease in the phagocytic activity. These data suggest that the downregulation of morphine is not limited to vertebrates but is also present in invertebrates.

Presence and role of nitric oxide in the central nervous system of the freshwater snail Planorbarius corneus: possible implication in neuron-microglia communication.

The aim of the present study was to investigate the involvement of nitric oxide (NO) as a messenger molecule in neuron-microglia communication in the central nervous system (CNS) of the freshwater snail Planorbarius corneus. The presence of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) was studied using NADPH-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry. The experiments were performed on whole ganglia and cultured microglial cells after different activation modalities, such as treatment with lipopolysaccharide and adenosine triphosphate and/or maintaining ganglia in culture medium till 7 days. In sections, nNOS immunoreactivity was found only in neurons and nNOS-positive elements were less numerous than NADPH-d-positive ones, with which they partially overlapped. The iNOS immunoreactivity was observed only after activation, in both nerve and microglial cells. We also found that the number of iNOS-immunoreactive neurons and microglia varied, depending on the activation modalities. In microglial cell cultures, iNOS was expressed in the first generation of cells only after activation, whereas a second generation, proliferated after ganglia activation, expressed iNOS even in the unstimulated condition.

Endoreplication: a molecular trick during animal neuron evolution.

The occurrence of endoreplication has been repeatedly reported in many organisms, including protists, plants, worms, arthropods, molluscs, fishes, and mammals. As a general rule, cells possessing endoreplicated genomes are largesized and highly metabolically active. Endoreplication has not been frequently reported in neuronal cells that are typically considered to be fully differentiated and nondividing, and which normally contain a diploid genome. Despite this general statement, various papers indicate that giant neurons in molluscs, as well as supramedullary and hypothalamic magnocellular neurons in fishes, contain DNA amounts larger than 2C. In order to study this issue in greater detail here, we review the available data about endoreplication in invertebrate and vertebrate neurons, and discuss its possible functional significance. As a whole, endoreplication seems to be a sort of molecular trick used by neurons in response to the high functional demands that they experience during evolution.

MDL 12330A inhibits the non-neuronal adenylyl cyclase from the freshwater snail Planorbarius corneus, but the neuronal enzyme is activated by this compound

N-(Cis-2-phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL 12330A), considered an inhibitor of adenylyl cyclase, has been tested on the enzyme activity of neuronal and non-neuronal tissues from the freshwater snail Planorbarius corneus. The drug dose-dependently activates the basal as well as agonist-stimulated adenylyl cyclase in the ganglionic preparations, while it exerts an inhibitory effect on the enzyme present in the non-nervous tissues examined. SQ 22536 and forskolin, respectively an inhibitor and activator of adenylyl cyclase, behave as generally reported both in central and peripheral tissues of the snail. This is, to our knowledge, the first report of a stimulatory action of MDL 12330A on an adenylyl cyclase system.

Effect of serotonin and neuropeptides on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus

The effect of serotonin, FMRFamide and the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. The amine and the cardioactive peptide stimulated the enzyme, although with different potencies, in all tissues studied and, when tested in combination, an additive activation was obtained. FMRFamide induced differential effects in the various targets: marked stimulation of adenylate cyclase, additive to that provoked by serotonin or SCPB, in salivary glands; inhibition of the enzyme, both alone and in combination with the other neuromediators, in the nervous tissue; whereas no influence was found in adenylate cyclase activity in the buccal mass. In the last of these tissues, the peptide might act through an intracellular second messenger other than cyclic AMP. The responsiveness of adenylate cyclase to these neuromediators in all the central ganglia suggested that they can exert an important role as neurotransmitters and/or neuromodulators in the central nervous system of the snail. Moreover, in the light of the differential sensitivity of adenylate cyclase in the salivary glands and buccal mass, we suggest that serotonin, FMRFamide and SCPB modulate the feeding behaviour of P. corneus in a complex way.