Mónica Silvina Sanchez

Melanin-Concentrating Hormone (MCH) Antagonizes the Effects of α-MSH and Neuropeptide E-I on Grooming and Locomotor Activities in the Rat

The intraventricular (i.c.v.) administration of the neuropeptide melanocyte stimulating hormone (alpha-MSH) is known to elicit a series of behaviors in the rat which include excessive grooming and other motor activities. In bony fish, the pigmentary effects of alpha-MSH can be antagonized by the neuropeptide melanin-concentrating hormone (MCH). We therefore examined whether MCH or its sister peptide neuro-peptide E-I (NEI), derived from the same precursor molecule, would modulate the effect of alpha-MSH on grooming and motor activity in the rat, or perhaps elicit some responses of their own. Rats were injected i.c.v. with either artificial cerebrospinal fluid, alpha-MSH, MCH, NEI, or with two peptides together, and behavioral responses were monitored over the next 65 min. The i.c.v. injection of 1 microgram MSH significantly enhanced grooming behavior. NEI at the same dose increased grooming, rearing, and locomotor activities. MCH alone had no behavioral effects but it annulled the behavioral responses induced by either alpha-MSH or NEI. alpha-MSH also antagonized the locomotor and rearing behavior induced by NEI. The physiological significance of these observations is discussed.

Correlation of Increased Grooming Behavior and Motor Activity with Alterations in Nigrostriatal and Mesolimbic Catecholamines After α-Melanotropin and Neuropeptide Glutamine–Isoleucine Injection in the Rat Ventral Tegmental Area

Abstract1. We wished to further study the behavioral effects of α-melanotropin (α-MSH), melanin-concentrating hormone (MCH), and neuropeptide glutamine–isoleucine (NEI).2. To this effect we administered α-MSH, MCH, and NEI in the ventral tegmental area of the rat, a structure where these neuropeptides are highly concentrated. To further elucidate the biochemical mechanisms of the behavioral effect of these neuropeptides, we determined the degree of grooming behavior and the levels of catecholamines, after neuropeptide administration.3. We preselected those animals responding to the central injection of α-MSH with excessive grooming behavior. We administered the neuropeptides at the dose of 1 μg/0.5 μL, in each side of the ventral tegmental area, bilaterally. We studied grooming behavior, locomotor activity, and total behavior scores, 30 and 65 min after administration of the peptides.4. Three groups of animals were decapitated immediately after the injection of the neuropeptides, and 30 or 65 min after injection. We measured dopamine (DA), noradrenaline (NA), and the dopac/dopamine ratio (DOPAC/DA) to determine steady state levels of catecholamines and an indirect measure of DA release and metabolism, respectively.5. Injections of α-MSH produced significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with significant decreases in DA content, increases in DOPAC content, and increases in the DOPAC/DA ratio. In the caudate putamen, changes in catecholamines occurred both at 30 and 65 min after injection. In the nucleus accumbens, changes were present at 65 min after injection. Conversely, there were no alterations in NA content, either in the caudate putamen or in the nucleus accumbens, at any time after the injection.6. Injections of NEI resulted in significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with increased DOPAC/DA ratio in the nucleus caudatus but not in the nucleus accumbens. Conversely, NEI produced increased NA concentrations in the nucleus accumbens, but not in the nucleus caudatus.7. Injections of MCH did not produce significant changes in behavior or significant changes in nucleus caudatus or nucleus accumbens catecholamines.8. Our results indicate (a) There is a correlation with alterations in behavior as induced for the neuropeptides injected here, and changes in extrapyramidal catecholamines. (b) There is a correlation between alterations in behavior and increases in DOPAC/DA ratio in the nucleus caudatus. (c) There is a correlation between alterations in behavior and alterations in catecholamines in the nucleus accumbens. In the nucleus accumbens, DOPAC/DA ratio is changed after α-MSH, and NA ratio is changed after NEI injection. (d) Absence of alterations in extrapyramidal catecholamines, and in particular in catecholamines in the nucleus accumbens, correlates with absence of behavioral alterations after neuropeptide administration to the ventral tegmental area.9. In conclusion, the behavioral effect of exogenous administration of neuropeptides in the ventral tegmental area is peptide-specific, and is probably associated with alterations in catecholamine metabolism and release in the nucleus caudatus and the nucleus accumbens. Both α-MSH and NEI seem to stimulate the nigrostriatal DA system. While α-MSH appears to stimulate the mesolimbic DA system as well, NEI may exert its actions not through the DA, but through the NA mesolimbic system. The precise contribution of DA and NA, and the relative role of the nucleus caudatus and nucleus accumbens in these behaviors remain to be elucidated.

Effects and interactions between α-MSH and MCH/NEI upon striatal cAMP levels

Abstract It is known that α-MSH augments cAMP levels in rat brain slices containing accumbens and caudate-putamen nuclei. In this study we examined: a) the effect of other neuropeptides: MCH and NEI, on this cyclic nucleotide; b) if the effects of α-MSH on cAMP production can be modulated by addition of MCH or NEI to the incubation medium. Both MCH and NEI (3.6 μM) increased the production of cAMP, whereas at doses of 0.6 μM exerted no effects. When α-MSH 0.6 μM was added with NEI or MCH (0.6 μM), only MCH blocked the increase in the cAMP induced by α-MSH. Neither MCH nor NEI at the highest dose used (3.6 μM) had any additive effect on AMPc when added together with α-MSH. We conclude that, at a high concentration, (MCH/NEI)-like peptides can use the intracellular signal transduction linked to cyclic nucleotides in the CNS.

Participation of the cholinergic system in the excessive grooming behavior induced by neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI).

The neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI) injected into the ventral tegmental area (VTA) or intraventricularly (icv) induces excessive grooming behavior (EGB) and motor activity (MA). Here, we studied whether the cholinergic system is involved in the NEI-induced behavior. The present results demonstrate that atropine, a general muscarinic antagonist, injected icv previous to NEI, suppresses the behavior provoked by icv injections of the peptide, whereas the prior icv injection of dyhidro-β-erythroidine, a general nicotinic antagonist, did not affect the EGB and MA induced by the peptide. From the experimental evidence, it is suggested that NEI may act specifically on a cholinergic afferent to dopaminergic cells. Also, the results appear to indicate that a neural target, different from the dopamine system, may be activated by the peptide to elicit behavioral changes, such as EGB.

α-melanocyte-stimulating hormone modulates lipopolysaccharide plus interferon-γ-induced tumor necrosis factor-α expression but not tumor necrosis factor-α receptor expression in cultured hypothalamic neurons.

In a previous work we showed that the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) exerts anti-inflammatory action through melanocortin 4 receptor (MC4R) in vivo in rat hypothalamus. In this work, we examined the effect of α-MSH on the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and their receptors in primary cultured rat hypothalamic neurons. We also investigated α-MSHs possible mechanism/s of action. α-MSH (5 μM) decreased TNF-α expression induced by 24h administration of a combination of bacterial lipopolysaccharide (LPS, 1 μg/ml) plus interferon-γ (IFN-γ, 50 ng/ml). Expression of TNF-α and IL-1β receptors TNFR1, TNFR2 and IL-1RI, was up-regulated by LPS+IFN-γ whereas α-MSH did not modify basal or LPS+IFN-γ-induced-TNFRs or IL-1RI expression. Both α-MSH and LPS+IFN-γ treatments increased CREB activation. α-MSH did not modify NF-κB activation induced by LPS+IFN-γ in hypothalamic neurons. In conclusion, our data show that α-MSH reduces TNF-α expression in hypothalamic neurons by a mechanism which could be mediated by CREB. The regulation of inflammatory processes in the hypothalamus by α-MSH might help to prevent neurodegeneration resulting from inflammation.

Hypo- and hyperthyroidism affect NEI concentration in discrete brain areas of adult male rats

To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic-pituitary-thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and l-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of l-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis.

Effect of Neuropeptide-EI on the Binding of [3H]SCH 23390 to the Dopamine D1 Receptor in Rat Striatal Membranes

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D1 receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D1 receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-3H] (R)-(+)-8 chloro-2,3,4,5- tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D1 receptor in a concentration-dependent manner.

Structure-activity studies of α-melanotropin fragments on cAMP production in striatal slices

Abstract In this work, we characterized the active site in the α-melanotropin hormone (α-MSH) sequence responsible for the enhancement of cAMP production in incubated striatal slices by using different α-MSH fragments. We also analyzed the effects of the co-incubation of the SCH23390, a dopaminergic D 1 antagonist, with the MSH fragments, to study the involvement of the D 1 receptor on this induction. A rise was observed in the levels of cAMP after addition of the 6 μM fragments MSH (1–10) , and 0.6 and 6 μM MSH (5–13) ; however, the values were lower than those induced by 6 μM α-MSH. On the contrary, the addition of MSH (9–13) , MSH (7–11) , or MSH (6–9) did not affect the cAMP content. The presence of 10 μM SCH23390 blocked the effect of the fragments on cAMP production. We conclude that the biologic activity of α-MSH, as observed through the levels of cAMP, declines when the length of its polypeptide chain is shortened, and that the presence of glutamic acid in the molecule, as well as the core sequence, are of importance for fragments′ activity.

The effect of melanotropic peptides on binding of [3H]dihydroalprenolol-hydrochloride to hypothalamic membranes

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.

Rotenone inhibits axonogenesis via an Lfc/RhoA/ROCK pathway in cultured hippocampal neurons

Rotenone, a broad‐spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone‐induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT‐associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 μM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone‐induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone‐induced MT de‐stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth.