Cristina Cocco

Oxytocin injected into the ventral tegmental area induces penile erection and increases extracellular dopamine in the nucleus accumbens and paraventricular nucleus of the hypothalamus of male rats.

The neuropeptide oxytocin (20–100 ng), induces penile erection when injected unilaterally into the caudal but not rostral mesencephalic ventral tegmental area (VTA) of male Sprague–Dawley rats. Such pro‐erectile effect started 30 min after treatment and was abolished by the prior injection of d(CH2)5Tyr(Me)2‐Orn8‐vasotocin (1 µg), an oxytocin receptor antagonist injected into the same caudal ventral tegmental area or of haloperidol (1 µg), a dopamine receptor antagonist, injected either into the nucleus accumbens shell (NAs) or into the paraventricular nucleus of the hypothalamus (PVN) ipsilateral to the injected ventral tegmental area. Penile erection was seen 15 min after the occurrence of, or concomitantly to, an increase in extracellular dopamine and its metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC) in the dialysate obtained from the nucleus accumbens or the paraventricular nucleus, which was also abolished by d(CH2)5Tyr(Me)2‐Orn8‐vasotocin (1 µg), injected into the ventral tegmental area before oxytocin. In the caudal ventral tegmental area oxytocin‐containing axons/fibres (originating from the paraventricular nucleus) appeared to closely contact cell bodies of mesolimbic dopaminergic neurons retrogradely labelled with Fluorogold injected into the nucleus accumbens shell, suggesting that oxytocin effects are mediated by the activation of mesolimbic dopaminergic neurons, followed in turn by that of incerto‐hypothalamic dopaminergic neurons impinging on oxytocinergic neurons mediating penile erection. As the stimulation of paraventricular dopamine receptors not only induces penile erection, but also increases mesolimbic dopamine neurotransmission by activating oxytocinergic neurons, these results provide further support for the existence of a neural circuit in which dopamine and oxytocin influence both the consummatory and motivational/rewarding aspects of sexual behaviour.

Oxytocin induces penile erection when injected into the ventral tegmental area of male rats: role of nitric oxide and cyclic GMP

Oxytocin (80 ng) injected into the caudal mesencephalic ventral tegmental area (VTA) of male rats induces penile erection. Such an effect occurs together with an increase in nitric oxide (NO) production, as measured by the augmented concentration of NO2− and NO3− found in the dialysate obtained from this brain area by means of intracerebral microdialysis. Both effects are abolished by d(CH2)5Tyr(Me)2‐Orn8‐vasotocin (1 μg), an oxytocin receptor antagonist, by S‐methyl‐l‐thiocitrulline acetate (20 μg), a neuronal NO synthase inhibitor, or by ω‐conotoxin GVIA (50 ng), a N‐type Ca2+ channel blocker, all injected into the VTA 15 min before oxytocin. In contrast, 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (40 μg), a guanylate cyclase inhibitor, given into the VTA 15 min before oxytocin, abolishes penile erection, but not the increase in NO production, while haemoglobin (40 μg), a NO scavenger, injected immediately before oxytocin reduces the increase in NO production, but not penile erection. 8‐Bromo‐cyclic guanosine monophosphate (0.5–10 μg) microinjected into the VTA induces penile erection with an inverted U‐shaped dose–response curve; the maximal effective dose being 3 μg. Immunohistochemistry reveals that in the caudal VTA oxytocin‐containing axons/fibres (originating from the paraventricular nucleus of the hypothalamus) contact cell bodies of mesolimbic dopaminergic (tyrosine hydroxylase‐positive) neurons containing both NO synthase and guanylate cyclase. These results suggest that oxytocin injected into the VTA induces penile erection by activating NO synthase in the cell bodies of mesolimbic dopaminergic neurons. NO in turn activates guanylate cyclase present in these neurons, thereby increasing cyclic GMP concentration.

Pro-VGF-derived peptides induce penile erection in male rats: possible involvement of oxytocin

The effect of five peptides derived from the C‐terminal portion of rat pro‐VGF (VGF577‐617, VGF588‐617, VGF599‐617, VGF556‐576 and VGF588‐597) on penile erection was studied after injection into the hypothalamic paraventricular nucleus of male rats. VGF577‐617, VGF588‐617, VGF599‐617 and, to a lower extent, VGF588‐597 (0.1–2 µg) induced penile erection episodes in a dose‐dependent manner when injected into the paraventricular nucleus, while VGF556‐576 was ineffective. VGF588‐617‐induced penile erection was reduced by nitroω‐l‐arginine methylester (L‐NAME; 20 µg), by morphine (5 µg) and by muscimol (1 µg), but not by dizocilpine [(+)MK‐801; 1 µg], nor by cis‐flupenthixol (10 µg) given into the paraventricular nucleus 10 min before the VGF peptide. d(CH2)5Tyr(Me)‐Orn8‐vasotocin (1 µg) effectively reduced VGF588‐617‐induced penile erection when given into the lateral ventricles but not when injected into the paraventricular nucleus. Immunocytochemistry with antibodies specific for the C‐terminal nonapeptide sequence of pro‐VGF (VGF609‐617) revealed numerous neuronal fibres and terminals within the paraventricular nucleus, including its parvocellular components. Here, many immunostained neuronal terminals impinged on parvocellular oxytocinergic neurons. The present results show for the first time that certain pro‐VGF C‐terminus‐derived peptides promote penile erection when injected into the paraventricular nucleus and suggest that, within this nucleus, these or closely related pro‐VGF‐derived peptides may be released to influence sexual function by activating paraventricular oxytocinergic neurons mediating penile erection.

Embedding media for cryomicrotomy: an applicative reappraisal.

We reassess here the formulation of cryoembedding media in connection with recent developments in commercial cryomicrotomes. Water-based solutions of polyvinyl alcohols were our starting media, and each of 2 different polymers (56–98, MW ∼195000; and 6–98, MW ∼47000) showed a critical concentration for optimum sectioning. At higher or lower polymer concentrations, wrinkles and folds became apparent in tissue areas of sections, or in the sectioned embedding medium areas between tissues, respectively. Addition of polyethylene glycol (MW 380–420) further facilitated and improved sectioning, resulting in frozen tissue blocks that cut well in the 2 to 100 &mgr;m range and further, using disposable blades throughout. Applying a wide temperature differential between tissue specimen (−11°C to −13°C) and cutting knife (−33°C to −35°C), serial adjacent sections were reproducibly obtained at a 2-&mgr;m setting, singly or in short ribbons. Embedding media of low and high viscosity were obtained, depending on the polyvinyl alcohol polymer used, and could be applied sequentially for tissue infiltration followed by embedding with precise sample orientation. When required, media were made semisolid by addition of carboxymethylcellulose.

Distribution of VGF peptides in the human cortex and their selective changes in Parkinson's and Alzheimer's diseases

VGF mRNA and its precursor‐derived products are selectively expressed in certain neurons and promptly respond to neurotrophins and to neural/electrical activity. Proteomic studies have previously revealed a reduction in some VGF peptides in the cerebrospinal fluid of patients affected by Alzheimer’s disease and other conditions, suggesting their potential diagnostic and clinical significance. As the presence of VGF peptides within the human cortex has been somewhat elucidated, they were studied postmortem in the frontal, parietal, and temporal cortex areas of control subjects and patients affected by Parkinson’s disease, and in parietal cortex samples from patients with Alzheimer’s disease. We raised antibodies to the C‐/N‐terminal portions of the proVGF precursor protein, to the TPGH and TLQP sequences and to the neuroendocrine regulatory peptide (NERP)‐1, all used for enzyme‐linked immunosorbent assay coupled with gel chromatography and for immunohistochemistry. In the control brain samples, the levels of TPGH and C‐terminus peptides were about 130–200 and 700–2000 pmol g−1, respectively, the N‐terminus and NERP‐1 peptides were less represented (about 10–30 and 4–20 pmol g−1, respectively), and the TLQP peptides were below detection limits. Upon gel chromatography, the VGF antisera mainly revealed small molecular weight forms (i.e. about 0.8–1.3 kDa), whereas VGF immunolocalisation was found within different types of neuron in rat and bovine brain cortices. In the Parkinson’s disease samples, a clear‐cut decrease was revealed in the parietal cortex only, exclusively for TPGH and NERP‐1 peptides, whereas in the Alzheimer’s disease samples, a reduction in all of the VGF peptides was shown. The results suggest the involvement of VGF in the physiological or pathophysiological mechanisms occurring in the parietal cortex of patients with Parkinson’s and Alzheimer’s diseases.

VGF Metabolic-related Gene: Distribution of Its Derived Peptides in Mammalian Pancreatic Islets

The vgf gene has been shown to be involved in several metabolic pathways. Because the pancreas is crucial to metabolism and food intake, we studied the VGF peptides in bovine, rat, and pig Langherans islets using antisera raised against specific sites along the primary sequence of the rat/mouse and human VGF protein precursor. Whereas almost all of the pancreatic endocrine cells expressed vgf mRNA, when using the VGF antisera a different staining pattern became apparent. VGF556–565 and VGF282–291 immunoreactivity were exclusively found in δ somatostatin-producing cells, whereas the human C-terminus antiserum selectively immunolabeled α glucagon and pancreatic polypeptide cells. The same cells were decorated with the VGF443–588 antiserum, which also weakly labeled β insulin-secreting cells. Finally, the VGF298–306 peptide and the rat C terminus were found in virtually all pancreatic endocrine cells. Using bovine, swine, and rat pancreatic extracts, data from chromatography and ELISA assay showed the presence of a high molecular mass form compatible with the proVGF and lower molecular mass fractions corresponding to short VGF peptides. In conclusion, selective VGF distribution may suggest a multifaceted cell type-specific processing of proVGF, resulting indifferent peptides probably involvedin neuroendocrine regulatory metabolic mechanisms.

Differential distribution of VGF-derived peptides in the adrenal medulla and evidence for their selective modulation

While vg f gene knockout mice are hyperactive and hypermetabolic, surprisingly the TLQP-21 brain VGF peptide increased energy consumption, suggesting that opposing regulatory effects could be exerted by peptides alternatively cleaved from the VGF precursor. Using antisera to the VGF precursor C-terminus and three cleavage products, we revealed a distinct differential distribution in adrenal, certain peptides (VGF(422-430): PGH peptides) being found throughout bovine and swine medulla, while C-terminus and TLQP peptides were confined to adrenaline cells in the above species and in rat and C-terminally shortened forms (VGF(604-612): HVLL peptides) to nor-adrenaline cells. Random abattoir samples of bovine and swine adrenal contained 520+/-40 and 450+/-60 pmol/g (mean+/-s.e.m. respectively) of C-terminus peptides and similar or lower amounts of others. Upon gel chromatography, bona fide VGF precursor, approximately 7.5 and approximately 3.5 kDa forms were revealed by C-terminus assays, HVLL peptides being limited to small fragments. TLQP peptides included ~7.5 kDa form and peaks accounting for TLQP-21 and predicted TLQP-30 and TLQP-42. Low molecular weight (MW) PGH peptides were revealed, together with a high MW form possibly encompassing the VGF precursor N-terminus. In acutely stressed swine, a striking increase was seen for C-terminus and TLQP peptides, with no significant differences for PGH peptides. A similar response was found in rat TLQP peptides showing a major increase upon an acute swimming stress and 30 min thereafter. A differential processing of the VGF precursor encompassing many areas of its primary sequence and selective modulations of its derived peptides occur in adrenal medullary cells, possibly relevant to adaptive homeostatic responses.

Selective expression of TLQP-21 and other VGF peptides in gastric neuroendocrine cells and modulation by feeding.

Although vgf gene knockout mice are hypermetabolic, administration of the VGF peptide TLQP-21 itself increased energy consumption. Agonist-antagonist roles are thus suggested for different VGF peptides, and the definition of their tissue heterogeneity is mandatory. We studied the rat stomach using antisera to C- or N-terminal sequences of known or predicted VGF peptides in immunohistochemistry and ELISA. TLQP (rat VGF(556-565)) peptide/s were most abundant (162±11 pmol/g, mean±s.e.m.) and were brightly immunostained in enterochromaffin-like (ECL) cells and somatostatin cells. A peptide co-eluting with TLQP-21 was revealed in HPLC of gastric and hypothalamic extracts, while the extended TLQP-62 form was restricted to the hypothalamus. Novel PGH (rat VGF(422-430)) peptide/s were revealed in ghrelin cells, mostly corresponding to low MW forms (0.8-1.5  kDa), while VGF C-terminus peptides were confined to neurons. VGF mRNA was present in the above gastric endocrine cell types, and was prominent in chief cells, in parallel with low-intensity staining for further cleaved products from the C-terminal region of VGF (HVLL peptides: VGF(605-614)). In swine stomach, a comparable profile of VGF peptides was revealed by immunohistochemistry. When fed and fasted rats were studied, a clear-cut, selective decrease on fasting was observed for TLQP peptides only (162±11 vs 74±5.3  pmol/g, fed versus fasted rats, mean±s.e.m., P<0.00001). In conclusion, specific VGF peptides appear to be widely represented in different gastric endocrine and other mucosal cell populations. The selective modulation of TLQP peptides suggests their involvement in peripheral neuro-endocrine mechanisms related to feeding responses and/or ECL cell regulation.

Oxytocin induces penile erection when injected into the ventral subiculum: Role of nitric oxide and glutamic acid

Oxytocin (100 ng) induces penile erection when injected unilaterally into the ventral subiculum of the hippocampus of male rats. The pro-erectile effect started mostly 30 min after treatment and occurred 15 min after an increase in both nitric oxide (NO) production, measured by the concentration of NO(2)(-) and NO(3)(-), the main metabolites of newly formed NO, and extra-cellular glutamic acid concentration in the dialysate obtained from the ventral subiculum by intracerebral microdialysis. These responses were abolished by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin (2 microg), an oxytocin receptor antagonist, S-methyl-L-thiocitrulline (SMTC), a selective inhibitor of neuronal NO-synthase (25 microg), and haemoglobin, a NO scavenger (25 microg), given into the ventral subiculum before oxytocin. Unlike d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin, SMTC and haemoglobin, (+)MK-801 (5 microg), a noncompetitive antagonist of NMDA receptors abolished oxytocin-induced penile erection, but reduced only partially the increase in NO production and extra-cellular glutamic acid. As NMDA (0.25-1 microg) injected into the ventral subiculum induces penile erection episodes, which also occurred with an increase of NO production and extra-cellular glutamic acid, and NMDA responses were abolished by (+)MK-801 (5 microg), but not by SMTC (25 microg) or haemoglobin (25 microg), injected into the ventral subiculum, these results show that oxytocin injected into the ventral subiculum increases NO production by activating its own receptors. NO in turn increases glutamic acid neurotransmission, leading to penile erection, possibly through neural (glutamatergic) efferent projections from the ventral subiculum to extra-hippocampal brain areas (e.g., prefrontal cortex) modulating the activity of mesolimbic dopaminergic neurons.

VGF peptides upon osmotic stimuli: Changes in neuroendocrine regulatory peptides 1 and 2 in the hypothalamic–pituitary-axis and plasma

VGF or VGF nerve growth factor inducible is a protein that has been found to play a role in regulating energy homeostasis and metabolism. From VGF precursor derive two neuroendocrine regulatory peptides NERP-1 and NERP-2 that, intracerebroventricular (icv) injected, modulate the antidiuretic hormone (ADH) release. Thus, we investigated possible modulations of the NERPs and other VGF peptides (namely VGF C-terminus, TLQP and PGH) in the hypothalamic-pituitary-axis, adrenal gland and plasma upon osmotic stimuli. The latter tissues were studied using water deprived (WD), salt loaded (SL), rehydrated after salt cargo and control rats by immunohistochemistry and immunoenzymatic assays. The high-performance liquid chromatography ensured the endogenous presence of the two NERPs in both plasma and hypothalamus. Upon dehydration, NERP-1 levels increased in the median eminence (M.E.) only, while using SL rats, the values of both NERPs increased in the M.E. and even in the hypothalamus. Conversely, in the blood of WD and SL rats, the levels of NERP-1 and NERP-2 decreased while, using pituitary from both rat groups, levels of NERP-2 increased and those of NERP-1 decreased. Reduction in the VGF C-terminus peptide levels was observed exclusively in the M.E. (using WD rats) and pituitary (using WD and SL rats), while PGH and TLQP peptide levels never changed in all tissues tested. By immunohistochemistry, the VGF peptides studied (apart from the TLQP peptides) were present in the hypothalamic and pituitary ADH containing neurons of the control rats, while using WD and SL rats, an immunostaining increase was selectively revealed for VGF C-terminus peptides in the hypothalamic neurons that produce ADH. All VGF changes found using SL rats disappeared after only 1h of rehydration. In conclusion, we hypothesize that NERPs may be involved in both autocrine and endocrine mechanisms important for the fluid balance.