Sandra Ceccatelli

Coexistence of peptides with classical neurotransmitters

In the present article the fact is emphasized that neuropeptides often are located in the same neurons as classical transmitters such as acetylcholine, 5-hydroxy-tryptamine, catecholamines, γ-aminobutyric acid (GABA) etc. This raises the possibility that neurons produce, store and release more than the one messenger molecule. The exact functional role of such coesisting peptides is often difficult to evaluate, especially in the central nervous system. In the periphery some studies indicate apparently meaningful interactions of different types with the classical transmitter, but other types of actions including trophic effects have been observed. More recently it has been shown that some neurons contain more than one classical transmitter, e.g. 5-HT plus GABA, further underlining the view that transfer of information across synapses may be more compex than perhaps hitherto assumed.

Neurotoxicity and molecular effects of methylmercury.

The neurotoxicity of high levels of methylmercury (MeHg) and the high susceptibility of the developing brain are well established both in humans and experimental animals. Prenatally poisoned children display a range of effects varying from severe cerebral palsy to subtle developmental delays. Still unknown is the lowest dose that impairs neurodevelopment. The primary source of human exposure is the fish. The data obtained so far from epidemiological studies on fish-eating populations are not consistent. A reference dose of 0.1 microg MeHg/kg per day has been established by the U.S. Environmental Protection Agency based on a study on Iraqi children exposed to MeHg in utero. However, these exposures occurred at high level for a limited period of time, and consequently were not typical of lower chronic exposure levels associated with fish consumption. Major obstacles for estimation of a threshold dose for MeHg include the delayed appearance of the neurodevelopmental effects following prenatal exposure and limited knowledge of cellular and molecular processes underlying these neurological changes. In this respect, a strategy which aims at identifying sensitive molecular targets of MeHg at environmentally relevant levels may prove particularly useful to risk assessment. Here some examples of MeHg molecular effects occurring at low doses/concentrations are presented.

Prenatal Dexamethasone Causes Oligonephronia, Sodium Retention, and Higher Blood Pressure in the Offspring

Recent reports have shown that low birth weight infants have a higher incidence of adult hypertension. These observations have stimulated a number of studies designed to evaluate the mechanisms of this phenomenon. In this study, fetal growth retardation was induced by treating pregnant rats with dexamethasone. After birth, pups whose mothers were treated with dexamethasone had a lower body and kidney weight and a lower number of glomeruli than control pups. Immunohistochemistry on treated kidneys demonstrated a marked reduction in the number of cells undergoing mitosis in the cortical nephrogenic zone. In the treated group, body and kidney weight normalized by 60 d of age, but blood pressure was significantly higher compared with controls (130 ± 4 versus 107 ± 1 mm Hg). In addition, GFR was significantly lower, albuminuria was higher, urinary sodium excretion rate and fractional sodium excretion were lower, and sodium tissue content was higher. In contrast, when pregnant rats were treated with a natural glucocorticoid (hydrocortisone) which is metabolized by the placenta, fetal development and adult blood pressure were normal. In conclusion, we found that high levels of maternal glucocorticoids impair renal development and lead to arterial hypertension in offspring. Even though renal mass eventually normalizes, glomerular damage as well as sodium retention occur and these factors may contribute to the development of hypertension.

Methylmercury-induced neurotoxicity and apoptosis

Methylmercury is a widely distributed environmental toxicant with detrimental effects on the developing and adult nervous system. Due to its accumulation in the food chain, chronic exposure to methylmercury via consumption of fish and sea mammals is still a major concern for human health, especially developmental exposure that may lead to neurological alterations, including cognitive and motor dysfunctions. Mercury-induced neurotoxicity and the identification of the underlying mechanisms has been a main focus of research in the neurotoxicology field. Three major mechanisms have been identified as critical in methylmercury-induced cell damage including (i) disruption of calcium homeostasis, (ii) induction of oxidative stress via overproduction of reactive oxygen species or reduction of antioxidative defenses and (iii) interactions with sulfhydryl groups. In vivo and in vitro studies have provided solid evidence for the occurrence of neural cell death, as well as cytoarchitectural alterations in the nervous system after exposure to methylmercury. Signaling cascades leading to cell death induced by methylmercury involve the release of mitochondrial factors, such as cytochrome c and AIF with subsequent caspase-dependent or -independent apoptosis, respectively; induction of calcium-dependent proteases calpains; interaction with lysosomes leading to release of cathepsins. Interestingly, several pathways can be activated in parallel, depending on the cell type. In this paper, we provide an overview of recent findings on methylmercury-induced neurotoxicity and cell death pathways that have been described in neural and endocrine cell systems.

Neurotransmitters, neuropeptides and binding sites in the rat mediobasal hypothalamus: effects of monosodium glutamate (MSG) lesions

SummaryIndirect immunofluorescence histochemistry and receptor autoradiography were used to study the localization of transmitter-/peptidecontaining neurons and peptide binding sites in the mediobasal hypothalamus in normal rats and in rats treated neonatally with repeated doses of the neurotoxin monosodium-glutamate (MSG). In the arcuate nucleus, the results showed a virtually complete loss of cell bodies containing immunoreactivity for growth hormone-releasing factor (GRF), galanin (GAL), dynorphin (DYN), enkephalin (ENK), corticotropin-like intermediate peptide (CLIP), neuropeptide Y (NPY), and neuropeptide K (NPK). Tyrosine hydroxylase(TH)-, glutamic acid decarboxylase(GAD)-, neurotensin(NT)- and somatostatin(SOM)-immunoreactive (IR) cells were, however, always detected in the ventrally dislocated, dorsomedial division of the arcuate nucleus. In the median eminence, marked decreases in numbers of GAD-, NT-, GAL-, GRF-, DYN-and ENK-IR fibers were observed. The numbers of TH-, SOM-and NPY-IR fibers were in contrast not or only affected to a very small extent, as revealed with the immunofluorescence technique. Biochemical analysis showed a tendency for MSG to reduce dopamine levels in the median eminence of female rats, whereas no effect was observed in male rats. Autoradiographic studies showed high to moderate NT binding sites, including strong binding over presumably dorsomedial dopamine cells. In MSG-treated rats, there was a marked reduction in GAL binding in the ventromedial nucleus. The findings implicate that most neurons in the ventrolateral and ventromedial arcuate nucleus are sensitive to the toxic effects of MSG, whereas a subpopulation of cells in the dorsomedial division of the arcuate nucleus, including dopamine neurons, are not susceptible to MSG-neurotoxicity. The results indicate, moreover that the very dense TH-IR fiber network in the median eminence predominantly arises from the dorsomedial TH-IR arcuate cells, whereas the GAD-, NT-, GAL-, GRF-and DYN-IR fibers in the median eminence to a large extent arise from the ventrolateral arcuate nucleus. Some ENK-and NPK-positive cells in the arcuate nucleus seem to project to the lateral palisade zone of the median eminence, but most of the ENK-IR fibers in the median eminence, located in the medial palisade zone, seem to primarily originate from an area(s) located outside the arcuate nucleus, presumably the paraventricular nucleus. The NPY-positive fibers in the median eminence contain to a large extent immunoreactive dopamine β-hydroxylase (DBH), and do not arise from the ventromedial arcuate nucleus. SOM-IR cells in the dorsal periventricular arcuate nucleus do not send major projections to the median eminence. The present findings thus show that MSG treatment represents a valuable tool to clarify the organization of chemically identified neuron populations in the arcuate nucleus-median eminence complex and provide further information for understanding the neuroendocrine effects of neonatal MSG treatment.

Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism

It is known that steroid hormones can affect neuronal susceptibility to different types of insults, including oxidative stress. Using an in vitro/ex vivo model, we have previously shown that cerebellar granule cells prepared from neonatal rats treated with a single dose of testosterone are less vulnerable to oxidative stress-induced cell death, via a mechanism involving an upregulation of the cellular antioxidant defenses. Whether the testosterone protective action on cerebellar granule cells was direct or indirect remained to be clarified. Therefore, in this study we have investigated the effects of in vitro testosterone treatment, to see whether it also protects cerebellar granule cells from oxidative stress-induced damage. Cerebellar granule cells treated with 10(-6) M testosterone for 48 h were found less susceptible to damage induced by 50 microM hydrogen peroxide, as shown by a 30% decrease in the number of cells with apoptotic morphology. The addition of the androgen receptor antagonist flutamide abolished the protective effect of testosterone, suggesting an androgen receptor-mediated mechanism. This hypothesis was further supported by the presence of the androgen receptor in cultured cerebellar granule cells. The activity of the antioxidant enzyme catalase was also measured, and a 2-fold increase was detected in the testosterone treated cells, but not in the cells co-treated with flutamide. The present results demonstrate that cerebellar granule cells treated in vitro with testosterone are protected from oxidative stress via a mechanism mediated by the androgen receptor. Similarly to what we observed after in vivo administration of testosterone, the potentiation of the antioxidant defences seems to play a major role in the protection afforded by testosterone.

Long-lasting depression-like behavior and epigenetic changes of BDNF gene expression induced by perinatal exposure to methylmercury.

Substantial evidence indicates that predisposition to diseases can be acquired during early stages of development and interactions between environmental and genetic factors may be implicated in the onset of many pathological conditions. Data collected over several decades have shown that chemicals are among the relevant factors that can endanger CNS. We previously showed that perinatal exposure to methylmercury (MeHg) causes persistent changes in learning and motivational behavior in mice. In this study, we report that the depression‐like behavior in MeHg‐exposed male mice is reversed by chronic treatment with the antidepressant fluoxetine. Behavioral alterations are associated with a decrease in brain‐derived neurotrophic factor (BDNF) mRNA in the hippocampal dentate gyrus and fluoxetine treatment restores BDNF mRNA expression. We also show that MeHg‐exposure induces long‐lasting repressive state of the chromatin structure at the BDNF promoter region, in particular DNA hypermethylation, an increase in histone H3‐K27 tri‐methylation and a decrease in H3 acetylation at the promoter IV. While fluoxetine treatment does not alter hypermethylation of H3‐K27, it significantly up‐regulates H3 acetylation at the BDNF promoter IV in MeHg‐exposed mice. Our study shows that developmental exposure to low levels of MeHg predisposes mice to depression and induces epigenetic suppression of BDNF gene expression in the hippocampus.

Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: An immunohistochemical study using experimental manipulations

Indirect immunofluorescence histochemistry was used to investigate the distribution and extent of co-localization of chemical messengers in magnocellular neurons of the supraoptic and paraventricular nuclei. In order to increase the number of neurons immunoreactive to the antisera used, experimental manipulations were employed. The homozygous Brattleboro (diabetes insipidus) rat was also investigated. In untreated rats, only vasopressin- and oxytocin-like immunoreactivities could be observed. Colchicine treatment alone resulted in appearance of galanin-, dynorphin-, cholecystokinin-, [Leu]enkephalin- and thyrotropin-releasing hormone-positive cells. In hypophysectomized rats, all these markers, except tyrosine hydroxylase, showed substantial further increases. In addition, peptide histidine-isoleucine-immunoreactive cell bodies could now be seen. After salt-loading alone, tyrosine hydroxylase-like immunoreactivity was markedly increased, whereas vasopressin- and oxytocin-like immunoreactivity were very weak or undetectable. When salt-loaded rats received colchicine, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity in addition increased, whereas galanin- and dynorphin-like immunoreactivity markedly decreased. The Brattleboro rats resembled untreated rats, except their lack of vasopressin-like immunoreactivity, the marked increase in tyrosine hydroxylase-like immunoreactivity, and smaller increase in galanin- and dynorphin-like immunoreactivity. Addition of colchicine to Brattleboro rats resulted in some distinct further changes in that dynorphin-like immunoreactivity decreased in some neurons and that [Leu]enkephalin-, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity increased substantially. Several similarities could be observed between the salt-loaded and Brattleboro rats, with or without colchicine. However, a marked difference in immunoreactive [Leu]enkephalin levels was observed with no difference in dynorphin-like immunoreactivity, and opposite changes in galanin-like immunoreactivity. The results confirm the traditional view that hypothalamic magnocellular neurons in the supraoptic and paraventricular nuclei contain two separate cell populations, characterized by vasopressin and oxytocin, respectively, and that they contain additional messenger molecules in specific patterns. Vasopressin-containing neurons primarily express tyrosine hydroxylase, galanin, dynorphin, [Leu]enkephalin and peptide histidine-isoleucine, and to a minor extent cholecystokinin and thyrotropin-releasing hormone. Oxytocin-containing neurons mainly have cholecystokinin and corticotropin-releasing factor, and to a minor extent galanin, dynorphin, [Leu]enkephalin and thyrotropin-releasing hormone. Furthermore, our results detail individual co-existence situations among these putative messenger molecules. Thus, magnocellular neurons respond in a differential way to various stimuli and they store multiple bioactive substances in specific combinations.

Distribution and Coexistence of Corticotropin-Releasing Factor-, Neurotensin-, Enkephalin-, Cholecystokinin-, Galanin- and Vasoactive Intestinal Polypeptide/Peptide Histidine Isoleucine-Like Peptides in the Parvocellular Part of the Paraventricular Nucleus

The distribution of several peptides, corticotropin-releasing factor (CRF), neurotensin (NT), enkephalin (ENK), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), cholecystokinin (CCK), thyrotropin-releasing hormone (TRH) and galanin (GAL) was studied in detail with immunohistochemistry in the various subdivisions of the parvocellular part of the rat hypothalamic paraventricular nucleus (PVN). Using a double-staining method and elution-restaining technique, the coexistence of CRF- and NT-like immunoreactivities (LI) with other neuropeptides was analyzed. Our results indicate that coexistence of two or more peptides in the PVN is common, and revealed that about 30% of the CRF neurons contain NT-LI and about 20% ENK-LI, whereas other peptides only occur in small fractions of the CRF cells. Thus, it seems possible to define three major subpopulations of CRF neurons, one containing NT-LI, another one containing ENK-LI and a third one apparently lacking these peptides. Conversely, about 60% of both NT- and ENK-immunoreactive neurons lacked CRF-LI. A large proportion of the small population of VIP/PHI neurons contained NT-LI. TRH neurons represented a neuron population completely distinct from the CRF neurons. Also, it did not seem to contain any of the other peptides studied with the rare exception of ENK-LI. Neuropeptides present in the PVN and presumably in nerve fibers of the external layer of the median eminence may participate in the control of the anterior pituitary hormone secretion. Whereas the role of CRF and TRH is well established, the physiological role of the other peptides studied here is still unclear.

Immunohistochemical evidence for a spinothalamic pathway co-containing cholecystokinin- and galanin-like immunoreactivities in the rat.

Using indirect immunofluorescence technique combined with retrograde tracing as well as surgical lesions, a system of spinothalamic neurons containing both galanin- and cholecystokinin-like immunoreactivity has been defined. The cell bodies are located in the lumbar segments L1-L5 with a preferential localization dorsal to the central canal at rostral levels and lateral to the canal at caudal levels. The cells project via the ventral part of the lateral funiculus to the most ventral and posterior parts of thalamus. Here a distinct, varicose terminal network was seen extending caudally from an area lateral to the medial lemniscus, running medially over the medial lemniscus, traversing the parafascicular nucleus and running dorsal to the fasciculus retroflexus into the periventricular gray matter. Transection of various parts of the spinal cord as well as retrograde tracing experiments indicate that the spinothalamic galanin cholecystokinin system represents a crossed pathway. The present results demonstrate that a spinothalamic system can be characterized by its content of galanin- and cholecystokinin-like peptides, two putative messenger molecules. It is only a minor component of the total spinothalamic projection.