Laura Soverchia

Variation at the rat Crhr1 locus and sensitivity to relapse into alcohol seeking induced by environmental stress

Alcoholism is a chronic relapsing disorder with substantial heritability. Uncovering gene–environment interactions underlying this disease process can aid identification of novel treatment targets. Here, we found a lowered threshold for stress-induced reinstatement of alcohol seeking in Marchigian–Sardinian Preferring (msP) rats genetically selected for high alcohol preference. In situ hybridization for a panel of 20 stress-related genes in 16 brain regions was used to screen for differential gene expression that may underlie this behavioral phenotype. An innate up-regulation of the Crhr1 transcript, encoding the corticotropin-releasing hormone receptor 1 (CRH-R1), was found in several limbic brain areas of msP rats genetically selected for high alcohol preference, was associated with genetic polymorphism of the Crhr1 promoter, and was accompanied by increased CRH-R1 density. A selective CRH-R1 antagonist (antalarmin, 10–20 mg/kg) was devoid of effects on operant alcohol self-administration in unselected Wistar rats but significantly suppressed this behavior in the msP line. Stress-induced reinstatement of alcohol seeking was not significantly affected by antalarmin in Wistar rats but was fully blocked in msP animals. These data demonstrate that Crhr1 genotype and expression interact with environmental stress to reinstate alcohol-seeking behavior.

Genetically selected Marchigian Sardinian alcohol-preferring (msP) rats: an animal model to study the neurobiology of alcoholism.

The present article provides an up‐to‐date review summarizing almost 18 years of research in genetically selected Marchigian Sardinian alcohol‐preferring (msP) rats. The results of this work demonstrate that msP rats have natural preference for ethanol characterized by a spontaneous binge‐type of drinking that leads to pharmacologically significant blood ethanol levels. This rat line is highly vulnerable to relapse and presentation of stimuli predictive of alcohol availability or foot‐shock stress can reinstate extinguished drug‐seeking up to 8 months from the last alcohol experience. The msP rat is highly sensitive to stress, shows an anxious phenotype and has depressive‐like symptoms that recover following ethanol drinking. Interestingly, these animals have an up‐regulated corticotrophin releasing factor (CRF) receptor 1 system. Clinical studies have shown that alcoholic patients often drink ethanol in the attempt to self‐medicate from negative affective states and to search for anxiety relief. We propose that msP rats represent an animal model that largely mimics the human alcoholic population that due to poor ability to engage in stress‐coping strategies drink ethanol as a tension relief strategy and for self‐medication purposes.

Multihormonal control of vitellogenesis in lower vertebrates.

The comparative approach on how and when vitellogenesis occurs in the diverse reproductive strategies displayed by aquatic and terrestrial lower vertebrates is presented in this chapter; moreover, attention has been paid to the multihormonal control of hepatic vitellogenin synthesis as it is related to seasonal changes and to vitellogenin use by growing oocytes. The hormonal mechanisms regulating vitellogenin synthesis are also considered, and the effects of environmental estrogens on the feminization process in wildlife and humans have been reported. It is then considered how fundamental nonmammalian models appear to be, for vitellogenesis research, addressed to clarifying the yolkless egg and the evolution of eutherian viviparity.

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

Background Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. Objectives We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Methods We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. Results Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. Conclusions The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Variation of the genetic expression pattern after exposure to estradiol-17β and 4-nonylphenol in male zebrafish (Danio rerio)

There is much concern about the increasing presence in the environment of synthetic chemicals that are able to disrupt the endocrine system. Among these compounds, 4-nonylphenol (4-NP) is one of the most studied xenoestrogens, due to its widespread accumulation in water sediment and consequent presence in fatty acid of aquatic organisms. Here, we have used a zebrafish microarray representing 16,399 genes to study the effects of 4-NP and estradiol-17beta (E2) in adult male zebrafish in order to elucidate the mechanism of action of 4-NP compared with that of E2. The microarray results showed that both 4-NP and E2 induced a strong expression of vitellogenin (VTG), the sex related precursor of the yolk proteins in oviparous vertebrates. Both treatments induced elevated protein turnover upregulating genes involved in proteolysis and those that are constituents of the ribosome. Many genes regulated by 4-NP and E2 are involved in energy metabolism, oxidative stress defense mechanisms, xenobiotic metabolism, and lipid metabolism. A different pattern of expression in the two treatments was found for genes involved in oxidative stress, since E2 seems to induce the mechanism of detoxification, while 4-NP seems to inhibit this protective mechanism of the cell. Overall, these findings demonstrate that the microarray approach can contribute significantly to the understanding of expression patterns induced by E2 and 4-NP in male zebrafish. The results also demonstrate that 4-NP is able to act through an alternative pattern to that of estradiol-17beta, modulating the expression of the same genes in a different manner.

Perinatal exposure to delta-9-tetrahydrocannabinol causes enduring cognitive deficits associated with alteration of cortical gene expression and neurotransmission in rats.

The aim of the present study was to investigate whether perinatal exposure to a moderate dose of delta‐9‐tetrahydrocannabinol (THC) alters cortical gene expression and neurotransmission, leading to enduring cognitive dysfunctions in rat offspring. To this purpose, rat dams were treated, from gestational day 15 to postnatal day 9, with THC at a daily dose (5 mg/kg, per os) devoid of overt signs of toxicity. THC did not influence reproduction parameters, whereas it caused subtle neurofunctional deficits in the adult offspring. Particularly, perinatal THC induced long‐lasting alterations of cortical genes related to glutamatergic and noradrenergic systems, associated with a decrease in the cortical extracellular levels of both neurotransmitters. These alterations may account, at least in part, for the enduring cognitive impairment displayed by THC‐exposed offspring. Taken together, the present results highlight how exposure to cannabinoids during early stages of brain development can lead to irreversible, subtle dysfunctions in the offspring.

Differential Splicing of Three Gonadotropin-Releasing Hormone Transcripts in the Ovary of Seabream (Sparus aurata)

Abstract Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

Neuropeptide S Receptor Gene Expression in Alcohol Withdrawal and Protracted Abstinence in Postdependent Rats

BACKGROUND Alcoholism is a chronic disease characterized by frequent intoxications followed by withdrawal episodes and relapse to alcohol use. Neuroplastic changes associated with these intoxication and withdrawal cycles are thought to play a key role in disease progression. Recently, it has been shown that neuropeptide S (NPS), a newly deorphanized neuropeptide receptor system, facilitates relapse to alcohol seeking in laboratory animals. Given that a history of ethanol intoxication may increase vulnerability to alcohol addiction, we sought to determine whether NPS receptor (NPSR) gene expression is altered during withdrawal. METHODS Rats were subjected to 1 week of intoxication by oral alcohol administration. NPSR gene expression was analyzed by in situ hybridization in rats 12 hours and 7 days after the last alcohol administration. To investigate the functional significance of NPSR system adaptation following protracted withdrawal 7 days after intoxication, we tested the anxiolytic-like properties of NPS in nondependent and postdependent rats using the shock probe defensive burying test (DB). RESULTS At both time points, increased NPSR gene expression was observed in several brain areas, including the endopiriform nucleus, the motor cortex, and the medial amygdaloid nucleus. Moderate increases in gene expression were also found in the lateral hypothalamus, paraventricular nucleus, basolateral and central amygdala. Differences from control animals were more pronounced after 7 days of abstinence. The upregulation of the NPSR system at this time point was confirmed by functional data indicating that intracerebroventricular (ICV) NPS administration (0.0, 0.3, and 0.1 nmol/rat) elicits more pronounced anxiolytic effects in postdependent animals than in controls subjected to the electric shock probe DB test. CONCLUSIONS Neuropeptide S receptor mRNA expression is increased in different brain areas of postdependent rats; as shown in the DB test, this expression change is functionally relevant.

Chronic THC during adolescence increases the vulnerability to stress-induced relapse to heroin seeking in adult rats

Cannabis derivatives are among the most widely used illicit substances among young people. The addictive potential of delta-9-tetrahydrocannabinol (THC), the major active ingredient of cannabis is well documented in scientific literature. However, the consequence of THC exposure during adolescence on occurrence of addiction for other drugs of abuse later in life is still controversial. To explore this aspect of THC pharmacology, in the present study, we treated adolescent rats from postnatal day (PND) 35 to PND-46 with increasing daily doses of THC (2.5-10mg/kg). One week after intoxication, the rats were tested for anxiety-like behavior in the elevated plus maze (EPM) test. One month later (starting from PND 75), rats were trained to operantly self-administer heroin intravenously. Finally, following extinction phase, reinstatement of lever pressing elicited by the pharmacological stressor, yohimbine (1.25mg/kg) was evaluated. Data revealed that in comparison to controls, animals treated with chronic THC during adolescence showed a higher level of anxiety-like behavior. When tested for heroin (20μg per infusion) self-administration, no significant differences were observed in both the acquisition of operant responding and heroin intake at baseline. Noteworthy, following the extinction phase, administration of yohimbine elicited a significantly higher level of heroin seeking in rats previously exposed to THC. Altogether these findings demonstrate that chronic exposure to THC during adolescence is responsible for heightened anxiety and increased vulnerability to drug relapse in adulthood.

Microarrays ‐ The Challenge of Preparing Brain Tissue Samples

Microarray experiments allow researchers to collect an amazing amount of gene expression data that have the potential to provide unique information to help interpretation of the biological functions of the central nervous system. These experiments are, however, technically demanding and present unique difficulties when used in the context of neuroscience research, in particular. Success or failure of microarray experiments are highly dependent on reproducible target preparations. This involves a relatively long chain of preparation steps, such as removal of tissue from experimental animals or from post‐mortem human brains, storage, selection, and excision of brain regions. This is followed by RNA extraction, reverse transcription, and labeling of target cDNAs or cRNAs. Additionally, it is emphasized that the quality of microarray data largely relies on the proper handling of animals throughout experiments and the time of the day when experiments are stopped. This article tries to provide hints for some basic rules to be observed in preparation of samples for expression profiling studies.