Stefano Puglisi-Allegra

Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder

Autistic disorder (MIM 209850) is currently viewed as a neurodevelopmental disease. Reelin plays a pivotal role in the development of laminar structures including the cerebral cortex, hippocampus, cerebellum and of several brainstem nuclei. Neuroanatomical evidence is consistent with Reelin involvement in autistic disorder. In this study, we describe several polymorphisms identified using RNA-SSCP and DNA sequencing. Association and linkage were assessed comparing 95 Italian patients to 186 ethnically-matched controls, and using the transmission/disequilibrium test and haplotype-based haplotype relative risk in 172 complete trios from 165 families collected in Italy and in the USA. Both case-control and family-based analyses yield a significant association between autistic disorder and a polymorphic GGC repeat located immediately 5′ of the reelin gene (RELN) ATG initiator codon, as well as with specific haplotypes formed by this polymorphism with two single-base substitutions located in a splice junction in exon 6 and within exon 50. Triplet repeats located in 5′ untranslated regions (5′UTRs) are indicative of strong transcriptional regulation. Our findings suggest that longer triplet repeats in the 5′UTR of the RELN gene confer vulnerability to autistic disorder.

Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitary-adrenocortical axis.

Microdialysis was employed to assess extracellular dopamine from medial prefrontal cortex, nucleus accumbens, nucleus caudatus, and acetylcholine from the hippocampus of conscious rats during and after 120 min restraint stress. Restraint stress rapidly stimulated the release and the metabolism of dopamine in the medial prefrontal cortex and in the nucleus accumbens, and acetylcholine release in the hippocampus. Fifty-sixty min later, although rats were still restrained, dopamine and acetylcholine release gradually returned to basal levels. When the animals were freed a considerable increase in the release of both neurotransmitters was observed. No changes in the striatum were observed throughout the experiments. The time-course of plasma corticosterone did not parallel that of dopamine and acetylcholine release, increasing during the whole stress procedure, and decreasing when the animals were released. Adrenalectomized rats responded to stress and liberation in much the same way as intact rats. The administration of exogenous corticosterone (0.5-1.5 mg/kg s.c.) did not change the release of dopamine from the prefrontal cortex and nucleus accumbens, and of acetylcholine from the hippocampus, while the dose of 3.0 mg/kg which stimulated them, raised plasma corticosterone to very high concentrations which had never been attained during stress. Moreover, RU 38486, an antagonist of brain glucocorticoid receptors, did not antagonize the stress-induced increase of neurotransmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress, depression and the mesolimbic dopamine system

Abstract The present review was aimed at re-evaluating results obtained from animal models of depression based on experimental stressors in the light of the most recent data on the effects of stress on mesolimbic dopamine (DA) functioning. The data reviewed reveal that the effects of stressful experiences on behaviour and on mesoaccumbens DA functioning can be very different or even opposite depending on the behavioural controllability of the situation, the genetic background of the organism and its life history. Exposure to a single unavoidable/uncontrollable aversive experience leads to inhibition of DA release in the accumbens as well as to impaired responding to rewarding and aversive stimuli. Moreover, the data reviewed indicate a strong relationship between these neurochemical and behavioural effects and suggest that they could model stress-induced expression and exacerbation of some depressive symptoms such as anhedonia and feeling of helplessness caused by life events as well as syndromal depression provoked by traumatic experiences in humans. Repeated and chronic stressful experiences can reduce the ability of stressors to disrupt behaviour, induce behavioural sensitisation to psychostimulants and promote adaptive changes of mesolimbic DA functioning. Opposite neural and behavioural changes, however, can be promoted in specific environmental conditions (repeated variable stressful experiences) or in genetically predisposed individuals. Thus, depressive symptoms may not represent the necessary outcome of stress experiences but be promoted by specific environmental conditions and by a genetically determined susceptibility.

Repeated stressful experiences differently affect limbic dopamine release during and following stress

The effects of repeated restraint stress exposures (daily 60 min, for 6 days) on extracellular dopamine in the nucleus accumbens, during and after the stress experience, have been investigated in rats by in vivo microdialysis. On the first day, restraint increased dopamine release during the first 40 min followed by a return to basal levels (50-60 min later). As soon as restraint ceased and the rats were set free, there was another increase in dopamine release lasting 40 min. On the second and third day, restraint produced only a slight increase in dopamine release, while no significant changes were evident from the fourth to the sixth day. By contrast, from the second to the sixth day the increase in dopamine release observed once rats were freed, was unchanged in comparison to the first day. The present results show that the activation of the mesolimbic dopaminergic system induced by aversive stimuli adapts to repeated experiences differently from that produced by pleasurable events, suggesting that aversive and rewarding experiences involve different neural systems.

Acute stress induces time-dependent responses in dopamine mesolimbic system

Exposure to either restraint or footshock (3-60 min) induced similar biphasic alterations of 3-methoxytyramine (3-MT) concentrations (initial increase followed by decrease below control levels) in the nucleus accumbens septi (NAS) of mice, as revealed by tissue analysis. The only difference between the two stressors was the earlier onset of the decrease phase in the restrained mice. In both stressful conditions acid metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) increased throughout stress, while no significant changes in dopamine (DA) concentrations occurred. These data suggest biphasic alteration of DA release during prolonged stress exposure. The analysis of release in restrained conscious rats by in vivo microdialysis (10-240 min) showed a similar biphasic DA evolution (initial increase followed by decrease below baseline levels) in the NAS. The only difference from the previous experiment was the delayed onset of the decrease phase. Similar changes in DOPAC and HVA were also evident. Moreover, freed rats showed an immediate increase of DA release over baseline levels, also indicating that depletion of the neurotransmitter cannot account for the reduction of released DA. Taken together, these results support the hypothesis that biphasic alteration of DA transmission in the mesolimbic system is a general response to stress and suggest that the initial increase of DA release represents an arousal response while the subsequent decrease in DA release may be related to coping failure.

Prefrontal/accumbal catecholamine system determines motivational salience attribution to both reward- and aversion-related stimuli

Recent evidence suggests that rewarding and aversive stimuli affect the same brain areas, including medial prefrontal cortex and nucleus accumbens. Although nucleus accumbens is known to respond to salient stimuli, regardless of their hedonic valence, with selective increased dopamine release, little is known about the role of prefrontal cortex in reward- and aversion-related motivation or about the neurotransmitters involved. Here we find that selective norepinephrine depletion in medial prefrontal cortex of mice abolished the increase in the release of norepinephrine by prefrontal cortex and of dopamine by nucleus accumbens that is induced by food, cocaine, or lithium chloride and impaired the place conditioning induced by both lithium chloride (aversion) and food or cocaine (preference). This is evidence that prefrontal cortical norepinephrine transmission is necessary for motivational salience attribution to both reward- and aversion-related stimuli through modulation of dopamine in nucleus accumbens, a brain area involved in all motivated behaviors.

The mesoaccumbens dopamine in coping with stress

Mesoaccumbens dopamine (DA) is involved in the stress response. Although neural mechanisms involved in stress are of paramount importance for both clinical and preclinical research, the results of studies on the stress response by mesoaccumbens DA have received little attention. Therefore, we aimed to review these results and propose a role for mesoaccumbens DA in coping with stress. The data reviewed support the view that fluctuations of tonic levels characterize the mesoaccumbens DA stress response. Stress-induced increase of tonic DA levels in nucleus accumbens (NAc) supports expression of responses aimed at removing and avoiding the stressor through activation of DA D2 receptors, whereas inhibition of DA is associated with cessation of active defensive responses. In novel unescapable/uncontrollable stressful conditions tonic levels of DA in NAc show an initial increase followed by a decrease below pre-stress levels that lasts as long as the stressful situation. This biphasic response fits with the dynamics of the primary and secondary appraisal of a stressor that cannot be removed, escaped or controlled by the organism. In fact, NAc DA fluctuations are controlled by the medial pre-frontal cortex, which is involved in stress appraisal. We propose that enhanced mesoaccumbens DA supports expression of active coping strategies against an event appraised as a stressor and that inhibition of DA is required for passive coping with stressful situations appraised as unescapable/uncontrollable.

Altered calcium homeostasis in autism-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1

Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient–control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca2+). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca2+ chelator ethylene glycol tetraacetic acid; neocortical Ca2+ levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca2+-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca2+ levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca2+ homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter × Environment Risk Factor for Anxiety and Depression

BACKGROUND A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT x environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse. METHODS We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene x environment effects. RESULTS Mice experiencing low maternal care showed deficient gamma-aminobutyric acid-A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT x environment risk factor. CONCLUSIONS These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor.

D1 and D2 receptor antagonists differently affect cocaine-induced locomotor hyperactivity in the mouse

Pretreament with small, per se ineffective doses of the selective D1 antagonist SCH 23390 inhibited hyperactivity induced by cocaine. On the other hand, the classic neuroleptic haloperidol and the selective D2 antagonist metoclopramide prevented the stimulatory effects of cocaine on locomotion only at hypokinetic doses, while the atypical neuroleptic (−)-sulpiride, a selective D2 antagonist, did not produce significant effects when administered at the hypokinetic dose of 12 mg/kg. Finally, at low doses (−)-sulpiride dose-dependently potentiated the locomotor-stimulating effects of cocaine, an effect that is not shared either with haloperidol or with metoclopramide. These results are discussed in terms of different roles of DA receptor subtypes in the modulation of the stimulant effects of cocaine on locomotion.