Maria Tattoli

Prenatal exposure to a cannabinoid agonist produces memory deficits linked to dysfunction in hippocampal long-term potentiation and glutamate release

To investigate the possible long-term consequences of gestational exposure to cannabinoids on cognitive functions, pregnant rats were administered with the CB1 receptor agonist WIN 55,212-2 (WIN), at a dose (0.5 mg/kg) that causes neither malformations nor overt signs of toxicity. Prenatal WIN exposure induced a disruption of memory retention in 40- and 80-day-old offspring subjected to a passive avoidance task. A hyperactive behavior at the ages of 12 and 40 days was also found. The memory impairment caused by the gestational exposure to WIN was correlated with alterations of hippocampal long-term potentiation (LTP) and glutamate release. LTP induced in CA3–CA1 synapses decayed faster in brain slices of rats born from WIN-treated dams, whereas posttetanic and short-term potentiation were similar to the control group. In line with LTP shortening, in vivo microdialysis showed a significant decrease in basal and K+-evoked extracellular glutamate levels in the hippocampus of juvenile and adult rats born from WIN-treated dams. A similar reduction in glutamate outflow was also observed in primary cell cultures of hippocampus obtained from pups born from mothers exposed to WIN. The decrease in hippocampal glutamate outflow appears to be the cause of LTP disruption, which in turn might underlie, at least in part, the long-lasting impairment of cognitive functions caused by the gestational exposure to this cannabinoid agonist. These findings could provide an explanation of cognitive alterations observed in children born from women who use marijuana during pregnancy.

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

Behavioural changes in the offspring of rats exposed to diazepam during gestation

Abstract Primiparous pregnant Sprague-Dawley dams were administered a single daily sc. injection of diazepam (0.1 and 1 mg/kg) or vehicle over gestation days 14–20. No differences in neonatal mortality and weight gain were found between the control and diazepam-exposed pups. Conversely, male pups prenatally treated with this benzodiazepine exhibited subtle behavioural alterations either during early postnatal life or during adulthood. In particular, a significant decrease in the locomotor activity of the diazepam-treated groups was found at the end of the second postnatal week (14–16 days). Furthermore, the administration of diazepam during gestation produced marked changes in the length of ultrasonic calls of rat pups removed from their nest. Finally, adult male rats (120 days of age) prenatally exposed to diazepam showed a notable impairment in copulatory activity as well as a significant decrease in the duration of ultrasonic (22 kHz) post-ejaculatory calls emitted during sexual behaviour. These findings suggest that late gestational exposure to diazepam induces both short- and long-term behavioural changes in rat offspring, changes characterized by altered activity patterns and emotional-motivational responsiveness to environmental challenges.

Prenatal Exposure to Carbon Monoxide Affects Postnatal Cellular Electrophysiological Maturation of the Rat Heart A Potential Substrate for Arrhythmogenesis in Infancy

Background—Maternal smoking is an independent risk factor for sudden infant death syndrome (SIDS). Carbon monoxide (CO) is a major component of smoke. No information is available about the effect of CO and/or smoking on postnatal maturation of the heart. The aim of this study was to investigate the effect of prenatal exposure to CO on cellular electrophysiological maturation in male Wistar rats. Methods and Results—The patch-clamp technique was used to measure action potential (AP) and ionic currents (Ito and ICa,L) from rat ventricular myocytes. During growth, AP duration measured at −20 and −50 mV (APD−20 and APD−50) decreased progressively in both groups; the process was significantly delayed in rats exposed prenatally to 150 ppm CO: At 4 weeks, APD−20 and APD−50 were 89.5±18.2 and 147.7±24.5 ms in CO (n=13) and 35.6±4.5 and 77.8±8.3 ms in control rats (Ctr; n=14; P <0.01 and P <0.05, respectively) and normalized at 8 weeks. At 4 weeks, the density of ICa,L was significantly higher (21.3±1.6 pA/pF, n=17, versus 15.9±1.6 pA/pF, n=22; P <0.05) and the density of Ito significantly lower (9.6±1.5, n=22, versus 15.2±2.2 pA/pF, n=19; P <0.01) in CO than in Ctr and normalized thereafter. Conclusions—Prenatal CO exposure affects the physiological shortening of APD in neonatal rats. We speculate that a prolonged myocyte repolarization induced by prenatal exposure to smoke may establish a period of vulnerability for life-threatening arrhythmias in infancy.

Prenatal exposure to low concentrations of carbon monoxide alters habituation and non-spatial working memory in rat offspring

Inhalation of low concentrations (75 and 150 ppm) of carbon monoxide (CO) by pregnant rats from days 0 to 20 of gestation leads to alterations in habituation and working memory in young adult male offspring subjected to the novel exploration object test. In particular, lack of habituation upon the second presentation of the objects and failure in the ability to discriminate between the novel and the familiar object were found in CO (75 and 150 ppm)-exposed offspring. These alterations were not accompanied by changes in spontaneous motor activity (open field test). The subtle behavioral deficits observed in the present study have been produced by prenatal exposure to CO levels resulting in maternal blood carboxyhaemoglobin (HbCO) concentrations equivalent to those observed in human cigarette smokers.

Genetic Factors Involved in the Effects of Developmental Low-Level Alcohol Induced Behavioral Alterations in Rats ☆

Behavioral and neurochemical effects of perinatal alcohol exposure (3% v/v solution from Day 15 of gestation to Day 7 after parturition) have been investigated in Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rat lines, selectively bred for opposite alcohol preference and consumption. In an elevated zero-maze model of anxiety, sucrose-exposed sP rats (sP-S): (i) spent significantly less time on the open arms (TO); (ii) exhibited a significantly lower number of head dips (HDIPS); and (iii) showed a higher number of stretched attend-postures (SAP) than sucrose-exposed sNP rats (sNP-S) at 90 and 180 days of age. The two rat lines displayed different emotional reactivity in response to alcohol exposure. Subtle differences in sexual behavior and ultrasonic emission (latency to the first intromission and to the first 50 kHz call) were observed between sP-S and sNP-S rats. sP-alcohol exposed (sP-A) offspring exhibited a higher latency to the first intromission than sNP-alcohol (sNP-A) treated rats. Moreover, a lower number of sP-A rats exhibited both intromission and ejaculation with respect to sNP-A animals. sP-S rats were significantly slower in recover of the righting reflex than sNP-S animals after a challenge dose of alcohol (3 g/kg, i.p.). Perinatal alcohol did not affect either onset or duration of sleep time in either line. Neurochemical experiments have shown that perinatal alcohol did not influence basal dopamine levels or amphetamine-induced dopamine increase in the prefrontal cortex of either sP or sNP offspring. These results, showing an endpoint-specific differential sensitivity of sP and sNP lines to perinatal low alcohol exposure, indicate that genetic factors could be responsible for selective susceptibility to behavioral alterations induced by developmental treatment with this drug of abuse.

Neurofunctional Effects of Developmental Alcohol Exposure in Alcohol-Preferring and Alcohol-Nonpreferring Rats

The neurofunctional effects of developmental alcohol exposure (3% v/v solution from day 15 of gestation to day 7 after parturition) have been investigated in Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rat lines, selectively bred for opposite alcohol preference and consumption. Alcohol exposure significantly decreased the rate of ultrasonic emission in sP male pups; whereas, it did not affect this indicator of emotional reactivity in sNP animals. Perinatal alcohol intake did not influence either learning of an active avoidance task or hippocampal long-term potentiation in both offspring lines. Significant differences in time spent exploring novel objects were observed between control sP and sNP rats subjected to the novel exploration object test. Alcohol exposed sP rats, but not alcohol exposed sNP rats, apparently lost the capacity to discriminate between the novel and the familiar object, even though this difference is difficult to interpret because of the large differences in the respective responses to the novel objects. Neurochemical experiments have shown that basal levels of dopamine (DA) and homovanillic acid (HVA) were significantly higher in the nucleus accumbens (NAC) of sP rats with respect to sNP animals. Perinatal alcohol did not affect basal DA and HVA concentrations or amphetamine-induced DA increase and HVA decrease in the NAC of either sP or sNP offspring. These results suggest that subtle behavioral alterations induced by developmental exposure to low doses of alcohol, which do not cause malformations and/or overt neurotoxicity, may be associated with genetic factors, although not necessarily those responsible for differences in alcohol preference.

Extracorporeal Shock-Wave Therapy for Supraspinatus Calcifying Tendinitis: A Randomized Clinical Trial Comparing Two Different Energy Levels

Background Extracorporeal shock-wave therapy (ESWT) represents a valid intervention in the treatment of people with supraspinatus calcifying tendinitis (SCT), but there is limited evidence for the useful range of ESWT doses. Objective The aim of this study was to compare 2 different ranges of energy flux density in treatment of SCT with ESWT. Design This study was designed as a single-blind randomized clinical trial. Setting This study was performed in a university hospital. Patients Forty-six patients with SCT were randomly assigned to 2 groups that received different therapeutic energy doses of ESWT: (1) group A received ESWT at an energy level of 0.20 mJ/mm2, and (2) group B received ESWT at an energy level of 0.10 mJ/mm2. Intervention The treatment protocol consisted of 4 sessions performed once a week. Measurements The change in mean Constant Murley Scale (CMS) scores at 3 and 6 months was the primary endpoint. The change in the mean visual analog scale (VAS) scores from baseline to 3 and 6 months after the intervention and radiographic change in size of calcium deposits were evaluated as secondary endpoints. At 12 months, pain relief was assessed using a numeric rating scale. Results Significant clinical improvement based on mean CMS scores was observed after 6 months in group A (X̅=79.43, SD=10.33) compared with group B (X̅=57.91, SD=6.53). Likewise, after 6 months, a significant decrease in VAS scores was found in group A (X̅=2.09, SD=1.54) compared with group B (X̅=5.36, SD=0.78). Calcific deposits disappeared in the same percentage of patients in both groups. Limitations The small sample size and lack of a control group were limitations of the study. Conclusions In ESWT for SCT, an energy level of 0.20 mJ/mm2 appears to be more effective than an energy level of 0.10 mJ/mm2 in pain relief and functional improvement.

Effects of prenatal exposure to low concentrations of carbon monoxide on sexual behaviour and mesolimbic dopaminergic function in rat offspring

1 Inhalation of low concentrations of carbon monoxide (CO) by pregnant rats (75 and 150 p.p.m. from day 0 to day 20 of gestation) leads to changes in mesolimbic dopaminergic transmission associated with an impairment of sexual behaviour in male offspring. 2 Eighty day old males exposed in utero to CO (150 p.p.m.) exhibited a significant increase in mount/intromission latency as well as a significant decrease in mount/intromission frequency. A significant decrease in ejaculation frequency was also found in CO (150 p.p.m.)‐exposed animals. 3 The acute administration of amphetamine, at a dose (0.5 mg kg−1 s.c.) stimulating copulatory activity in control rats, failed to reduce mount/intromission latency and did not increase mount frequency in 80‐day offspring exposed to CO (150 p.p.m.) during gestation. 4 These behavioural alterations were paralleled by neurochemical changes (in vivo microdialysis) showing that prenatal CO exposure, at concentrations (150 p.p.m.) that did not affect basal extracellular levels of dopamine in the nucleus accumbens, blunted the amphetamine (0.5 mg kg−1 s.c.)‐induced increase in dopamine release in 80‐day old male rats. 5 No significant changes in either behavioural or neurochemical parameters were observed in 10‐month old rats exposed prenatally to CO. 6 Since the alterations in sexual behaviour and dopaminergic transmission have been produced by prenatal exposure to CO levels resulting in maternal blood carboxyhaemoglobin concentrations equivalent to those maintained by human cigarette smokers, the present data further point out the large risk that the smoking mother poses for her offspring.

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.