Pietro Borracci

Acute exposure to methylmercury at two developmental windows: Focus on neurobehavioral and neurochemical effects in rat offspring

The neurobehavioral and neurochemical effects produced by prenatal methylmercury exposure (8 mg/kg, gestational-days 8 or 15), were investigated in rats. On postnatal day 40, animals exposed to methylmercury and tested in the open field arena, showed a reduction in the number of rearings, whereas the number of crossings and resting time was not altered with respect to the age-matched control rats. The methylmercury-exposed groups showed a lower level of exploratory behavior as well as an impairment in habituation and working memory when subjected to the novel object exploration task. The neophobia displayed by methylmercury-exposed rats is unlikely to be attributed to a higher degree of anxiety. Prenatal methylmercury exposure did not affect motor coordination or motor learning in 40-day-old rats subjected to the balance task on a rotating rod, and it did not impair the onset of reflexive behavior in pups screened for righting reflex, cliff aversion and negative geotaxis. In cortical cell cultures from pups exposed to methylmercury during gestation, basal extracellular glutamate levels were higher, whereas the KCl-evoked extracellular glutamate levels were lower than that measured in cultures from rats born to control mothers. In addition, a higher responsiveness of glutamate release to N-methyl-D-aspartic acid receptor activation was evident in cortical cell cultures from pups born from methylmercury-treated dams than in cultures obtained from control rats. The present results suggest that acute maternal methylmercury exposure induces, in rat offspring, subtle changes in short-term memory as well as in exploratory behavior. These impairments seem to be associated to alterations of cortical glutamatergic signaling.

Developmental omega-3 supplementation improves motor skills in juvenile-adult rats.

Long‐chain polyunsaturated fatty acids are critical for brain growth spurt during both foetal and postnatal period. They play important roles in the expression of genes regulating cell differentiation and neuronal growth, as well as in the development of synaptic processing of neural cell interaction. Foetus and placenta are dependent on maternal supply for their growth and development, and supplemented infants show significantly greater mental and psychomotor scores. In particular, it has been shown that if mothers take omega‐3 supplements, their babies are smarter and better physically coordinated. On these grounds, the aim of the present study was to investigate, in the Sprague–Dawley rat, the effects of perinatal treatment with omega‐3 on motor activity, motor coordination, motor learning and memory. From gestational day 8 throughout the lactation period, dams received either an emulsion of 0.05 g/kg body weight omega‐3 in fruit juice, or an emulsion of 1 g/kg body weight omega‐3 in fruit juice or just the fruit juice (control). Omega‐3 formula was made of 27% docosahexaenoic acid and 53% eicosapentaenoic acid. On the day of birth (postnatal day 1), all pups were weighed, and then randomly culled to eight pups per litter. Pups were weaned at 21 days of age. One male pup per litter from each litter (control, n = 6; omega‐3 0.05 g/kg, n = 5; omega‐3 1 g/kg, n = 6) was used. Both control and treated rats were tested for (i) locomotor activity using the open field paradigm, (ii) motor coordination and motor learning using the rotarod/accelerod task and (iii) memory using the passive avoidance paradigm. Rats were tested on postnatal day 21 and re‐tested on postnatal day 90. As a result, docosahexaenoic acid and eicosapentaenoic acid supplementation significantly improved motor coordination. In particular, the latency to fall at the first speed was significantly increased in the treated rats as compared to the control animals. This benefit was observed with both doses at each tested age. The rat performance in accelerating rotation speed mode, which provides an indication of motor learning ability, was not modified by the omega‐3 supply. Finally, the omega‐3 treatment did not influence motor activity in the open field‐tested rats, nor the memory ability in the passive avoidance task. In conclusion, perinatal omega‐3 supplementation exerts a long lasting beneficial effect on the rotarod performance indicating improvement in balance and motor coordination and, possibly, in the functioning of pathways governing this task.

Dietary Choline Deprivation Impairs Rat Brain Mitochondrial Function and Behavioral Phenotype

Dietary choline deprivation (CD) is associated with behavioral changes, but mechanisms underlying these detrimental effects are not well characterized. For instance, no literature data are available concerning the CD effects on brain mitochondrial function related to impairment in cognition. Therefore, we investigated brain mitochondrial function and redox status in male Wistar rats fed a CD diet for 28 d. Moreover, the CD behavioral phenotype was characterized. Compared with rats fed a control diet (CTRL), CD rats showed lower NAD-dependent mitochondrial state III and state IV respiration, 40% lower complex I activity, and significantly higher reactive oxygen species production. Total glutathione was oxidatively consumed more in CD than in CTRL rats and the rate of protein oxidation was 40% higher in CD than in CTRL rats, reflecting an oxidative stress condition. The mitochondrial concentrations of cardiolipin, a phospholipid required for optimal activity of complex I, was 20% lower in CD rats than in CTRL rats. Compared with CTRL rats, the behavioral phenotype of CD rats was characterized by impairment in motor coordination and motor learning assessed with the rotarod/accelerod test. Furthermore, compared with CTRL rats, CD rats were less capable of learning the active avoidance task and the number of attempts they made to avoid foot shock was fewer. The results suggest that CD-induced dysfunction in brain mitochondria may be responsible for impairment in cognition and underline that, similar to the liver, the brain also needs an adequate choline supply for its normal functioning.

Gestational all-trans retinoic acid treatment in the rat: Neurofunctional changes and cerebellar phenotype

Neurofunctional effects produced by gestational all-trans retinoic acid (all-trans RA) treatment were investigated in the offspring of Sprague-Dawley rats. Reproduction data, onset of reflexive behavior, locomotor activity, motor coordination and motor learning were examined. Moreover, possible changes in size and morphology of the cerebellum were evaluated. The results show that all-trans RA treatment (2.5 mg/kg, by gavage) on gestational days (GD) 11-13 significantly increased postnatal mortality and decreased pup weight gain. Moreover, all-trans RA-treated rats showed a significant delay in eyes opening, hair growth as well as in the maturation of righting reflex, cliff aversion and pole grasping. All-trans RA treatment significantly impaired the ambulatory activity in adult rats without altering the number of rearings. All-trans RA-treated rats subjected to the rotarod/accelerod task showed significant impairment in both motor coordination and motor learning ability. The morphological analysis revealed a significant reduction in the cerebellar size and impairment in foliation profile, at PND 3 with subsequent recovery at PNDs 8 and 40. The evidence that functional alterations increase with age and persist in adulthood whereas the morphological changes decline with age, strongly supports the view that, besides the cerebellum morphology, the organization of the cerebellar circuitry, and in particular of cortico-cerebellar connections, are also affected by all-trans RA treatment.

Effects of early gestational all-trans retinoic acid treatment on motor skills: A longitudinal study in the offspring of Sprague–Dawley rats

The purpose of the present study was to investigate the behavioral outcomes of all-trans retinoic acid (RA) treatment in the period spanning gestational day (GD) 8-10. A sublethal dose (2.5mg/kg b.w.) compatible with high neonatal survival, sufficient to supply male offspring for later behavioral testing, was used. Indeed, the mortality rate at birth was 7.8%. Reproduction parameters (body weight gain of dams during gestation, number of dams giving birth, pregnancy length, litter size at birth), offspring body weight gain and the development of their somatic characteristics (ear unfolding, auditory conduit opening, eyes opening, hair growth) were not altered by RA. Instead, the onset of righting reflex and negative geotaxis were delayed by 2 days, suggesting vestibular involvement and abnormal functioning of the cerebellum. Then, the performance of RA-treated rats on open field and rotarod/accelerod tasks was assessed from postnatal day (PND) 21 to 90. Similar to the previously investigated GD 11-13 RA treatment, the GD 8-10 RA treatment impaired the open field activity and rotarod/accelerod performance in young adult rats, thus suggesting a task-specific rather than a stage-specific effect of low-dose retinoids during brain development. The delayed appearance of these outcomes underlines the relevance of longitudinal studies to sort out specific RA-targeted neurochemical-behavioral pathways that could be labelled as having no phenotype based on standard examination at birth.

Prenatal exposure model simulating CO inhalation in human cigarette smokers: sphingomyelin alterations in the rat sciatic nerve.

Prenatal exposure to low concentrations of carbon monoxide (CO, 150 ppm) causes long-term alterations in sphingomyelin (SM) homeostasis in peripheral nervous system, but not brain of male rat offspring. In particular, unlike sphinganine (intermediate of complex sphingolipid biosynthesis de novo), the concentrations of sphingosine (intermediate of complex sphingolipid turnover) were increased by 2.35-fold in the sciatic nerve of CO-exposed offspring with respect to controls (P<0.05, overall one-way ANOVA). These subtle alterations were not accompanied by changes in motor activity (F=0.25, df=1/10, n.s., overall one-way-ANOVA). The results suggest that the SM homeostasis in the sciatic nerve is particularly susceptible to prenatal CO exposure resulting in maternal carboxyhaemoglobin (HbCO) levels equivalent to those found in human cigarette smokers.

Rat embryo exposure to all-trans retinoic acid results in postnatal oxidative damage of respiratory complex I in the cerebellum

The results of the present work show that the exposure of pregnant rats to low doses of all-trans-retinoic acid (ATRA) (2.5 mg/kg body weight) results in postnatal dysfunction of complex I of the respiratory chain in the cerebellum of the offspring. ATRA had no effect on the postnatal expression of complex I and did not exert any direct inhibitory effect on the enzymatic activity of the complex. The ATRA embryonic exposure resulted, however, in a marked increase in the level of carbonylated proteins in the mitochondrial fraction of the cerebellum, in particular of complex I subunits. The postnatal increase of the carbonylated proteins correlated directly with the inhibition of the activity of complex I. ATRA had, on the other hand, no effect on oxygen free-radical scavengers. It is proposed that embryonic exposure to ATRA results in impairment of protein surveillance in the cerebellum, which persists after birth and results in accumulation of oxidatively damaged complex I.

Late embryonic exposure to all-trans retinoic acid induces a pattern of motor deficits unrelated to the developmental stage.

The present study extends previous investigations examining the behavioral outcomes of all-trans retinoic acid (RA) exposure at embryonic (E) days 14-16. A sublethal dose (2.5 mg/kg b.w.) compatible with high neonatal survival sufficient to supply offspring for later behavioral testing, was used. The results show that E14-16 RA exposure, similar to E8-10 or E11-13 (previous studies), impairs locomotor activity (open field test) as well as motor coordination and motor learning (rotarod/accelerod task) in young-adult rats. The results provide further evidence that RA exposure induces a pattern of motor deficits which are not strictly related to the embryonic stage, compatible with the protracted developmental profile of the cerebellum.

Smoking during Pregnancy: A Risk Factor for Peripheral Neuropathy?

Studies dealing with the outcomes of developmental carbon monoxide (CO) exposure on myelination in rat offspring are reviewed. Prenatal CO exposure from gestational day 0 to gestational day 20 impairs myelin deposition around peripheral axons resulting in a significant hypomyelination in juvenile and adult rats. Myelin protein patterns analyzed by SDS-polyacrylamide gel electrophoresis and lipid patterns analyzed by the HPTLC method are not altered in both peripheral and central nervous systems of CO-exposed offspring. Interestingly, when sphingomyelin is extracted and purified, the derivatization by OPA reagent and analysis by reversed-phase HPLC reveal a significant increase in sphingosine levels in peripheral nervous system but not in central nervous system of CO-exposed rats. The above morphological and biochemical alterations are not accompanied by motor disabilities.