Pharmacogenetics of myotonic hNav1.4 sodium channel variants situated near the fast inactivation gate.

Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel.

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.

Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins.

Significance of F3/Contactin gene expression in cerebral cortex and nigrostriatal development.

F3/Contactin is a neuronal surface glycoprotein, which plays a general role in neural development and, in particular, in neuronal and oligodendrocyte differentiation. In a previous study using the F3/EGFP transgenic mice, which express an EGFP reporter under control of the regulatory region from the mouse F3/Contactin gene, the activation of the F3/Contactin promoter was found to correlate with granule and Purkinje neuron differentiation in developing cerebellar cortex. Here we report that in developing cerebral cortex and basal ganglia the F3/Contactin gene is mostly activated during early commitment of neuronal precursors, thus indicating a region-specific profile of its developmental activation. We also report that, in the same structures of F3/EGFP mice, a downregulation of the endogenous F3/Contactin gene occurs, which correlates with upregulation of the dopaminergic phenotype and with locomotor pattern abnormalities. Therefore, F3/EGFP transgenic mice exhibit morphological and functional phenotypes recapitulating those arising from imbalance of the striatal dopaminergic pathway. As for the underlying mechanisms, we postulate that in F3/EGFP mice F3/Contactin downregulation results from the ability of transgene promoter sequences to interfere with the activation of the endogenous gene, thus realizing an F3/Contactin knockdown model, while dopaminergic upregulation is consistent with a general F3/Contactin inhibitory effect on the neuronal phenotype.

Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-α.

BACKGROUND: In addition to cytotoxic mechanisms directly impacting neurons, ß-amyloid (Aß)-induced glial activation also promotes release of proinflammatory molecules that may self-perpetuate reactive gliosis and damage neighbouring neurons, thus amplifying neuropathological lesions occurring in Alzheimer's disease (AD). Palmitoylethanolamide (PEA) has been studied extensively for its anti-inflammatory, analgesic, antiepileptic and neuroprotective effects. PEA is a lipid messenger isolated from mammalian and vegetable tissues that mimics several endocannabinoid-driven actions, even though it does not bind to cannabinoid receptors. Some of its pharmacological properties are considered to be dependent on the expression of peroxisome proliferator-activated receptors-α (PPARα). FINDINGS: In the present study, we evaluated the effect of PEA on astrocyte activation and neuronal loss in models of Aß neurotoxicity. To this purpose, primary rat mixed neuroglial co-cultures and organotypic hippocampal slices were challenged with Aß1-42 and treated with PEA in the presence or absence of MK886 or GW9662, which are selective PPARα and PPARγ antagonists, respectively. The results indicate that PEA is able to blunt Aß-induced astrocyte activation and, subsequently, to improve neuronal survival through selective PPARα activation. The data from organotypic cultures confirm that PEA anti-inflammatory properties implicate PPARα mediation and reveal that the reduction of reactive gliosis subsequently induces a marked rebound neuroprotective effect on neurons. CONCLUSIONS: In line with our previous observations, the results of this study show that PEA treatment results in decreased numbers of infiltrating astrocytes during Aß challenge, resulting in significant neuroprotection. PEA could thus represent a promising pharmacological tool because it is able to reduce Aß-evoked neuroinflammation and attenuate its neurodegenerative consequences.

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.

Emerging Roles for Ion Channels in Ovarian Cancer: Pathomechanisms and Pharmacological Treatment.

Ovarian cancer (OC) is the deadliest gynecologic cancer, due to late diagnosis, development of platinum resistance, and inadequate alternative therapy. It has been demonstrated that membrane ion channels play important roles in cancer processes, including cell proliferation, apoptosis, motility, and invasion. Here, we review the contribution of ion channels in the development and progression of OC, evaluating their potential in clinical management. Increased expression of voltage-gated and epithelial sodium channels has been detected in OC cells and tissues and shown to be involved in cancer proliferation and invasion. Potassium and calcium channels have been found to play a critical role in the control of cell cycle and in the resistance to apoptosis, promoting tumor growth and recurrence. Overexpression of chloride and transient receptor potential channels was found both in vitro and in vivo, supporting their contribution to OC. Furthermore, ion channels have been shown to influence the sensitivity of OC cells to neoplastic drugs, suggesting a critical role in chemotherapy resistance. The study of ion channels expression and function in OC can improve our understanding of pathophysiology and pave the way for identifying ion channels as potential targets for tumor diagnosis and treatment.

Increased sarcolemma chloride conductance as one of the mechanisms of action of carbonic anhydrase inhibitors in muscle excitability disorders.

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 µM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 µM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50-100 µM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 µM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.

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.

Pathomechanisms of a CLCN1 Mutation Found in a Russian Family Suffering From Becker's Myotonia.

Objective: Myotonia congenita (MC) is a rare muscle disease characterized by sarcolemma over-excitability inducing skeletal muscle stiffness. It can be inherited either as an autosomal dominant (Thomsen's disease) or an autosomal recessive (Becker's disease) trait. Both types are caused by loss-of-function mutations in the CLCN1 gene, encoding for ClC-1 chloride channel. We found a ClC-1 mutation, p.G411C, identified in Russian patients who suffered from a severe form of Becker's disease. The purpose of this study was to provide a solid correlation between G411C dysfunction and clinical symptoms in the affected patient. Methods: We provide clinical and genetic information of the proband kindred. Functional studies include patch-clamp electrophysiology, biotinylation assay, western blot analysis, and confocal imaging of G411C and wild-type ClC-1 channels expressed in HEK293T cells. Results: The G411C mutation dramatically abolished chloride currents in transfected HEK cells. Biochemical experiments revealed that the majority of G411C mutant channels did not reach the plasma membrane but remained trapped in the cytoplasm. Treatment with the proteasome inhibitor MG132 reduced the degradation rate of G411C mutant channels, leading to their expression at the plasma membrane. However, despite an increase in cell surface expression, no significant chloride current was recorded in the G411C-transfected cell treated with MG132, suggesting that this mutation produces non-functional ClC-1 chloride channels. Conclusion: These results suggest that the molecular pathophysiology of G411C is linked to a reduced plasma membrane expression and biophysical dysfunction of mutant channels, likely due to a misfolding defect. Chloride current abolition confirms that the mutation is responsible for the clinical phenotype.

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 µM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 µM for closed channels and 9 µM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 µM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 µM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets.

Micro- and macrovascular complications are major causes of disability and death in patients with diabetes mellitus. Functional impairment of endothelial activity precedes the development of morphological alterations during the progression of diabetes. This endothelial dysfunction results from reduced bioavailability of the vasodilator nitric oxide (NO), mainly due to accelerated NO degradation by reactive oxygen species (ROS). Although hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia independently contribute to endothelial dysfunction via several distinct mechanisms, increased oxidative stress seems to be the first alteration triggering several others. Mechanisms proposed to explain glucose- and lipid-induced vascular alterations in diabetes include accelerated formation of advanced glycation end-products (AGEs), protein kinase C activation, inflammatory signaling and oxidative stress. Insulin resistance with impaired PI 3-kinase effects decreases insulin mediated production of NO and reduces vasodilation, capillary recruitment and antioxidant properties of endothelium. Compensatory hyperinsulinemia enhances activation of intact MAP-kinase pathways and contributes to pro-atherogenic events by increasing secretion of endothelin-1 (ET-1), stimulating expression of adhesion molecules such as VCAM-1 and E-selectin, and inducing production of ROS. Conventional therapies to reduce hyperglycemia, dyslipidemia and insulin resistance may effectively improve endothelial function and delay the onset of vascular complications. Novel therapeutic approaches designed to inhibit AGEs formation, reduce PKC activation, decrease inflammatory signals and restore the ox/redox balance of endothelium may be predicted to ameliorate vascular function in diabetic state. This review summarizes the current knowledge on the most important mechanisms involved in endothelial dysfunction during diabetes. In addition, novel therapeutic strategies that may result from recently identified targets are also described.

Dexamethasone minimizes the risk of cranial nerve injury during CEA.

OBJECTIVE: The incidence of cranial and cervical nerve injury during carotid endarterectomy (CEA) ranges from less than 7.6% to more than 50%. Lesions are mainly due to surgical maneuvers such as traction, compression, tissue electrocoagulation, clamping, and extensive dissections. The use of dexamethasone (DEX) and its beneficial effects in spinal cord injuries have already been described. We investigated whether DEX could also be beneficial to minimize the incidence of cranial and cervical nerve injury during CEA. PURPOSE: To evaluate whether dexamethasone is able to reduce the incidence of cranial nerve injuries. MATERIALS AND METHODS: From March 1999 through April 2006, 1126 patients undergoing CEA because of high-grade carotid stenosis were enrolled and randomized by predetermined randomization tables into two groups. The first group, "A", included 586 patients that all received an intravenous administration of dexamethasone following a therapeutic scheme. The second group, "B", included 540 control subjects that received the standard pre- and postoperative therapy. All patients were submitted to a deep cervical plexus block, eversion carotid endarterectomy, and selective shunting. Three days after the operation, an independent neurologist and otorhinolaryngologist evaluated the presence of cranial nerve deficits. All patients (group A and group B) showing nerve injuries continued the treatment (8 mg of dexamethasone once in the morning) for 7 days and were re-evaluated after 2 weeks, 30 days, and every 3 months for 1 year. Recovery time took from 2 weeks to 12 months, with a mean time of 3.6 months. The chi(2) test was used to compare the two groups and to check for statistical significance. RESULTS: The incidence of cranial nerve dysfunction was higher in group B and the statistical analysis showed a significant effect of dexamethasone in preventing the neurological damage (P = .0081). The incidence of temporary lesions was lower in group A and the chi(2) test yielded a P value of .006. No statistically significant differences were found when comparing the effect of dexamethasone in men and women. In addition, dexamethasone had no statistically significant effect on the incidence of permanent cranial nerve injuries. Finally, no adverse effect related to the administration of dexamethasone was observed. CONCLUSION: Perioperative administration of dexamethasone is effective in minimizing the incidence of temporary cranial nerve injuries during CEA.

Oxytocin/Osteocalcin/IL-6 and NGF/BDNF mRNA Levels in Response to Cold Stress Challenge in Mice: Possible Oxytonic Brain-Bone-Muscle-Interaction.

Oxytocin (Oxt), osteocalcin (Ost), and NGF/BDNF have a role in bone homeostasis, reproduction, and cognition. Oxt/Ost is required for muscle repair. We investigated gene response of muscle and the inter-organ communication following cold stress (CS). The mRNA quantity of Ngf, Ost, Oxt, Bdnf, p75ntr, Ntrk1, Gprc6a, Oxtr, Ntrk2, UCP1, and Il-6 genes in bone, brain, soleus (SOL), and tibialis anterior (TA) muscles from adult mice following CS were investigated. The myosin heavy-chain Mhc2b, Mhc1, Mhc2x, and Mhc2a gene expression were investigated. Mice were maintained at T = 23°C or 4°C for 6 h and 5-days (5d). CS mice did not show signs of muscle degeneration. An upregulation of Ucp1 and Ngf genes by 2 and 1.5 folds, respectively, in TA after 6 h CS and Ntrk1 by 4 and 22 folds in SOL muscle after 6 h and 5d CS, respectively, was observed; while after 6 h CS p75Ntr was downregulated in either muscle. Bdnf was unaffected, while after 5d CS Ntrk2 was upregulated in TA. Ost was downregulated in SOL by 0.9-folds at 5d. Following 5d CS, Oxtr and Il-6 genes were upregulated, respectively, by 1 and 1.5 folds in SOL. A downregulation of Mhc2b, respectively, by 0.96 and 0.88-folds after 6 h and 5d CS in SOL and Mhc2a was also downregulated by 0.88-fold after 5d CS in TA. Mhc1 and Mhc2x were not affected. Changes in the expression levels of genes in TA and SOL muscles, bone, and brain following CS were regulated by IL6 and Oxt. CS potentiates the slow-twitch phenotype of SOL which is in line with the metabolic need of this muscle, and the potentiation of the slow-twitch phenotype in TA. Oxt and IL6 coordinate a phenotype-dependent tonic effect of slow-twitch muscle and Oxt regulates the inter-organ interaction between brain and SOL muscle. Muscle tropism is maintained by NGF signaling following CS.

Effects of perinatal exposure to delta-9-tetrahydrocannabinol on the emotional reactivity of the offspring: a longitudinal behavioral study in Wistar rats.

RATIONALE: The endocannabinoid system plays a crucial role in the control of emotionality and recent clinical findings have shown that heavy prenatal exposure to cannabis is significantly associated with self-reported anxiety symptoms in exposed children. However, the long-term neurobehavioral consequences of in utero exposure to low-moderate doses of cannabinoid compounds have never been investigated. OBJECTIVE: The objective of this study was to investigate whether perinatal exposure to moderate doses of the active constituent of cannabis, the CB(1) cannabinoid receptor agonist delta-9-tetrahydrocannabinol (THC), influences the emotional reactivity of rat offspring. METHODS: Primiparous Wistar rats were treated during pregnancy and lactation with doses of THC equivalent to the current estimates of moderate cannabis consumption in humans (2.5-5 mg kg(-1), per os, from gestational day 15 to postnatal day 9). The emotional reactivity of infant, adolescent, and adult offspring was investigated using the isolation-induced ultrasonic vocalization, social interaction, and elevated plus-maze tests, respectively. RESULTS: Perinatal THC treatment did not affect parameters of reproduction; however, at the dose of 5 mg kg(-1), it increased the number of ultrasounds emitted by rat pups removed from the nest, inhibited social interaction and play behavior in the adolescent offspring, and induced an anxiogenic-like profile in the adult offspring tested in the elevated plus-maze test. CONCLUSION: These results suggest that the endocannabinoid system is involved in the control of emotionality since early developmental stages. Thus, even moderate doses of cannabinoid compounds, when administered during the perinatal period, can have profound consequences for brain maturation, leading to long-lasting neurodevelopmental alterations.

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.

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.

Prenatal methylmercury exposure: effects on stress response during active learning.

The long-term impact of prenatal methylmercury (MeHg) exposure on the stress response during active learning was investigated. Pregnant rats were gavage fed MeHg (8 mg/kg) on gestational day 15. Ninety-day-old rats born to both MeHg- and saline-treated dams were subjected to an active avoidance test. The active avoidance-experienced rats (AAERs) with prenatal exposure to MeHg showed significant impairment in learning ability and exhibited higher levels of corticosterone than the untreated AAERs. The present findings suggest that the abnormal increase in plasma corticosterone levels could contribute to the poor performance of MeHg-treated AAERs in this learning task.

Effects of low dose methylmercury administration during the postnatal brain growth spurt in rats.

Male Sprague-Dawley rats from eight litters were orally administered 0.75 mg/kg/day methylmercury (MeHg) chloride from postnatal day (PD) 14 to PD 23. One male pup per litter from eight different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once. The MeHg dose level resulted in Hg brain concentrations of 0.82+/-0.05 microg/g tissue (n=4). Locomotor behavior was studied in the Opto-Varimex apparatus by testing rats (n=8) weekly from PD 24 to PD 45. Performance of rats (n=8) on learning paradigm was analysed on PD 90. MeHg treatment induced a significant reduction in the number of rearings without altering the distance travelled, the resting time and the time spent in the central part of the arena. Results of conditioned avoidance task showed that, unlike control rats, MeHg-treated animals did not show improvement over blocks and never reached a level of performance that would indicate significant learning had taken place. The present results show that low level exposure to MeHg during late brain growth spurt induces subtle and persistent motor and learning deficits, further underlining the serious potential hazard for the exposed children.