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.

Mapping ligand binding pockets in chloride ClC-1 channels through an integrated in silico and experimental approach using anthracene-9-carboxylic acid and niflumic acid.

BACKGROUND AND PURPOSE: Although chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is limited. ClC-1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC-1 blockers have been discovered, such as anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA), whereas no activator exists. The absence of a ClC-1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators. EXPERIMENTAL APPROACH: Here we provide an in-depth characterization of drug binding pockets in ClC-1 through an integrated in silico and experimental approach. We first searched putative cavities in a homology model of ClC-1 built upon an eukaryotic CLC crystal structure, and then validated in silico data by measuring the blocking ability of 9-AC and NFA on mutant ClC-1 channels expressed in HEK 293 cells. KEY RESULTS: We identified four putative binding cavities in ClC-1. 9-AC appears to interact with residues K231, R421 and F484 within the channel pore. We also identified one preferential binding cavity for NFA and propose R421 and F484 as critical residues. CONCLUSIONS AND IMPLICATIONS: This study represents the first effort to delineate the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins.

Neurofunctional effects of developmental sodium fluoride exposure in rats.

Contrasting studies on the toxic effects of sodium fluoride (NaF) during developmental stages of Wistar rats, lead us to investigate the neurofunctional effects caused by its perinatal exposure, devoid of any overt sign of toxicity and/or gross malformation. NaF solution was administered to pregnant rats by intragastric gavage at a daily dose of 2.5 and 5.0 mg/kg from gestational day 0 to day 9 after parturition. Developmental NaF exposure caused sex and dose specific behavioural deficits which affected males more than females in the majority of the evaluated end-points. In particular, the perinatal exposure to NaF 5.0 mg/kg, significantly affected learning, memory, motor coordination and blood pressure only in male rats. Conversely, a 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 observed only in NaF 5.0 mg/kg female rats. Finally, a significant impairment of sexual behaviour was observed in male rats at both NaF dose levels. The present data indicate that perinatal rat exposure to NaF results in long lasting functional sex-specific alterations which occur at fluoride levels approaching those experienced by offspring of mothers.

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.

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures.

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.

Block of sodium current in myelinated nerve fibre with enkephalins.

The effects of leu- and met-enkephalin were investigated on the node of Ranvier of isolated nerve fibers of frog under current and voltage clamp conditions. When added to the external solutions, enkephalins (1--5 mM) caused a slight decrease in peak Na+ and steady state K+ currents. The action potential was not significantly affected. When added to the internal medium (by diffusion from the two cut ends of the fibre), enkephalins (less than 5 mM) drastically reduced the peak Na+ current without significantly affecting the steady state K+ current. The block of Na+ current was greatly accelerated and enhanced by repetitive depolarizations. The sodium current slowly recovered after the end of pulsing. The extent of the block and rate of accumulation increased with increasing magnitude and frequency of the depolarizing pulses. Internal applications of enkephalins induced a blockade of the action potential when the fibre was stimulated at frequencies above 0.1 Hz. The results suggest that during depolarizations, enkephalin molecules plug the inner end of Na+ channels or immobilize the channel gates leaving the channels in a closed configuration, and remain in or near the pores for a long time after the end of depolarizations. Possible physiological significance and molecular mode of action of enkephalins on myelinated nerve fibres are discussed.

Long-term effects on cortical glutamate release induced by prenatal exposure to the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone: an in vivo microdialysis study in the awake rat.

The aim of the present in vivo microdialysis study was to investigate whether prenatal exposure to the CB(1) receptor agonist WIN55,212-2 mesylate (WIN; (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone), at a dose of 0.5 mg/kg (s.c. from the fifth to the 20th day of gestation), that causes neither malformations nor overt signs of toxicity, influences cortical glutamate extracellular levels in adult (90-day old) rats. Dam weight gain, pregnancy length and litter size at birth were not significantly affected by prenatal treatment with WIN. Basal and K(+)-evoked dialysate glutamate levels were lower in the cerebral cortex of adult rats exposed to WIN during gestation than in those born from vehicle-treated mothers. In both group of animals WIN (0.1 mg/kg, i.p.) increased dialysate glutamate levels. However, while the blockade of the CB1 receptors with the selective receptor antagonist SR141716A completely counteracted the WIN-induced increase in those rats exposed to vehicle during gestation, it failed to antagonise the increase in those born from WIN-treated dams. These findings suggest that prenatal exposure to the CB1 receptor agonist WIN, at a concentration which is not associated with gross malformations and/or overt signs of toxicity, induces permanent alterations in cortical glutamatergic function. The possibility that these effects might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users is discussed.

Interactions between molecules of a steroid anaesthetic (alphaxalone) and ionic channels of nodal membrane in voltage-clamped myelinated nerve fibre.

1. The effects of the anaesthetic alphaxalone (0.05 to 1 mM) on the node of Ranvier of isolated myelinated nerve fibres of the frog were studied under voltage-clamp conditions. 2. When added to the solution bathing voltage-clamped nodes, alphaxalone modified neither linear leakage nor capacitative currents but rapidly and reversibly blocked K and Na currents. The blocking effects of the anaesthetic on both types of current were not dependent on the frequency of stimulation of the nerve fibres between 0.7 and 10 Hz. 3. The kinetics of the Na current were not modified by alphaxalone but, in the presence of the drug, the K current showed an apparent fast inactivation. 4. Alphaxalone rapidly and reversibly shifted towards negative voltages both the steady-state K conductance-voltage and the peak Na steady-state inactivation-voltage relationships, without noticeable modification of their shape. In contrast, the anaesthetic reversibly decreased the slope of the peak Na conductance-voltage curve. 5. The reduction of the K current induced by alphaxalone was voltage-dependent with an apparent dissociation constant first decreasing from about 0.25 to 0.08 mM between -20 mV and +20 mV and then remaining constant above +20 mV. In contrast, the apparent dissociation constant for the Na current was almost constant with increasing voltages and equalled about 0.30 mM. Hill coefficient values for both K and Na currents were noticeably less than one. 6. It is concluded that, at higher concentrations than those attainable in the brain or in the plasma during surgical anaesthesia in man, alphaxalone has a 'local anaesthetic-like' action on the peripheral nervous system in that it specifically and differentially interacts with K and Na channel gating systems: it is suggested that the anaesthetic would preferentially modify open K and inactivated Na channels.

Effects of some antiepileptic drugs on the repetitive activity of the node of Ranvier.

1 Effects of some antiepileptic drugs on the repetitive activity of the node of Ranvier have been tested on frog myelinated nerve fibres. 2 Nerve fibres were stimulated by supraliminal direct current pulses of long duration. Motor fibres responded with a single action potential; sensory fibres responded with repetitive firing at a frequency of about 300/s. Chlordiazepoxide hydrochloride (0.1 mM), phenobarbitone sodium (0.25 mM) or diphenylhydantoin sodium (0.5 mM) suppressed the repetitive activity. 3 Sensory and motor nerve fibres stimulated by a 10 kHz alternating current of strength twice the threshold responded with repetitive firing at a frequency of 400-500/s. Superfusion of the node with chlordiazepoxide hydrochloride (0.2 mM), phenobarbitone sodium (0.5 mM) or diphenylhydantoin sodium (1.7 mM) reduced the frequency of firing by 50% either in sensory or in motor fibres activated by a.c. stimulation; at the same concentrations, the drugs altered amplitude of the action potential and threshold for electric excitability by less than 10%. 4 Unlike local anaesthetics, chlordiazepoxide, phenobarbitone and diphenylhydantoin are more selective in inhibiting repetitive firing than in reducing the amplitude of the action potential or increasing the threshold for electric excitability. 5 Trimethadione (up to 5 mM) was ineffective on repetitive firing elicited either with direct or with alternating current.

Mechanism of action of ketamine in the current and voltage clamped myelinated nerve fibre of the frog.

The effects of the general anaesthetic ketamine, on the frog isolated node of Ranvier, were studied under current and voltage clamp conditions. Ketamine (0.5 and 1 mM) reversibly decreased the amplitude of the action potential and increased both the duration of the action potential and the threshold potential. When the K current was blocked, spontaneous action potentials appeared after washout of the drug. Ketamine rapidly blocked the Na current and more slowly modified a fraction of Na channels (about 10%) to give rise to a non-inactivatable (late) Na current. After washout of the drug, the block reversed more rapidly than the ketamine-induced late Na current disappeared. Steady-state outward, peak Na and ketamine-induced late Na currents were rapidly and reversibly blocked by ketamine with an apparent dissociation constant of 0.7 mM. Both peak Na and ketamine-induced late Na currents were reversibly blocked by procaine.

Inhibition of ionic currents in frog node of Ranvier treated with naloxone.

1 Myelinated nerve fibres of frog sciatic nerve were investigated under current and voltage clamp conditions. In the presence of 68 microM external naloxone, the action potential was completely, though progressively, blocked within 15 min of drug superfusion. The resting potential remained constant. 2 Under voltage clamp conditions both peak Na+ and steady-state K+ currents were decreased reversibly by external naloxone. Both currents were reduced in a dose-dependent manner but, whereas sodium current was affected by the smallest concentrations of naloxone (1.3 up to 12.5 microM), potassium current was decreased only by higher concentrations (25 up to 112 microM). 3 The time-course of development of the effect on both Na+ and K+ currents after exposure to 112 microM naloxone (a concentration giving more than 50% of decrease) showed that the effect develops quickly within the first 2 min of exposure to the drug, but afterwards both currents continue to fall more slowly, though progressively. 4 Experiments with constant test pulses to Em = - 10 mV and conditioning prepulses of various amplitudes, showed that the Na inactivation curve, h infinity (Em), was shifted in a negative direction along the potential axis; the shape of the curve was also slightly changed in the presence of naloxone since the shift was larger near the top of the curve. All the observed effects were reversible after returning to the standard Ringer solution. 5 Internal naloxone (less than 0.2 mM) reduced the amplitude of the action potential as well as peak Na+ and steady-state K+ currents; the sodium inactivation curve, h infinity (Em), was shifted to more negative potentials. 6 A possible anaesthetic-like activity of naloxone on the nodal membrane is discussed.

Electrophysiological studies of the effects of the general anaesthetic etomidate on frog myelinated nerve fibre.

The effects of the general anaesthetic etomidate (0.1 to 1 mM) upon the node of Ranvier of frog isolated nerve fibres were investigated under current and voltage clamp conditions. When added to the external solution, etomidate reversibly decreased the amplitude of the action potential. The action potential block, induced by the drug, was reversed by increasing the membrane potential. Etomidate rapidly and reversibly blocked the Na current with an apparent dissociation constant of 0.6 mM. In the presence of the drug, the steady-state inactivation-voltage curve of the Na current was shifted towards negative voltages. The block of Na current by etomidate was partially removed by repetitive depolarization preceded by a 50 ms period of hyperpolarization. In contrast, the block was enhanced when the repetitive depolarization was not preceded by hyperpolarization. This suggests that Na channels were preferentially blocked by the drug in the inactivated state. The K current was reversibly blocked by etomidate with an apparent dissociation constant of 0.2 mM. In the presence of the drug, the K current showed an apparent fast inactivation suggesting that K channels were blocked in the open state. We conclude that at higher concentrations than those attainable in the mammalian brain following single anaesthetic doses the general anaesthetic etomidate has a "local anaesthetic-like' action on the peripheral nervous system.

Alterations in the ontogeny of rat pup ultrasonic vocalization produced by prenatal exposure to nitrogen dioxide.

Wistar female rats were exposed to low concentrations of nitrogen dioxide, NO2 (1.5 and 3 ppm) from day 0 to day 20 of pregnancy. The results show that prenatal exposure to this oxidant gas produced significant changes in the duration pattern of ultrasonic vocalizations emitted by male pups removed from their nest. In particular, a significant decrease in the length of ultrasonic calls was found in both 10- and 15-day-old rats exposed to NO2 (3 ppm) during gestation. These alterations were found at dose levels which did not significantly affect reproduction parameters, body weight gain and motor activity development. These findings suggest that gestational exposure to NO2, at concentrations below those associated with overt signs of toxicity, induces in rat offspring subtle behavioral changes characterized by altered ontogeny of ultrasonic emission.

Irreversible impairment of active avoidance behavior in rats prenatally exposed to mild concentrations of carbon monoxide.

Wistar female rats were exposed to relatively mild concentrations of carbon monoxide (75 and 150 ppm) from day 0 to day 20 of pregnancy. The results show that prenatal exposure to CO (150 ppm) significantly impairs the acquisition of a two-way active avoidance task in 3-month-old male rats as well as the acquisition and reacquisition of this schedule in 18-month-old animals subjected to six daily 20-trial sessions. These deficits do not seem to be attributable to alterations of a non-associative nature, as the intertrial activity and the escape response latencies in CO exposed animals were not significantly affected with respect to controls. These findings, showing that gestational exposure to CO induces in rat offspring permanent learning and memory impairment, confirm that the offspring of smoking mothers may be at considerably greater risk than current epidemiological studies on birthweight and neonatal mortality suggest.

Role of polysialic acid in peripheral myelinated axons.

Polysialic acid (PSA), generally lost from the vertebrate nervous system during maturation, may regulate developmental differences in axon growth, bundling, and sprouting. Changes in polysialic levels on the axon surface seem to be involved during development in establishing normal pattern of muscle innervation. Besides the well-established role of PSA as a regulator of cell-cell interactions during development, PSA expression in myelinated axons may be related to reparative events in response to chemically induced injuries. Histochemical staining method using lectins with well-characterized binding specificities shows that glycoconjugates of the node of Ranvier undergo a rearrangement during exposure to 2,5-hexanedione, known to induce a peripheral neuropathy characterized by giant axonal swelling and retrograde demyelination. In particular, neutral glycoproteins with terminal galactose are replaced by sialoglycoproteins, consistent with the proposed role of PSA as a regulator of axonal behaviour during regeneration.

Ontogenesis of autonomic receptors in detrusor muscle and bladder sphincter of human fetus.

An investigation was made of the distribution and the ontogenesis of various types of receptors in the bladder detrusor muscles and sphincter isolated from human fetuses at different stages of pregnancy. Cholinergic receptors appear very early in the fetal urinary bladder since contractile responses to bethanechol, competitively blocked by atropine, are observed in detrusor muscle preparations at three months and in the sphincter at four months. Later on, the density of cholinergic receptors increases in the detrusor muscle whereas there is a progressive reduction in the sphincter.

Functional evaluation of new experimental model of microsurgical tubulovasostomy in rat.

The function of a new experimental microsurgical model of tubulovasostomy is evaluated. The model is represented by an end-to-end tubulodeferential anastomosis after removal of the proximal (juxtaepididymal) half of the vas deferens and segmental unfolding of the distal part of the epididymal tubule. Both anastomosed segments are evaluated for responsiveness to norepinephrine and field stimulation. The results are discussed in terms of accurate selection of segments to be anastomosed on the basis of their morphofunctional correlates; in fact, it seems worth replacing the segments removed only with others endowed with equivalent properties.

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.

Mechanism of action of a structural analog of alphaxalone on myelinated nerve fibre.

The action of alphadolone acetate (0.05-5 mM), a steroid anaesthetic and structural analog of alphaxalone, was investigated on frog myelinated axons under voltage-clamp conditions. When applied externally, alphadolone acetate reduced K and Na currents, with apparent dissociation constants of 0.70 and 1.74 mM, respectively, and without noticeable modification in their time course. In addition, Na conductance-voltage and steady-state inactivation-voltage curves were shifted towards negative voltages. This effect was more pronounced on the steady-state inactivation-voltage relationship. These results suggest that alphadolone acetate blocked K channels indifferently in their resting or open state, and Na channels preferentially in their inactivated state. Alphaxalone has been shown to preferentially block open K and inactivated Na channels (Benoit et al., 1988, Br. J. Pharmacol. 94, 635). Thus, a structural change of a steroid molecule can lead to differences in its mechanism of action. This supports the hypothesis of direct interactions between steroid molecules and target membrane proteins with resulting anaesthetic activity.

Prenatal exposure to low levels of carbon monoxide alters sciatic nerve myelination in rat offspring.

Prenatal exposure to low concentrations of carbon monoxide (CO, 75 and 150 ppm from day 0 to day 20 of gestation), resulting in maternal blood HbCO concentrations equivalent to those maintained by human cigarette smokers, leads to subtle myelin alterations in the sciatic nerve of male rat offspring. The rapid growth spurt in pup body weight was related to the period of maximal increase in myelin sheath thickness in both control and CO-exposed animals. A significant reduction in myelin sheath thickness of sciatic nerve fibers, paralleled by changes in the frequency distribution, occurred in both 40- and 90-day-old rats exposed in utero to CO (75 and 150 ppm). Myelin deficit observed in 75 and 150 ppm CO-exposed animals showed up only after the major spurt in myelination but not early during development. The subtle myelin alterations observed in CO-exposed offspring were not accompanied by changes in developmental pattern of axon diameters and did not result in a gross impairment of motor activity. These results suggest that the myelination process is selectively targeted by a prenatal exposure model simulating the CO exposure observed in human cigarette smokers.