Variation in Mitochondrial Cytochrome c Oxidase I DNA Can Successfully Identify Culex (Melanoconion) pedroi (Diptera: Culicidae) and Culex (Melanoconion) ribeirensis (Diptera: Culicidae).

Culex (Melanoconion) pedroiSirivanakarn & Belkin 1980 and Culex (Melanoconion) ribeirensisForattini & Sallum 1985 are two morphologically very similar species of the Pedroi subgroup of mosquitoes in the Spissipes section of the subgenus Melanoconion of the genus Culex L. 1758. We carried out an analysis of the mitochondrial cytochrome c oxidase I (COI) DNA variation between these two species. The recent observation of sympatric coexistence in a forested area of Rio de Janeiro State (Brazil) triggered the need to validate these two species previously identified based on morphology. We concluded that the COI is a useful tool for identification of Cx. pedroi and Cx. ribeirensis.

Animal models for glutaryl-CoA dehydrogenase deficiency.

In vitro studies suggest that excitotoxic cell damage is an underlying mechanism for the acute striatal damage in glutaryl-CoA dehydrogenase (GCDH) deficiency. It is believed to result from an imbalance of glutamatergic and GABAergic neurotransmission induced by the accumulating organic acids 3-hydroxyglutaric acid (3-OH-GA) and to a lesser extent glutaric acid (GA). Stereotaxic administration of 3-OH-GA and GA into the rat striatum have confirmed these results, but may not truly represent the effect of chronic exposure to these compounds. In an attempt to better understand the pathophysiology of GCDH deficiency in vivo , two animal models have been utilized. A mouse that lacks GCDH activity in all tissues was generated by gene targeting in embryonic stem cells. These animals develop the characteristic biochemical phenotype of the human disease. Pathologically, these mice have a diffuse spongiform myelinopathy similar to that in human patients; however, there is no evidence for acute striatal damage or sensitivity to acute encephalopathy induced by catabolism or inflammatory cytokines. A naturally occurring animal model, the fruit-eating bat Rousettus aegypticus, lacks hepatic and renal GCDH activity, but retains cerebral enzyme activity. Like the mouse, these bats develop the characteristic biochemical phenotype of glutaryl-CoA dehydrogenase deficiency, but lack overt neurological symptoms such as dystonia. It is not known whether they also develop the spongiform myelinopathy seen in the Gcdh-deficient mice. Otherwise, these constellations would suggest that cerebral GCDH deficiency is responsible for the development of neuronal damage. The lack of striatal damage in these two rodent models may also be related to species differences. However, they also highlight our lack of a comprehensive understanding of additional factors that might modulate the susceptibiliy of neurons to accumulating 3-OH-GA and GA in GCDH deficiency. Unravelling these mechanisms may be the key to understanding the pathophysiology of this unique disease and to the development of neuroprotective strategies.

Modulation of glutamatergic and GABAergic neurotransmission in glutaryl-CoA dehydrogenase deficiency.

Although the precise mechanisms underlying the CNS degeneration of patients with glutaryl-CoA dehydrogenase (GCDH) deficiency are still the subject of intense debate, many studies have highlighted that excitotoxicity plays a fundamental role in the neuropathology of this disease, particularly involving the N-methyl-D-aspartate receptor subtype of ionotropic glutamate receptors. Modulation of the glutamatergic system by these compounds involves an inhibition of glutamate uptake into synaptosomes and synaptic vesicles, and a decrease in glutamate binding. Furthermore, glutaric and 3-hydroxyglutaric acids inhibit glutamate decarboxylase, the key enzyme of GABA synthesis, and striatal GABAergic medium-spiny neurons are highly vulnerable to 3-hydroxyglutaric acid-induced neurotoxicity. In conclusion, glutaric acid and 3-hydroxyglutaric acid induce an imbalance in glutamatergic and GABAergic neurotransmission.

Influence of the test situation on pup retrieval behavior of normal and undernourished lactating rats

Undernutrition of dams and pups disrupts the retrieval efficiency of mothers. However, if the mothers are assessed in their home cages, they spend more time with their litters. In the present study the effect of test conditions on pup retrieval behavior of mothers receiving a 25 percent (well-nourished group) and 8 percent casein diet (undernourished group) was examined. In agreement with previous studies, undernourished mothers spent more time with their litters than well-nourished dams as lactation proceeded. Pup retrieval behavior varied with test conditions. In the first experiment, the maternal behavior of dams was assessed by the standard procedure (pups were separated from their mother and scattered over the floor of the home cage). The mother was then returned and the number of retrieved pups was recorded. From day 3 to 8, the retrieval efficiency of undernourished dams decreased, while the retrieval efficiency of well-nourished mothers did not vary. In the second experiment, mothers were subjected to a single retrieval test (on day 9 of lactation) using the procedure described for experiment 1. No difference between well-nourished and undernourished mothers was observed. In the third experiment, seven-day-old pups were separated from the mothers and returned individually to a clean home cage. Dietary treatment did not affect the retrieval efficiency. However, undernourished dams reconstructed the nest more slowly than did well-nourished dams. Taken together, these results suggest that pup retrieval behavior of the undernourished mother is not impaired by dietary restriction when the maternal environment is disturbed minimally

Influence of the test situation on pup retrieval behavior of normal and undernourished lactating rats.

Undernutrition of dams and pups disrupts the retrieval efficiency of mothers. However, if the mothers are assessed in their home cages, they spend more time with their litters. In the present study the effect of test conditions on pup retrieval behavior of mothers receiving a 25% (well-nourished group) and 8% casein diet (undernourished group) was examined. In agreement with previous studies, undernourished mothers spent more time with their litters than well-nourished dams as lactation proceeded. Pup retrieval behavior varied with test conditions. In the first experiment, the maternal behavior of dams was assessed by the standard procedure (pups were separated from their mother and scattered over the floor of the home cage). The mother was then returned and the number of retrieved pups was recorded. From day 3 to 8, the retrieval efficiency of undernourished dams decreased, while the retrieval efficiency of well-nourished mothers did not vary. In the second experiment, mothers were subjected to a single retrieval test (on day 9 of lactation) using the procedure described for experiment 1. No difference between well-nourished and undernourished mothers was observed. In the third experiment, seven-day-old pups were separated from the mothers and returned individually to a clean home cage. Dietary treatment did not affect the retrieval efficiency. However, undernourished dams reconstructed the nest more slowly than did well-nourished dams. Taken together, these results suggest that pup retrieval behavior of the undernourished mother is not impaired by dietary restriction when the maternal environment is disturbed minimally.

Intrastriatal administration of 3-hydroxyglutaric acid induces convulsions and striatal lesions in rats.

Glutaryl-CoA dehydrogenase deficiency is an inherited neurometabolic disease complicated by precipitation of acute encephalopathic crises during a vulnerable period of brain development. These crises result in bilateral striatal damage and subsequently a dystonic dyskinetic movement disorder. In previous in vitro studies neuronal damage in this disease has been linked to an excitotoxic mechanism mediated in particular by one of the accumulating metabolites, 3-hydroxyglutaric acid. However, nothing is known about the in vivo effects of this organic acid. In the present study, we used a stereotaxic intrastriatal injection technique to investigate the behavioral and neurotoxic effects of 3-hydroxyglutaric acid exposure in rats. Here, we report that 3-hydroxyglutaric acid induced an increase in convulsion frequency and duration as determined by open field measurement. Nissl-stained coronal sections from treated rats revealed a pale lesion in the striatum following 3-hydroxyglutaric acid exposure. N-methyl-D-aspartate (NMDA) receptor blockade by MK-801 and stimulation of GABA(A) receptors by muscimol prevented the induction of convulsions and striatal damage by 3-hydroxyglutaric acid, whereas blockade of non-NMDA receptors by 6,7-dinitroquinoxaline-2,3-dione (DNQX) was not protective. We conclude that 3-hydroxyglutaric acid induces convulsions and striatal damage via initiation of an imbalance in the excitatory glutamatergic and the inhibitory GABAergic neurotransmission, resulting in an enhanced excitatory input in striatal neurons. These results support the hypothesis of NMDA receptor-mediated excitotoxic cell damage in glutaryl-CoA dehydrogenase deficiency and represent the basis for the development of new neuroprotective treatment strategies.

3-Methyl-5-hydroxy-5-trichloromethyl-1H-1-pyrazolcarboxyamide induces antinociception.

The antinociceptive action of a novel pyrazole-derived compound, 3-methyl-5-hydroxy-5-trichloromethyl-1H-1-pyrazolcarboxyamide (MPCA) was evaluated using the formalin and tail-immersion tests in mice. Anti-inflammatory activity was assessed by paw plethysmometry in adult rats using the carrageenin-induced paw edema test. Subcutaneous administration of MPCA (22, 66, and 200 mg/kg) induced a dose-dependent decrease in the time spent licking during the neurogenic and inflammatory phases of the formalin test, and preadministration of naloxone (1 mg/kg, sc) did not prevent MPCA-induced (200 mg/kg, sc) antinociception. Naloxone decreased the spontaneous locomotor activity of mice, while MPCA had no effect on locomotion. In contrast, administration of the opioid antagonist caused a significant increase in the locomotor behavior of mice previously injected with MPCA. MPCA was devoid of antinociceptive action by the tail-immersion test and of anti-inflammatory activity. Moreover, MPCA had no effect on the motor performance of mice in the rotarod test. These results suggest that MPCA induces antinociception in the neurogenic and inflammatory phases of the formalin test, an effect that does not involve opioid receptors.

Pharmacological evidence that alpha-ketoisovaleric acid induces convulsions through GABAergic and glutamatergic mechanisms in rats.

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the pathophysiology of this disorder are poorly known. In the present study we investigated the effect of intrastriatal administration of the alpha-keto acids accumulating in MSUD on the behavior of adult rats. After cannula placing, rats received unilateral intrastriatal injections of alpha-ketoisocaproic acid (KIC, 8 micromol), alpha-ketoisovaleric acid (KIV, 8 micromol), alpha-keto-beta-methylvaleric acid (KMV, 6 micromol) or NaCl. KIV elicited clonic convulsions in a dose-response manner, whereas KIC and KMV did not induce seizure-like behavior. Convulsions provoked by KIV were prevented by intrastriatal preadministration of muscimol (46 pmol) and MK-801 (3 nmol), but not by the preadministration of DNQX (8 nmol). These results indicate that among the keto acids that accumulate in MSUD, KIV is the only metabolite capable of causing convulsions in the present animal model and indicates that KIV is an important excitatory metabolite. Moreover, the participation of GABAergic and glutamatergic NMDA mechanisms in the KIV-induced convulsant behavior is suggested, since KIV-induced convulsions are attenuated by muscimol and MK-801. The authors suggest that KIV may play an important role in the convulsions observed in MSUD, and highlight its relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.

Intrastriatal administration of 5-aminolevulinic acid induces convulsions and body asymmetry through glutamatergic mechanisms.

The involvement of glutamatergic and GABAergic mechanisms in the behavioral effects induced by the intrastriatal injection of 5-aminolevulinic acid (ALA) (1-8 mgr;mol/2 mgr;l), a metabolite that accumulates in porphyrias, was evaluated. ALA administration to adult female rats increased locomotor activity, induced clonic convulsions and elicited dose-dependent body asymmetry assessed by the elevated body swing test. ALA-induced convulsions were prevented by intrastriatal preadministration of the glutamate antagonists, 6, 7-dinitroquinoxaline-2,3-dione (8 nmol/0.5 microl) or dizocilpine (2. 5 nmol/0.5 microl), but not by the GABA agonist, muscimol (46 pmol/0. 5 microl). Body asymmetry was prevented only by 6, 7-dinitroquinoxaline-2,3-dione pretreatment. A higher dose of muscimol (92 pmol/0.5 microl) prevented both ALA-induced convulsions and body asymmetry. However, this dose of muscimol induced motor biases, which make difficult to ascertain the involvement of GABA(A) receptors in ALA-induced behavioral effects. This study suggests that glutamatergic mechanisms underlie the ALA-induced convulsions and body asymmetry. The present results may be of value in understanding the physiopathology of the neurological dysfunction occurring in acute porphyrias.

Ascorbic acid and alpha-tocopherol attenuate methylmalonic acid-induced convulsions.

The effects of chronic administration of alpha-tocopherol or melatonin, or acute ascorbic acid administration on the convulsant action of methylmalonic acid (MMA) were investigated in adult male rats. Animals were chronically injected with alpha-tocopherol (40 mg kg(-1), i.p.), melatonin (5 mg kg(-1), i.p.) or vehicle for 7 days. Buffered MMA (6 micromol/2 microl) or NaCl (9 micromol/2 microl) was injected intrastriatally and the animals were observed for the appearance of clonic or tonic-clonic convulsions and rotational behavior. Ascorbic acid (100 mg kg(-1), s.c.) was administered 30 min before MMA injection. Alpha-tocopherol and ascorbic acid pretreatment decreased the duration of the convulsive episodes and the rotational behavior elicited by MMA. This study provides evidence that free radical generation may participate in the convulsant effects of methylmalonic acid.

Pharmacological evidence for GABAergic and glutamatergic involvement in the convulsant and behavioral effects of glutaric acid.

The effect of intrastriatal administration of glutaric acid (GTR), a metabolite that accumulates in glutaric acidemia type I (GA-I), on the behavior of adult male rats was investigated. After cannula placing, rats received unilateral intrastriatal injections of GTR buffered to pH 7.4 with NaOH or NaCl. GTR induced rotational behavior toward the contralateral side of injection and clonic convulsions in a dose-dependent manner. Rotational behavior was prevented by intrastriatal preadministration of DNQX and muscimol, but not by the preadministration of MK-801. Convulsions were prevented by intrastriatal preinjection of muscimol. This study provides evidence for a participation of glutamatergic non-NMDA and GABAergic mechanisms in the GTR-induced behavioral alterations. These findings may be of value in understanding the physiopathology of the neurological dysfunction in glutaric acidemia.

Effects of methylmalonate and propionate on [3H]glutamate binding, adenylate cyclase activity and lipid synthesis in rat cerebral cortex.

The effects of methylmalonic (MMA) and propionic acid (PPA), metabolites that accumulate in methylmalonic and propionic acidemia respectively, on [3H]glutamate binding, adenylate cyclase activity and [U-14C]acetate incorporation into lipids were investigated in rat cerebral cortex. Neither acid effected [3H]glutamate binding, regardless of the presence of sodium in the incubation medium. Also, the acids had no effect on basal or GMP-PNP-stimulated adenylate cyclase activity. These results suggest that MMA and PPA do not interact with glutamate binding sites and have no effect on basal or guanine nucleotide-stimulated adenylate cyclase activity. In contrast, [U-14C]acetate incorporation into brain lipids was significantly blocked by both acids, the effects being more pronounced with PPA, indicating an inhibition of brain lipid biosynthesis caused by MMA and PPA. These results may explain at least in part the hypomyelinization and/or demyelinization characteristic of patients affected by methylmalonic acidemia and propionic acidemia.

Intrahippocampal GMP administration improves inhibitory avoidance performance through GABAergic and glutamatergic mechanisms in rats.

Guanidino compounds play specific physiological and pathological roles in the central nervous system. We investigated the effect of an intrahippocampal infusion of GMP (a guanidino compound) administered immediately post-training on the inhibitory avoidance learning paradigm in rats. Bilateral intrahippocampal micro-injection of GMP (0-30 nmol) caused a dose-dependent increase in test step-down latencies which was completely reversed by intrahippocampal co-administration of muscimol or baclofen (GABA agonists) or preadministration (15 min pre-training, i.p.) of MK-801 (an NMDA antagonist). These results provide evidence for a participation of GABA and NMDA receptors in the GMP-induced increase in the test step-down latencies.

GMP reverses the facilitatory effect of glutamate on inhibitory avoidance task in rats.

Previous studies have demonstrated that post-training intrahippocampal glutamate administration improves inhibitory avoidance task performance in rats. Antagonism of the agonist actions of glutamate by guanine nucleotides has been shown at the molecular and behavioural level. In the present investigation we demonstrate that intrahippocampal co-administration of GMP (guanosine 5'-monophosphate) reverses the facilitatory effect of glutamate on the inhibitory avoidance learning paradigm and inhibits [3H]glutamate binding in hippocampal synaptic plasma membranes. These results suggest that guanine nucleotides may modulate glutamate actions.

Intrastriatal methylmalonic acid administration induces rotational behavior and convulsions through glutamatergic mechanisms.

The effect of intrastriatal administration of methylmalonic acid (MMA), a metabolite that accumulates in methylmalonic aciduria, on behavior of adult male Wistar rats was investigated. After cannula placing, rats received unilateral intrastriatal injections of MMA (buffered to pH 7.4 with NaOH) or NaCl. MMA induced rotational behavior toward the contralateral side of injection and clonic convulsions in a dose-dependent manner. Rotational behavior and convulsions were prevented by intrastriatal preadministration of MK-801 and attenuated by preadministration of succinate. This study provides evidence for a participation of NMDA receptors in the MMA-induced behavioral alterations, where succinate dehydrogenase inhibition seems to have a pivotal role.

Neurochemical effects of L-pyroglutamic acid.

The effect of L-pyroglutamic acid, a metabolite that accumulates in pyroglutamic aciduria, on different neurochemical parameters was investigated in adult male Wistar rats. Glutamate binding, adenylate cyclase activity and G protein coupling to adenylate cyclase were assayed in the presence of the acid. L-pyroglutamic acid decreased Na(+)-dependent and Na(+)-independent glutamate binding. Basal and GMP-PNP stimulated adenylate cyclase activity were not affected by the acid. Furthermore, rats received unilateral intrastriatal injections of 10-300 nmol of buffered L-pyroglutamic acid. Vehicle (0.25 M Tris-Cl, pH 7.35-7.4) was injected into the contralateral striatum. Neurotoxic damage was assessed seven days after the injection by histological examination and by weighing both cerebral hemispheres. No difference in histology or weight could be identified between hemispheres. These results suggest that, although capable of interfering with glutamate binding, pyroglutamate did not cause a major lesion in the present model of neurotoxicity.

Handling during suckling alters rat behavior but does not reverse the deleterious effects of undernutrition on naltrexone-induced inhibition of exploratory activity.

The effects of undernutrition during suckling, early environmental stimulation and naltrexone administration on the locomotor activity test were investigated in adult rats. Young rats were undernourished from delivery until weaning (23 days) by feeding their dams a diet containing 7% casein diet. Mothers of well-nourished rats were fed a 25% casein diet. After weaning, both groups of rats received standard lab chow. From day 2 to day 8 after delivery the animals were stimulated by being placed individually in plastic containers for 4 minutes. From day 12 to 19 the litters were removed from their home cages and placed for 10 min in plastic cages. Exploratory behavior was assessed using a biocompartmentalized open field. The number of crossings from one compartment to the other and the number of rearings were recorded. Undernourished animals crossed less frequently than control animals and presented a higher latency to the first crossing response. Early handling increased the total number of rearing responses regardless of nutritional treatment. Naltrexone administration caused a dose-dependent reduction in the number of rearing responses only in the well-nourished group.

Lead exposure and latent learning ability of adult female rats.

The present report investigates the effects of lead exposure during the pre- and postnatal period on the latent learning ability in a maze. Rats were submitted to lead acetate intoxication by giving their dams 0.5, 1.0, or 4.0 mM lead acetate or deionized water. Rats were weaned at 21 days, and the treatment was continued by giving the same solution of lead acetate in the drinking water until behavioral testing. Under a nonappetitive condition, rats were exposed either to an open field or to a maze apparatus. They were thereafter deprived of food and tested in the maze apparatus. Lead treatment induced an increase in locomotor activity in the open field during training sessions, and animals failed to habituate to the environment. Nevertheless, both lead-treated and control rats that had been previously exposed to the maze performed better than those exposed to the open field. These results suggest that rats intoxicated with lead are able to learn about the environment when no immediate reinforcement is involved.