Methamphetamine mimics the neurochemical profile of aging in rats and impairs recognition memory.

Brain neurochemistry and cognition performance are thought to decline with age. Accumulating data indicate that similar events occur after prolonged methamphetamine (MA) exposure. Using the rat as a model, the present study was designed to uncover common alteration patterns in brain neurochemistry and memory performance between aging and prolonged MA exposure. To this end, animals were treated with a chronic binge MA administration paradigm (20mg/kg/day from postnatal day 91 to 100). Three-age control groups received isovolumetric saline treatment and were tested at the MA age-matched period, and at 12 and 20 months. We observed that both MA and aged animals presented a long, but not short, time impairment in novelty preference and an increased anxiety-like behavior. Neurochemical analysis indicated similar MA- and age-related impairments in dopamine, serotonin and metabolites in the striatum, prefrontal cortex and hippocampus. Thus, the present data illustrate that MA may be used to mimic age-related effects on neurotransmitter systems and advocate MA treatment as a feasible animal model to study neuronal processes associated with aging.

Oxidative stress response in the adult rat retina and plasma after repeated administration of methamphetamine.

Methamphetamine (MA) is a psychostimulant that target the sensory systems, with the neurosensory retina having been shown to be affected. In the brain, MA-related toxicity can be linked to oxidative stress; the same relationship has yet to be established for the retina. The aim of this study, therefore, was to evaluate the effects of repeated exposure to MA on oxidative stress parameters in the rat retina. Oxidative stress parameters in the blood plasma were also assessed. Male Wistar rats were given 5mg/kg MA every 2h for a period of 6h (i.e., 4 injections) daily between postnatal day (PND) 91 and 100. Evolution of body weight was registered. Rats were sacrificed at PND 110. Blood plasma was collected and immediately frozen for storage at -70 degrees C. The eyes were enucleated, and the retina and choroids rapidly dissected on ice under dim light also to be stored at -70 degrees C. Lipid peroxidation activity was measured by the thiobarbituric acid (TBA) test. Total antioxidant status, superoxide dismutase (SOD) activity, catalase (Cat) activity, and nitrogen oxides contents were also determined. Lipid peroxidation was significantly higher in the retina and blood plasma of the MA-treated rats. Total antioxidant levels were significantly lower in both retina and blood plasma of the MA-treated rats. The activity of SOD was significantly increased in the retina and blood plasma of MA-treated rats. Catalase activity did not differ between groups in either the retina or the blood plasma. Nitric oxide production was significantly higher in both the retina and blood plasma in the MA-treated animals. The overall findings show that the oxidative stress defence mechanisms in the retina are compromised by MA toxicity. The results are similar to those found in the brain, and, moreover, showed some correlation with the blood plasma.

Ecstasy-induced oxidative stress to adolescent rat brain mitochondria in vivo: influence of monoamine oxidase type A.

The administration of a neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') to the rat results in mitochondrial oxidative damage in the central nervous system, namely lipid and protein oxidation and mitochondrial DNA deletions with subsequent impairment of the correspondent protein expression. Although these toxic effects were shown to be prevented by monoamine oxidase B inhibition, the role of monoamine oxidase A (MAO-A) in MDMA-mediated mitochondrial damage remains to be evaluated. Thus, the aim of the present study was to clarify the potential interference of a specific inhibition of MAO-A by clorgyline, on the deleterious effects produced by a binge administration of a neurotoxic dose of MDMA (10 mg MDMA/kg of body weight, intraperitoneally, every 2 hours in a total of four administrations) to an adolescent rat model. The parameters evaluated were mitochondrial lipid peroxidation, protein carbonylation and expression of the respiratory chain protein subunits II of reduced nicotinamide adenine dinucleotide dehydrogenase (NDII) and I of cytochrome oxidase (COXI). Considering that hyperthermia has been shown to contribute to the neurotoxic effects of MDMA, another objective of the present study was to evaluate the body temperature changes mediated by MDMA with a MAO-A selective inhibition by clorgyline. The obtained results demonstrated that the administration of a neurotoxic binge dose of MDMA to an adolescent rat model previously treated with the specific MAO-A inhibitor, clorgyline, resulted in synergistic effects on serotonin- (5-HT) mediated behaviour and body temperature, provoking high mortality. Inhibition of MAO-A by clorgyline administration had no protective effect on MDMA-induced alterations on brain mitochondria (increased lipid peroxidation, protein carbonylation and decrease in the expression of the respiratory chain subunits NDII and COXI), although it aggravated MDMA-induced decrease in the expression of COXI. These results reinforce the notion that the concomitant use of MAO-A inhibitors and MDMA is counter indicated because of the resulting severe synergic toxicity.

Exploratory behavior in rats postnatally exposed to cocaine and housed in an enriched environment.

Exposure to cocaine in early periods of postnatal life is usually associated with changes in development of neurotransmitter systems and structure of the central nervous system. Such changes are most likely correlated with behavioral alterations. Environmental enrichment conditions (EC) in early stages is a factor that affects structural and behavioral development. The purpose of this study is to examine the effects of EC on rats postnatally exposed to cocaine on exploratory behavior. Wistar rats were assigned to four groups-Group 1: pups exposed to cocaine hydrochloride (15 mg/kg body weight/day) s.c., in two daily doses, from postnatal day (PND) 1 to 28 and reared in EC; Group 2: pups exposed to cocaine as previously described and reared in a standard environmental conditions (SC); Group 3: pups saline-injected and reared in EC; and Group 4: pups saline-injected and reared in SC. On PND 21, 24, and 28, groups of four rats (to reduce anxiety) were placed for 10 minutes into an arena with several objects. The following exploratory behavioral categories were examined: object interaction, exploration, manipulation, approximation, and total time of object contact. Animals from Group 2 showed decreased object interaction and total contact on PND 21. Control offspring reared in EE showed decreases in exploratory behavior at all ages analyzed compared with the control SE group, while cocaine-exposed animals reared in EC showed decreased object interaction, object approximation, and total exploratory behavior. The results in this group suggest that EC improved information acquisition and memory processes in animals postnatally exposed to cocaine.

Hormonal, neurochemical, and behavioral response to a forced swim test in adolescent rats throughout cocaine withdrawal.

The use of cocaine in adults has been linked to depression and/or anxiety. Several studies have shown an association between cocaine-primed craving and depressive symptoms. In animal models, the forced swim test (FST) is frequently used for screening depressive-like behavior. This study aimed to verify the presence of depression-like symptoms in adolescent rats after chronic cocaine exposure by analyzing behavior in a FST. The subsequent alterations in neurotransmitters and hypothalamus-pituitary-adrenal axis activity induced by this test were also analyzed. Both male and female adolescent Wistar rats were submitted to a chronic "binge" pattern of administration of cocaine hydrochloride, and subjects were tested in a forced swim test 2 days after cocaine's last administration. At the end of the behavioral test, trunk blood was collected for quantification of corticosterone plasma levels, and hypothalamus, prefrontal cortex, amygdala, and hippocampus were dissected for neurochemical determinations. No significant differences were found in the behavior on the FST of both males and females after withdrawal from chronic cocaine administration. Nevertheless, plasma levels of corticosterone were increased in cocaine-treated males, although not significantly (P= 0.065). In females cocaine failed to affect corticosterone levels. Of interest, neurochemical analyses showed that dopamine turnover was decreased in amygdala in cocaine-treated males (not significantly, P= 0.055). No significant differences were found on neurotransmitter levels in the other brain regions analyzed. Withdrawal from chronic cocaine administration during adolescence did not have a significant effect on stress-induced behavioral alterations, although the neurochemical response to the stressful situation provided by FTS seemed to be affected.

Correlation of axon size and myelin occupancy in rats prenatally exposed to methamphetamine.

The abuse of methamphetamine (MA) and other psychostimulants is a social and medical problem. In particular, the use of these drugs by pregnant women results in an increased number of children exposed prenatally to psychostimulants. Our previous work has demonstrated that prenatal exposure to MA affects the normal development of the rat visual system due to alterations of biochemical mechanisms and oxidative stress. It was also demonstrated that prenatal exposure to MA affects the dopaminergic system of the rat retina and optic nerve (ON) myelination. The present work was conducted to evaluate the effects of prenatal exposure to MA on the development of the ON in terms of axon growth and the myelin sheath. Pregnant female rats were given 5 mg/kg/day MA, subcutaneously (s.c.), in 0.9% saline from gestational day (GD) 8 to 22. The pair-fed control group was injected s.c. with an isovolumetric dose of 0.9% saline. Qualitative analysis was performed using representative electron ultramicrographs. Quantitative analysis was performed at an electron microscopic level on ON cross sections; parameters measured included myelinated/unmyelinated ratio, outer axon mean area, inner axon mean area, myelin mean area, myelin occupancy and distribution of axons by size. The ON of prenatally MA-exposed rats presented a higher rate of deformed axons and slighter lamellar separation. At PND 21, the average outer axon area of MA-treated males was significantly reduced. The average inner axon area only showed a significant difference between MA and control males for axons with an area of less than 0.3 microm(2). The average myelin area of MA-treated males was significantly reduced, and in MA-treated females was only significantly reduced in axons with an area of less than 0.3 microm(2). The percentage of myelin occupancy was significantly affected in MA-treated males, and in MA-treated females in the group of axons with an area of more than 0.3 microm(2). At PND 14 no significant differences were found between MA and control groups. The spectrum of ON myelinated axon size of MA-treated animals was shifted to the left at PND 14 and PND 21 for both genders. These results are in agreement with previous animal studies of prenatal and perinatal exposure to drugs of abuse. Taken together, these data indicate that the ON is vulnerable to early exposure to MA which causes developmental changes and may interfere with the functioning of the visual system.

Monoamine oxidase-B mediates ecstasy-induced neurotoxic effects to adolescent rat brain mitochondria.

3,4-Methylenedioxymethamphetamine (MDMA)-induced neurotoxicity and the protective role of monoamine oxidase-B (MAO-B) inhibition were evaluated at the mitochondrial level in various regions of the adolescent rat brain. Four groups of adolescent male Wistar rats were used: (1) saline control, (2) exposed to MDMA (4 x 10 mg/kg, i.p.; two hourly), (3) treated with selegiline (2 mg/kg, i.p.) 30 min before the same dosing of MDMA, and (4) treated with selegiline (2 mg/kg, i.p.). Body temperatures were monitored throughout the whole experiment. Animals were killed 2 weeks later, and mitochondria were isolated from several brain regions. Our results showed that "binge" MDMA administration causes, along with sustained hyperthermia, long-term alterations in brain mitochondria as evidenced by increased levels of lipid peroxides and protein carbonyls. Additionally, analysis of mitochondrial DNA (mtDNA) revealed that NDI nicotinamide adenine dinucleotide phosphate dehydrogenase subunit I and NDII (nicotinamide adenine dinucleotide phosphate dehydrogenase subunit II) subunits of mitochondrial complex I and cytochrome c oxidase subunit I of complex IV suffered deletions in MDMA-exposed animals. Inhibition of MAO-B by selegiline did not reduce hyperthermia but reversed MDMA-induced effects in the oxidative stress markers, mtDNA, and related protein expression. These results indicate that monoamine oxidation by MAO-B with subsequent mitochondrial damage may be an important contributing factor for MDMA-induced neurotoxicity.

Postnatal exposure to cocaine in rats housed in an enriched environment: effects on social interactions.

This study was undertaken to evaluate the effects of environmental enrichment (EE) in rats exposed to cocaine during the first month of postnatal life by examining several categories of social behaviour (play fighting, social investigation, comfort behaviours and invitation to play). Wistar rats were divided in four groups: pups exposed to cocaine hydrochloride (15 mg/kg body weight/day), sc, in two daily doses, from postnatal day (PND) 1 to 28 and reared in EE; exposed to cocaine as previously described and reared in standard environment (SE); saline-exposed and reared in EE; pups saline-exposed and reared in SE. On PND 21, 24 and 28, social interactions were examined for 10 min. Results show that cocaine animals reared in SE decreased the frequency of play solicitation. Control animals reared in EE exhibited decreased play fighting and social investigation behaviours compared to SE-reared rats. Animals postnatally exposed to cocaine when reared in EE displayed more comfort and invitation to play behaviours and decreased social investigation compared with SE-reared animals. The results suggest that in rats postnatally exposed to cocaine, EE rearing elicited differences in both processing of environmental stimuli and a response to social challenges.

Effects of prenatal exposure to methamphetamine on the development of the rat retina.

In recent years there has been growing use of methamphetamine (METH) by pregnant women, resulting in an increasing number of children exposed prenatally to this drug of abuse. METH is known to be potentially neurotoxic to human adults, but there is minimal information with respect to the consequences of such exposure to the fetus. The purpose of this study was to ascertain external parameters of animal development, as well as neurochemical and immunohistochemical alterations at three key points of retinal development (postnatal day [PND] 7, 14, and 30). Rats of the Wistar strain were used in this experimental model. Pregnant females received a dose of 5 mg/kg body weight per day of METH-HCl in 0.9% saline, from gestational day (GD) 8 to 22. The control group to be used was pair fed and saline injected. Litters were randomly culled at PND 1 to 8 pups. Analysis of maternal body weight gain during pregnancy showed that females treated with METH had lower body weights than control-treated females. The body weight on PND 1, showed that animals treated with METH prenatally had smaller body weights than the control-treated animals and also that females weighed less than males. Prenatal exposure to METH did not alter the retinal levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the male group and the level of dopamine (DA) in both female and male groups when compared with their respective pair fed control groups during the first month of life. Correlating with the neurochemical data, no obvious changes on the localization of TH immunoreactivity in the rat retina at PND 7, 14, and 30 could be detected between control and METH-treated animals. Thus, exposure to METH disrupted this pattern in a gender-dependent manner. These data confirm previous observation that developing rats are protected against the adult type of METH-induced neurotoxicity. Therefore, conventional markers used for adult animals appear to be unsatisfactory to demarcate boundaries of the PND 1 to 30 critical periods.

Effects of postnatal exposure to methamphetamine on the development of the rat retina.

Since the development of different cell types in the retina occurs at different rates, it is possible that exposure to an exogenous substance may produce effects during one time period, but not during another. This study aims to analyze the effects of methamphetamine (METH) in the growth pattern of an experimental model as well as neurochemical and immunohistochemical parameters of the dopaminergic system of the rat retina. The three development stages chosen in this study are key markers in rat eye development. Rats were given 15 mg/kg body weight per day of METH as subcutaneous injections in 0.9% saline (3 mL/kg weight/day) from the day after birth PND 1 to PND 6, PND 13, and PND 29. Each daily dose was split into two. The control group was injected subcutaneously with saline. Both the schedule and volume for injecting saline in the control group were the same as for the METH-treated group. There were no significant differences in the total number of offspring per litter among treatment groups. All offspring had similar body weight at birth. Analysis of body weight on PND 1, showed that animals treated with METH had similar body weights to control-treated animals and females had smaller weights than males. For growth evolution, only litters with a sex ratio of four males and four females were used. Animals treated with METH had smaller body weights than the control-treated animals for all ages studied (PND 7, 14, and 30). Within the control group at PND 30, a significant difference was found in the body weight of females, which was lower when compared with males. For the postnatal model, 7 deaths occurred for the METH-exposed group. No deaths occurred in the control group in a total of 16 saline-injected litters comprising 186 pups. Although the levels of dopamine (DA) was within normal values for the postnatally exposed METH group when compared with its respective control group at PND 7 and 30, at PND 14 this was not the case: in this experimental group, the level of DA was lower than in the control group for both females and males. Support for this result was not evident from the TH immunoreactivity studies, probably because the methodology lacks the sensitivity to distinguish any mild effects, such as that observed in the postnatal model at PND 14. The level of the DA metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) remained unaffected at all ages studied, for both females and males. The results obtained in this study support the view that, during the critical periods in which the catecholamines can influence the development of neurones, METH transiently affects the pattern of the dopaminergic system in the developing retina.

Prenatal exposure to cocaine and enriched environment: effects on social interactions.

Exposure to cocaine throughout gestation may produce several deleterious outcomes in the offspring that include effects on neurotransmitter systems and structure of the central nervous system. Such changes are most likely correlated with behavioral alterations. Environmental enrichment (EE) in early stages is a factor that affects structural and behavioral development. This article examines the effects, upon social interactions, of EE during the first month of life in rats prenatally exposed to cocaine. Wistar dams were subcutaneously exposed to 60 mg/kg of cocaine divided in two daily doses from gestational day (GD)8 to GD22. Pair-fed controls were given saline vehicle in the same protocol. Offspring were distributed to the different environments in four experimental groups. Group 1: offspring from dams prenatally exposed to cocaine as previously described and reared in EE from postnatal day (PND)1 to PND28; Group 2: pups from cocaine-exposed dams and reared in a standard environment (SE); Group 3: pups from pair-fed saline-exposed dams and reared in EE; Group 4: offspring from saline-exposed dams and reared in SE. On PND21, 24, and 28, rats were examined in several social behavioral categories (play fighting, social investigation, comfort behaviors, and solicitation to play) for 10 min. Animals reared in SE do not display any differences due to treatment in the behavioral categories analyzed. Control offspring reared in EE presented decreased play fighting, decreased solicitation to play, and decreased social investigation compared to the control SE group, while cocaine-exposed animals reared in EE did not present these variations. These results suggest that EE rearing may unmask hidden effects of prenatal cocaine exposure.

Myelination changes in the rat optic nerve after prenatal exposure to methamphetamine.

The use of psychostimulants during adolescence and early adult life has increased in recent years. It is known that these substances affect the sensory systems, and the optic nerve has been shown to be a target tissue. This work was conducted to evaluate the effects of prenatal exposure to methamphetamine (MA) on the developmental pattern of the rat optic nerve. Pregnant female rats were given 5 mg/kg body weight/day MA, s.c., in 0.9% saline from gestational days 8 to 22. The control group was injected with an isovolumetric dose of 0.9% saline. Animal model parameters, such as gestational body weight evolution, food intake and pups parameters were registered. The offspring were sacrificed at postnatal days (PND) 7, 14 and 21. Morphometric analyses were performed at light and electron microscopic levels on optic nerve cross sections; parameters measured included optic nerve diameter and area, axonal density, total number of axons and myelin thickness. Myelin basic protein (MBP) was measured by western blotting in optic nerve samples at PND14 and PND21. The animal model parameters, such as maternal and pup weight, showed no significant differences between MA and control groups. Optic nerve diameter was smaller at PND7 in the male MA group and in both male and female MA groups at PND21. The mean cross-sectional area was smaller at PND14 in the male MA group and in both male and female groups at PND21. The total number of myelinated axons did not vary between groups at any of the studied ages. The myelin thickness of the axons in MA-treated females was thinner when compared with the respective control group at PND21. No other differences were found concerning myelin thickness. There was a reduction of MBP protein expression in MA-injected females at PND14 and PND21. The combined results suggest that prenatal exposure to MA affects the myelination process.

Prenatal cocaine exposure accelerates morphological changes and transient expression of tyrosine hydroxylase in the cochlea of developing rats.

Prenatal cocaine exposure causes alterations in auditory brainstem response in children and experimental animals and has adverse effects on auditory information processing and language skills in children. These effects may result from lesions in the cochlea since this organ is particularly sensitive to chemical insults during the development. We have thus studied here the effect of prenatal cocaine exposure on the maturation of the rat cochlea using the transient non-catecholaminergic expression of tyrosine hydroxylase in spiral ganglion neurons as an index of cochlear maturation and morphometry to evaluate the maturation of primary auditory neurons and the organ of Corti. We showed that prenatal cocaine exposure accelerated the cochlear maturation. In the basal coil of cochleas from PND8 cocaine-treated pups, the Kölliker's organ had disappeared, the tunnel of Corti was opened, and the stria vascularis no longer contained undifferentiated marginal cells. The maximum expression of tyrosine hydroxylase in type I primary auditory neurons occurred at PND8 instead of PND12 in pair-fed controls. On the other hand, the prenatal cocaine exposure had no effect on the width and height of the organ of Corti, spiral ganglion volume and number and size of primary auditory neurons. In conclusion, our data suggest that prenatal cocaine exposure, though not lethal to primary auditory neurons, accelerates aspects of the cochlear sensorineural maturation. This accelerated cochlear maturation in cocaine-treated rat pups could cause auditory dysfunctions by desynchronizing the development of the whole auditory pathway.

Methamphetamine and lipid peroxidation in the rat retina.

BACKGROUND: The use of psychoactive drugs during adolescence and early adult life has increased in the last few decades. It is known that developmental exposure to psychostimulants affects the sensory systems, and the retina has been shown to be a target tissue. This work was conducted to evaluate the pattern of lipid peroxidation in the rat retina following prenatal exposure to methamphetamine (MA). METHODS: Pregnant female Wistar rats were given MA (5 mg/kg of body weight/day; SC, in 0.9% saline) from GD 8 to 22. Offspring were sacrificed at postnatal days (PNDs) 7, 14, and 21. The retinas were homogenized, and both the total antioxidant and superoxide dismutase (SOD) activities were measured by enzymatic-colorimetric methods. The lipid peroxidation byproducts (malondialdehyde [MDA] and MDA-like metabolites) were measured by the thiobarbituric acid test. RESULTS: Total antioxidant levels were lower in the MA group at PND 21 in both males and females. The activity of SOD was higher in PND 7 females from the MA group. MDA levels were higher in the MA group at PND 21 in both genders. CONCLUSIONS: These findings suggest that prenatal-induced MA toxicity in the retina may be related to lipid peroxidation processes and oxidative stress.

Prenatal cocaine exposure: effects on locomotor activity in rat offspring.

This study examines the developmental effects of prenatal exposure to cocaine in the rat, evaluated during the first month of life through open-field behavior. The offspring of Wistar dams that received 60mg/kg of cocaine, from gestational day 8 to 22, were examined in the open-field during the second, third and fourth weeks of postnatal life in three consecutive 15-min daily sessions, starting on postnatal day (PND) 14, (PND 14-16), PND 21 (PND 21-23) and PND 28 (PND 28-30). Results show that prenatal exposure to cocaine increased total activity and rearing behavior on PND 22 and PND 29. Also, on PND 14, cocaine-exposed animals reared significantly more than control rats. There were no significant differences in the frequency of center and peripheral ambulation, nor in the defecation rate. The present results evidence alterations in the emotional behavior of rats prenatally exposed to cocaine. The delayed onset of exploration in the open-field observed in cocaine-exposed animals suggests that they take more time to become habituated to a novel and open environment.

Effects of postnatal cocaine exposure and environmental enrichment on rat behavior in a forced swim test.

This study examined the effects of environmental enrichment on rats exposed to cocaine during the first month of life, in several categories of behavior observed in a forced swim test. Wistar rats were divided in four groups. The first included pups that were subjected to injections of cocaine hydrochloride (15 mg/kg body weight/day, subcutaneously, in two daily doses, from postnatal days 1 to 27) and reared in an enriched environment (CocEE); the second, pups that were subjected to injections of cocaine (as previously described) and reared in a standard environment (CocSE); the third, pups that were subjected to saline injections and reared in an enriched environment (SalEE); the fourth, pups that were subjected to saline injections and reared in a standard environment (SalSE). On postnatal days 26 and 27, rats were tested in a swimming pool in two 5-min sessions. The categories of behavior studied in this work were: fast swim, slow swim, struggling, diving, and immobility. Results showed that postnatal cocaine exposure decreased the time spent on fast swim during the two sessions and increased the immobility behavior during the second session in CocSE pups compared with SalSE pups. SalEE pups increased the time spent in fast swim, slow swim, and diving, and decreased the time spent in struggling and immobility during the two sessions compared with SalSE pups. CocEE animals spent more time in fast swim and struggling and less the time in immobility compared with CocSE pups. The present results suggest that postnatal cocaine exposure affects the ability of these animals to cope with stressful situations, and that environmental enrichment seems to enable the rats to adopt a more active strategy, one that allows them to better cope with this particular stress situation.

Abnormal immunoreactivity to serotonin in cerebellar Purkinje cells after neonatal cocaine exposure.

Neonatal cocaine is known to affect the developing serotonergic system in many brain structures, including the cerebellum. Changes in the cerebellar Purkinje cells after drug exposure are well documented and result in impairment of movement and other cerebellar disorders such as ataxia. These cells have a major postnatal developmental pattern; therefore, neonatal exposure to cocaine is likely to affect them. In this work, male and female Wistar rats were injected with 15 mg of cocaine hydrochloride/kg body weight/day, subcutaneously, in two daily doses, from postnatal day 1 (PND1) to PND29. Controls were given 0.9% of saline. On PND14, PND21, and PND30, rats were transcardially perfused, and brains removed and cryoprotected. Coronal sections from the cerebellum were processed for immunocytochemistry of cells containing serotonin (5-hydroxytryptamine, or 5-HT). At the same postnatal age, rats from at least three different litters were sacrificed by decapitation, and brains were dissected for determination of 5-HT in the cerebellum by high-performance liquid chromatography with electrochemical detection. Upon the expected distribution of immunoreactivity to 5-HT, an abnormal immunoreactivity to 5-HT was observed in the Purkinje cells of six cocaine-exposed animals, but not in control animals. Also, levels of cerebellar 5-HT in cocaine-exposed rats were significantly increased on PND21. These results, together with previously reported observations of altered patterns of motor behavior, indicate that neonatal cocaine exposure affects the serotonergic cerebellar system, altering the standard development of Purkinje cells and possibly compromising the motor function.

Catecholamine-independent transient expression of tyrosine hydroxylase in primary auditory neurons is coincident with the onset of hearing in the rat cochlea.

During the last stages of neuronal maturation, tyrosine hydroxylase is transiently expressed in the absence of the other catecholamine-synthesizing enzymes. We show here that it is expressed in rat spiral ganglion neurons between postnatal days 8 and 20, with a peak of expression at postnatal day 12. These tyrosine hydroxylase-immunoreactive neurons did not display aromatic amino acid decarboxylase- or dopamine-beta-hydroxylase-immunoreactivities, ruling out the possibilities of dopamine or noradrenaline synthesis. They also did not display peripherin- or intense neurofilament 200-kDa-immunoreactivities, two indicators of type II primary auditory neurons. Tyrosine hydroxylase-immunoreactive dendrites were seen in synaptic contact with the inner hair cells and expressed the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, further confirming the type I nature of the neurons transiently expressing the enzyme. The end of the tyrosine hydroxylase expression was not due to cell death because the immunoreactive neurons did not show TUNEL-labelled nuclei. Finally, all the type I neurons expressed the tyrosine hydroxylase mRNA at postnatal day 12, suggesting that the expression of the enzyme is a maturational step common to all these neurons and that the expression of the protein is not synchronized. Because the period of transient expression of tyrosine hydroxylase in type I neurons parallels the periods of maturation of evoked exocytosis in inner hair cells and of appearance and maturation of the cochlear potentials, we propose that the expression of the enzyme indicates the onset of hearing in individual type I primary auditory neurons. This enzyme expression could rely on a Ca2+ activation of its encoding gene subsequent to a sudden and massive Ca2+ entry through voltage-activated Ca2+ channels.

Neonatal exposure to cocaine: altered dopamine levels in the amygdala and behavioral outcomes in the developing rat.

The amygdala is a brain region that is known to be implicated in the development of behavioral sensitization to cocaine. This area is often related to conditioned associations, stress responses, and anxiety; and these behaviors are usually posited to be due to altered dopamine levels. This study aimed to evaluate the effects of neonatal exposure to cocaine on the levels of neurotransmitters in the amygdala of developing rats and to relate these levels with open-field observations, mainly rearing behavior, that is regarded to reflect emotional components. Male and female Wistar rats were given 15 mg of cocaine hydrochloride/kg body weight, subcutaneously, in two daily doses, from postnatal day 1 (PND1) to PND30. Controls were given 0.9% saline. Open-field activity was registered on PND14, 21, and 30 in three sessions of 15 min each. In PND30, rats were decapitated, and the amygdala dissected from both brain hemispheres and processed for determination of dopamine (DA) and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-EC). Results show that in PND14 and 21 all registered activity behaviors were increased in male and female cocaine-exposed animals. In PND30, there was a significant decrease in rearing and in global activity in the group exposed to cocaine, and DA levels were significantly decreased in the amygdala of the same group. No differences were found between the left and right amygdala. These results suggest that chronic neonatal cocaine administration leads to depletion of DA levels in the amygdala, which is consistent with previous findings. Furthermore, the lower levels of DA are associated with decreased rearing behavior, which may indicate emotional depression. These results can help to clarify the role of amygdala in cocaine-induced behavioral sensitization in the developing rat.

Structural and functional cellular alterations underlying the toxicity of methamphetamine in rat retina and prefrontal cortex.

The consumption of illicit drugs is an increasing problem in contemporary societies, and is one of the major causes of death and illness all over the world. Methamphetamine is among the drugs more widely used. Although evidence for a role of reactive species--especially reactive oxygen species (ROS) and apoptotic events--has been shown, the mechanism(s) underlying the cellular toxicity induced by this drug is not yet fully identified. In this context the elucidation of the cytotoxic effects induced by methamphetamine in rat frontal cortex and retina, which compromise cell viability and ultimately result in cell death, can further contribute to the understanding of its mechanism of action. This knowledge may provide new insights into the development of new therapeutic approaches to prevent or ameliorate deleterious alterations of the nervous system. The use of epifluorescence microscopy associated with different fluorescent probes, markers of structural and/or functional cell parameters, can be used as a powerful tool to carry out those studies, in particular, the viability probes propidium iodide (PI) to assess plasma membrane integrity and fluorescein diacetate (FDA), which can monitor intracellular esterase activity and/or pH. In a preliminary study, the kinetic assessment of cellular changes induced by different drug concentrations (0, 1.2, 3, and 6 mM) allowed detection of dose-dependent alterations that are observed earlier in the retina. In fact, in the retina it was possible to monitor alterations (at 4 h of incubation) both in plasma membrane integrity and in esterase activity and/or pH for the lowest drug concentration (1.2 mM). In the prefrontal cortex these changes were only visible for drug concentrations > or = 3 mM. This work is a novel approach to the mechanisms of action of illicit drugs in the central nervous system and will provide the foundations and guidelines for further investigations in the context of tolerance, dependence, and addiction.