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Efficient reuptake of synaptically released glutamate is essential for preventing glutamate receptor overstimulation and neuronal death. Glutamate transporters play a vital role in removing extracellular glutamate from the synaptic cleft. This study analyzed the expression of the glial (GLAST) and neuronal (EAAC1) subtypes of glutamate transporter in the cerebellum of male and female offspring exposed pre- and postnatally to Delta9-tetrahydrocannabinol (THC, the main component of marijuana). Pregnant rats were administered saline or THC from gestational day 5 to postnatal day 20 (PD20). The expression of glutamate transporters was examined at PD20, PD30 and PD70 (10 and 50 days after THC withdrawal) to analyze the short- and long-term effects of prenatal THC exposure. The expression of the glutamate transporter GLAST in astroglial cells and EAAC1 in Purkinje neurons decreased in THC-exposed offspring compared to controls. This reduction was observed at all ages but mainly in males. Moreover, the glial glutamate transporter level in THC-exposed rats (quantified by Western blot) was lower than in control rats. These results suggest that THC exposure during cerebellar development may alter the glutamatergic system not only during the period of drug exposure but in the postnatal stage following withdrawal. The down-regulation reported here might reflect an abnormal maturation of the glutamatergic neuron-glia circuitry., (2004 S. Karger AG, Basel.)

In this study we analyzed the responses of cerebellar astroglial cells to pre- and perinatal delta(9)-tetrahydrocannabinol (THC) exposure in three postnatal ages and both sexes. To determine whether THC during development directly modifies astroglial growth, this study investigated the effects of THC on astroglial morphological changes and on the expression of specific astroglial markers (glial fibrillary acidic protein: GFAP and glutamine synthetase: GS). Our results demonstrated that the administration of THC during development has deleterious effects on astroglial maturation in the cerebellum. These results also indicate that THC might interfere with astroglial differentiation in a way dependent on sex. The effect of cannabinoids on the development of cerebellar astroglial cells (astrocytes and Bergmann glial cells) is to reduce protein synthesis, since both GFAP and GS decreased in astroglial cells, not only during THC exposure but also in adult ages. Our data suggest that pre- and perinatal THC exposure directly interferes with astroglial maturation by disrupting normal cytoskeletal formation, as indicated by the irregular disposition of GFAP and the lower GFAP expression observed at all the ages studied. THC exposure during development may also modulate glutamatergic nervous activity since GS expression is reduced in THC-exposed brains. GS expression increased progressively after THC withdrawal, but GS expression had still not reached control values two months after THC withdrawal. This indicates that glutamate uptake is lower in glial cells exposed to THC, since GS expression is lower than in older controls. Consequently, glutamatergic neurotransmission may be affected by cannabinoid exposure during gestation. Therefore, cannabinoids exert developmental toxicity, at least on astroglial cells, which could contribute to fetal brain growth retardation., (Copyright 2002 S. Karger AG, Basel)

The responses of neurons and astroglial cells to pre- and perinatal exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) were evaluated in the substantia nigra (SN) of male and female rats, at three postnatal ages (PD21, PD30 and PD70), by immunohistochemical detection of tyrosine hydroxylase (TH) in dopaminergic neurons and of glial fibrillary acidic protein (GFAP) in astrocytes. Our results showed that the effects of pre- and perinatal exposure to Delta(9)-THC on neuronal and astroglial immunoreactivities in the SN (compacta and reticulata) varied with sex, with male rats being more susceptible than females. Prenatal exposure to Delta(9)-THC decreased TH immunoreactivity in the SN of males on PD21 when compared to both their controls and Delta(9)-THC-exposed females of the same age. Furthermore, the TH expression decreased with age in Delta(9)-THC-exposed males in the SNc pars compacta, whereas it increased in controls. On the contrary, TH expression was maintained stable in the SN pars compacta of Delta(9)-THC-exposed females from PD21. These differences in neuronal development caused by prenatal Delta(9)-THC exposure were associated with significant differences in GFAP expression by astroglial cells in both sexes. On PD21, GFAP immunoreactivity decreased in the SN in Delta(9)-THC-exposed male rats. Although GFAP expression increased in Delta(9)-THC-exposed males with age, it did not reach control levels by PD70. On the contrary, significantly increased GFAP expression in the Delta(9)-THC-exposed females on PD21 was observed, compared to their controls and also to Delta(9)-THC-exposed male rats; however, the GFAP expression shown by Delta(9)-THC-exposed females stabilized from PD21. These Delta(9)-THC-induced changes in the glial development could indicate that Delta(9)-THC accelerated the maturation of astrocytes in female rats, whereas Delta(9)-THC delayed astrocytic maturation in Delta(9)-THC-exposed males. These findings suggest that pre- and perinatal exposure to Delta(9)-THC can lead to long-term effects in both neurons and glial cells., (Copyright 2000 S. Karger AG, Basel.)

We have extensively reported that delta9-tetrahydrocannabinol (delta9-THC) exposure results in changes in the adult functionality of dopaminergic neurons, in particular, mesotelencephalic pathways, although some changes are evident only after pharmacological challenges. In the present study, we have examined whether similar changes might be observed in gamma-aminobutyric acid (GABA) activity, in particular, in those regions where cannabinoid receptors have been reported to be located in GABA-containing neurons. To this end, we first examined GABA content and glutamic acid decarboxylase (GAD) activity in several brain regions of adult male and female rats that had been perinatally exposed to delta9-THC or oil. Delta9-THC exposure did not modify either GAD activity or GABA content in the ventral-tegmental area, nucleus accumbens, substantia nigra, caudate-putamen, and globus pallidus, thus suggesting no changes in the basal presynaptic activity of GABA-containing neurons. Second, we tested the motor response in the open-field test of these animals after a single injection of muscimol, a GABA(A) receptor agonist, baclofen, a GABA(B) receptor agonist, or vehicle. We observed that the motor inhibition caused by baclofen, in terms of decreased ambulation and stereotypy and increased inactivity, was more marked in magnitude in delta9-THC-exposed males and females. This was not observed for the GABA(A) receptor agonist, muscimol, indicating a receptor specificity. To extend this observation, we also examined whether the potential differences in the behavioral response found in the above experiment might be due to changes at the level of the efficiency of the activation of these receptors by measuring basal and baclofen-stimulated [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]-GTPgammaS) binding in adult male and female rats that had been perinatally exposed to delta9-THC or oil. However, our results were negative, because perinatal delta9-THC exposure did not increase baclofen-stimulated [35S]-GTPgammaS binding in the areas studied; in particular, in the substantia nigra, an area of interest for the interactions GABA(B) receptor/cannabinoid receptor. Collectively, the present results indicate that although perinatal delta9-THC did not produce any changes in GABA content and GAD activity in limbic and motor areas in adulthood, it did increase the behavioral response to GABA(B) receptor agonists. However, this increase was not due to changes in GABA(B) receptor activation of signal transduction mechanisms, as revealed the analysis of the percentage of stimulation by baclofen of [35S]-GTPgammaS binding in the substantia nigra and other structures of males and females perinatally exposed to delta9-THC.

Opiates and cannabinoids are among the most widely consumed habit-forming drugs in humans. Several studies have demonstrated the existence of interactions between both kind of drugs in a variety of effects and experimental models. The present study has been focused to determine whether perinatal delta9-tetrahydrocannabinol (Delta9-THC) exposure affects the susceptibility to reinforcing effects of morphine in adulthood and whether these potential changes were accompanied by variations in mu opioid receptor binding in brain regions related to drug reinforcement. Adult female rats born from mothers that were daily treated with delta9-THC during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle-exposed mothers, an effect that did not exist in delta9-THC-exposed male offspring. This increase was significantly greater on the last day of acquisition period. There were not significant differences when the subjects were lever pressing for food. In parallel, we have also examined the density of mu opioid receptors in the brain of adult male and female offspring that were exposed to Delta9-THC during the perinatal period. Collectively, perinatal exposure to delta9-THC produced changes in mu opioid receptor binding that differed regionally and that were mostly different as a function of sex. Thus, delta9-THC-exposed males exhibited a lower density for these receptors than their respective oil-exposed controls in the caudate-putamen area as well as in the amygdala (posteromedial cortical nucleus). On the contrary, delta9-THC-exposed females exhibited higher density of these receptors than their respective oil-exposed controls in the prefrontal cortex, the hippocampus (CA3 area), the amygdala (posteromedial cortical nucleus), the ventral tegmental area and the periaqueductal grey matter, whereas the binding was lower than control females only in the lateral amygdala. These results support the notion that perinatal delta9-THC exposure alters the susceptibility to morphine reinforcing effects in adult female offspring, in parallel with changes in mu opioid receptor binding in several brain regions., (Copyright 1998 Elsevier Science B.V.)

We have recently demonstrated that the magnitude of L-3,4-dihydroxyphenylacetic acid (DOPAC) lowering effect caused by amphetamine in midbrain dopaminergic neurons of adult rats was lesser in animals that had been perinatally exposed to delta9-tetrahydrocannabinol (delta9-THC) than controls. In the present study, we have examined whether this loss in the responsiveness to amphetamine might be due to changes at the level of dopamine transporter (DAT), the main molecular site for the action of amphetamine, following the perinatal exposure to delta9-THC. To this end, we have analyzed DAT mRNA levels, by using in situ hybridization, in the substantia nigra and ventral tegmental area, the areas where cell bodies of DAT-containing midbrain neurons are located, of adult male and female rats that had been perinatally exposed to delta9-THC. In addition, we also analyzed mRNA levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis. Results were as follows. Both adult male and female rats that had been perinatally exposed to delta9-THC exhibited similar mRNA levels to controls for both DAT and TH in the substantia nigra as well as in the ventral tegmental area. This observation makes it difficult to support the idea that the differences found in adulthood after pharmacological challenges were caused by irreversible changes at the level of gene expression for these two key proteins.

The exposure of pregnant rats to delta 9-tetrahydrocannabinol (delta 9-THC), the main psychoactive constituent of Cannabis sativa, during the perinatal period affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brains of their offspring at peripubertal and adult ages. In the present work we explored whether these effects also appear during fetal and early neonatal periods, when TH expression plays an important role in neural development. To this end, the mRNA amounts for TH and the amounts and activity of this enzyme, in addition to catecholamine (CA) contents, were analyzed in the brain of fetuses at different gestational days (GD) and of newborns at two postnatal ages, which had been daily exposed to delta 9-THC or vehicle from d 5 of gestation. Results were as follows. The exposure to delta 9-THC markedly affected the expression of the TH gene in the brain of fetuses at GD 14. Thus, the amounts of its mRNA at this age were higher in delta 9-THC-exposed fetuses than in controls. This corresponded with a marked rise in the amounts of TH protein and in the activity of this enzyme at this age. Normalization was found in these parameters at GD16. However, a marked sexual dimorphism in the response of TH gene to cannabinoid exposure appeared from GD18 and was particularly evident at GD21, when TH-mRNA amounts increased in developing female brains, but decreased in developing male brains exposed to delta 9-THC, effects that were mostly prolonged to early postnatal ages. However, these changes did not correspond always with parallel changes in the amounts and activity of TH and in CA contents, as occurred in GD14, suggesting that delta 9-THC would not be affecting the basal capability to synthesize CAs in TH-containing neurons, but would affect the responsiveness of TH gene. We found only a marked increase in the production of L-3,4-dihydroxyphenylacetic acid, the main intraneuronal dopamine metabolite, in female newborns exposed to delta 9-THC. Collectively, our results support the belief that the perinatal exposure to delta 9-THC affects the expression of the TH gene and, sometimes, the activity of this enzyme in brain catecholaminergic neurons in certain critical periods of fetal and early neonatal brain development. These results support the notion that cannabinoids are able to affect the gene expression of specific key proteins for catecholaminergic development, and that these alterations might be the origin of important long-term neurobehavioral effects caused by perinatal cannabinoid exposure at peripubertal and adult ages.

The present study has been designed to explore further the existence of a persistent, but 'silent' alteration in the adult functionality of midbrain dopaminergic neurons following perinatal cannabinoid exposure. To this end, we evaluated the responsiveness of these neurons, measured at the neurochemical or behavioral levels, to pharmacological challenges with a variety of dopaminergic drugs administered to adult male and female rats that had been exposed to delta 9-tetrahydrocannabinol (THC) or vehicle during the perinatal period. Results were as follows: In the first experiment, we tested the magnitude of motor inhibition caused by administration of dopaminergic receptor antagonists. The most interesting observation was that the administration of SCH 23390, a D1 antagonist, produced a more marked motor inhibition, reflected by a greater decrease in the ambulation measured in an open-field test, in adult animals of both sexes when they had been exposed perinatally to THC. This did not occur with the motor inhibition caused by sulpiride, a D2 antagonist. In the second experiment, we evaluated the sensitivity of midbrain dopaminergic neurons to amphetamine (AMPH), which causes, through different mechanisms, a decrease in dopamine (DA) metabolism. The most interesting observation was that adult females, when exposed perinatally to THC, exhibited a trend to lesser response to AMPH, in terms of decreasing DA metabolism, than oil-exposed females. This was observed in dopaminergic terminals reaching the limbic forebrain area, but not in those terminals reaching the striatum, and was a specific effect for THC-exposed adult females because it was not observed in THC-exposed adult males. In the third experiment, we evaluated the in vivo synthesis of DA in midbrain dopaminergic neurons by analyzing the magnitude of L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation caused by the blockade of L-DOPA decarboxylase with NSD 1015. The most worthy finding was that, as occurred in the above experiment, adult females, when exposed perinatally to THC, tended to exhibit a higher ability to synthesize DA in vivo in the limbic forebrain but not in the striatum, as reflected by the increased L-DOPA accumulation observed after NSD 1015 administration. As in the above experiment, this was not seen in males. In summary, our results are consistent with the possible existence of subtle and sexually dimorphic changes in the sensitivity of midbrain dopaminergic neurons in adulthood caused by the exposure to THC during perinatal development. These silent changes could be revealed after the administration of drugs which specifically act on key processes of dopaminergic neurotransmission, such as the synthesis, reuptake and catabolism of DA and its binding to receptors.

We have previously reported that the exposure of pregnant female rats to delta 9-tetrahydrocannabinol (THC) during the perinatal period affected the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their male offspring. Those studies were done in animals perinatally exposed to THC but tested at peripubertal and adult ages. In the present work, we explored whether these effects also appear during early fetal brain development, when TH expression plays an important role in neuronal development. To this end, TH-mRNA concentrations were measured by Northern blot analysis with a specific TH probe in the brain of fetuses at gestational days 14 and 16 which had been prenatally exposed to THC or vehicle from day 5 of gestation. In parallel, measurements of TH activity and catecholamine contents by HPLC were also done. The results obtained were as follows. The prenatal exposure to THC markedly affected the expression of the TH gene in the brain of fetuses at gestational day 14. Thus, the amounts of TH-mRNA at this age were higher (2-fold) in THC-exposed fetuses than in controls. This corresponded with a marked increase in the activity of this enzyme (3-fold) at this age. Normalization was found in both parameters at gestational day 16. In summary, the prenatal exposure to THC affected the expression of the TH gene and the activity of this enzyme in brain catecholaminergic neurons during early fetal brain development. These results support the notion that cannabinoids are able to act at the level of the gene expression of specific key proteins for brain development.

The changes induced by maternal exposure to cannabinoids in the maturation of nigrostriatal, tuberoinfundibular and mesolimbic dopaminergic activities of rat offspring 15-40 days old were studied. In the striatum, tyrosine hydroxylase activity was constantly decreased during cannabinoid exposure in males. This decrease was correlative to increased number of D1 and D2 dopaminergic receptors. Both effects were also observed after the drug withdrawal caused by weaning on day 24. In females, the most consistent effect appeared on day 20, when decreased dopamine content and number of D1 receptors were observed. Both effects disappeared after drug withdrawal, but the reduction in the number of D1 receptors was again observed 40 days after birth. In the limbic area, cannabinoid exposure caused a decrease in the number of D1 receptors in 15-day-old females, along with decreases in the content of dopamine and its metabolite, L-3,4-dihydroxyphenylacetic acid. Changes in receptors disappeared on subsequent days, but increases in L-3,4-dihydroxyphenylacetic acid content and in its ratio with dopamine (L-3,4-dihydroxyphenylacetic acid/dopamine) were observed on day 20 followed by a decrease in the neurotransmitter content on day 30. In males, tyrosine hydroxylase activity increased on day 30, followed by an increase in L-3,4-dihydroxyphenylacetic acid content and L-3,4-dihydroxyphenylacetic acid/dopamine ratio on day 40. In the hypothalamus, the cannabinoid effects were always manifested after the cessation of drug exposure. Thus, a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio was observed in 30-day-old females, and it was followed by a decrease on day 40, accompanied by a decrease in the anterior pituitary content of dopamine. Rise in prolactin release was not significant. In males, tyrosine hydroxylase activity was increased 30 days after birth, while L-3,4-dihydroxyphenylacetic acid content decreased. On day 40, L-3,4-dihydroxyphenylacetic acid content increased, paired to a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio and anterior pituitary content of dopamine and to a decrease in the prolactin release. Perinatal exposure to cannabinoids altered the normal development of nigrostriatal, mesolimbic and tuberoinfundibular dopaminergic neurons, as reflected by changes in several indices of their activity. These changes were different regarding the sex and brain areas. Cannabinoid effects were more marked and constant in the striatum of males, while alterations in limbic neurons were mostly transient and those in hypothalamic neurons occurred after drug withdrawal. A long-term impact of these early changes on the neurological processes of adulthood is plausible.