perinatal Δ 9-tetrahydrocannabinol exposure in rats modifies the responsiveness of midbrain...

Drug and Alcohol Dependence 42 (1996) 155.-166

Perinatal A9-tetrahydrocannabinol exposure in rats modifies the responsiveness of midbrain dopaminergk neurons in adulthoud to a

variety of challenges with dopaminergic drugs’

Lucia Garcia, Rosario de Miguel, Jo& A. Ramos, J. Javier Fernkndez-Ruiz*

Institutn Comphplzrtense de Drogodependencias, Department of Biochemistry, Facult~~ of Medicine. Complutense ~~niw~sit~~, ?KO4(l-Mndrid, Spain

Received IO April 1996; revised 10 July 1996; accepted IO July 1996

Abstract

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 A9-tetrahydrocarmabinol (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 D, 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 D, 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 dopdminergic 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.

Keywords: Cannabinoids; AY-tetrahydrocannabinol; Midbrain dopaminergic neurons: Dopamine: Tyrosine hydroxylase; D, and D, receptors: Motor behavior: SCH 23390; Sulpiride; Amphetamine: NSD 1015

1. Introduction * Corresponding author. Tel.: + 34 1 3941450; fax: + 34 I

3941691; e-mail: [email protected] ! Parts of this work have been previously presented in abstract

form and published in the Proceedings of 1995 Meeting of Interna- tional Cannabis Research Society (Scottsdale, AZ, USA).

We have demonstrated extensively that p&natal exposure to cannabinoids markedly afrects the maturation of several dopaminergic systems (Rodriguez de

0376-8716’96/$35.00 c 1996 Elsevier Science Ireland Ltd. All rights reserved PII S037h-G7 16(96)0 1276.8

156 L. Garcia et al. 1 Drug and Alcol~ol Dependence 42 (1996) 155-166

Fonseca et al., 1991; Fernandez-Ruiz et al., 1992, 1994; Bonnin et al., 1994). These effects appear early, even before the complete differentiation and maturation of dopaminergic projections into their target areas (Ro- driguez de Fonseca et al., 1992), in particular during the last third of gestation and first days of postnatal life, when cannabinoids seem to alter the expression of key genes for development of dopaminergic neurotransmission (Bonnin et al., 1995; Fernandez-Ruiz et al., 1996). Among the dopaminergic pathways affected most by the perinatal exposure to cannabinoids are the midbrain dopaminergic neurons (Rodriguez de Fonseca et al., 1991), in particular those neurons whose fibers reach the striatum and constitute the nigrostriatal dopaminergic system (Bonnin et al., 1994; Navarro et al., 1994), although we have also found changes in the other major component of midbrain dopaminergic fibers that reach the limbic forebrain nuclei and constitute the mesolimbic system (Rodriguez de Fonseca et al., 1991). The most characteristic effect, by its magnitude and persistence, was a decrease in the activity of tyrosine hydroxylase (TH) in the striatum of males perinatally exposed to cannabinoids (Rodriguez de Fonseca et al., 1991; Fernandez-Ruiz et al., 1994). This effect was observed permanently along the peripubertal period (15-40 days after birth), in parallel with a slight decrease in motor activity (Navarro et al., 1994). Wal- ters and Carr (1986) also found decreases in the activity of this enzyme at immature ages after perinatal exposure to marihuana extracts. Accompanying this modifi- cation in TH activity, we have also found changes in gene expression of this enzyme in the midbrain (Bonnin et al., 1994), the area where cell bodies of nigrostriatal and mesolimbic TH-containing terminals are clustered, and increases in the density of both D, and D, receptors in the striatum (Rodriguez de Fonseca et al., 1991; Fernandez-Ruiz et al., 1994). As occurred with TH, this last effect was observed exclusively in males and tended to disappear after the withdrawal of the drug at wean- ing and, particularly, in adulthood (Navarro et al., 1994), presumably due to the appearance of compensa- tory mechanisms during the course of further development.

Despite the disappearance in adulthood of cannabinoid-induced presynaptic and postsynaptic changes observed over the course of postnatal development, we suspect the persistence at this age of some irreversible alterations in the functionality of these neurons, result- ing in behavioral disturbances. These alterations might not be evident in basal conditions, but may be mani- fested in particular circumstances and in a sexually dimorphic manner. We had previously formulated this hypothesis (Navarro et al., 1994) based on a set of facts involving different key processes for dopaminergic neurotransmission. Thus: (i) the levels of TH-mRNA in the midbrain of adult males perinatally exposed to

cannabinoids were lesser than in controls (Bonnin et al., 1994), although TH activity was unaffected in the regions receiving terminals from midbrain dopaminergic cell bodies (Navarro et al., 1994; Fernandez-Ruiz et al., 1994); (ii) these animals exhibited significant changes in the motor response executed when exposed to novel stimuli in an open-field test (Navarro et al., 1994), in particular, in the frequency of stereotypy whose rela- tion to specific dopaminergic receptors seems to be clearly defined (Arnt, 1987); these changes did not exist in females (Navarro et al., 1994); (iii) the activity of midbrain dopaminergic neurons in adult male rats, that had been perinatally exposed to cannabinoids, was affected by a challenge with A9-tetrahydrocannabinol (THC), the prototypical psychoactive cannabinoid, to a lesser extent than naive rats (Fernandez-Ruiz et al., 1994) whereas adult females, when exposed perinatally to cannabinoids, responded to an adult challenge with THC as did naive rats (Fernandez-Ruiz et al., 1994). Recently, we have provided the first evidence to sup- port the hypothesis of the existence of an irreversible, although ‘silent’, alteration in the adult functionality of midbrain dopaminergic neurons originated as a consequence of perinatal THC exposure. We have observed that perinatal exposure to THC alters the ability of these neurons in adulthood to respond, particularly in males, to drugs which deplete dopamine (DA) stores and induce TH gene expression, such as reserpine and a-methyl-p-tyrosine (Garcia et al., 1996).

The present work has been designed to complete these first results. Thus, we have examined the responsiveness, tested at neurochemical or behavioral levels, of these neurons when challenged pharmacologically with a variety of drugs, injected at well-defined active doses, that affect key processes for dopaminergic neurotransmission, such as DA metabolism and reuptake or D, and D, DA receptors. To this end, we have performed three different experiments. In the first experiment, we evaluated the degree of motor inhibition (measured in the open-field test) caused by the administration of specific antagonists for either D, or D, DA receptors. As mentioned previously, both receptors were affected by perinatal THC exposure during the course of postnatal development (Rodriguez de Fon- seca et al., 1991). DA antagonists have been largely reported to produce a set of motor deficits (catalepsy, inactivity), through the blockade of these receptors in the areas receiving terminals from midbrain dopaminergic neurons, when injected to adult rats (Campbell and Baldessarini, 1981; McDougall et al., 1990; Meyer, 1993; Kostrzewa and Kastin, 1993; Garrett and Holtz- man, 1994) and also when administered during the perinatal period (Scalzo et al., 1989).

In the second experiment, we evaluated the responsiveness of midbrain dopaminergic neurons to the administration of amphetamine (AMPH). This drug has

been reported to produce dopaminergic hyperactivity when injected to adult rats: (i) by reducing the DA reuptake process; (ii) by inhibiting the activity of monoamine oxidase; or (iii) via its stimulant effects on the release of an extravesicular pool of newly synthesized DA. In the present study, we have paid special attention to a common consequence, from a neurochemical point of view, of this triad of effects of AMPH: the decrease in DA intraneuronal metabolism reflected in the loss of L-3,4-dihydroxyphenylacetic acid (DOPAC) production (Elverfors and Nissbrandt, 1992). This process has been reported to be affected signih- cantly by THC during development (Rodriguez de Fon- seca et al., 1991). In the third experiment, we tested the ability of midbrain dopaminergic neurons to synthesize DA in vivo, in view of the important effects of perinatal THC exposure on TH gene expression and activity. Thus, we analyzed L-3,4-dihydroxyphenylalanine (L- DOPA) accumulation following NSD 1015induced blockade of L-DOPA decarboxylase, a classical test for analyzing in vivo synthesis of DA (Steger et al., 1989; Rowlett et al., 1993).

2. Methods

2.1. Animals

Female virgin Wistar rats were housed in a room with a controlled photoperiod (08.00-20.00 h light) and temperature (23 + 1’C). They had free access to standard food (Panlab, Barcelona, Spain) and water. At adult age ( > 8 weeks of life; 150-200 g), daily vaginal smears were taken between lO:OO-12:00 h, and only those animals exhibiting three or more consistent 4-day cycles were used in this study. Females in the proestrous phase were allowed to stay with a male for mating, and a new vaginal smear was taken on the next day. Those animals showing the presence of sperm cells were accepted as probably pregnant and used for the cannabinoid exposure studies. The day on which sperm plugs were found was designated as the first day of gestation.

2.2. Perinu fat THC e.pmwe

THC, kindly supplied by the National Institute on Drug Abuse (USA), was prepared in a sesame oil solution for administration. Pregnant females received a daily oral dose of THC (5 mg/kg) from the day 5 of gestation, as in our previous studies (Bonnin et al., 1995; Fernandez-Ruiz et al., 1996; Garcia et al., 1996). This dose is an extrapolation from current estimates of moderate exposure to this compound in humans, cor- recting for differences in route of administration and body surface area (Nahas, 1984). Control rats were fed

with vehicle alone. This treatment was maintained until day 24 after birth, the day on which rats were weaned. Then, males and females were housed separately (4 per cage) and left to reach adult age ( > 10 weeks). During the whole treatment period, we recorded a set of gestational and lactational parameters, such as maternal and neonatal weight gain, maternal food and water intake, gestational length, placental and fetal weights, litter size and maternal plasma THC concentrations, in order to show that potential adult changes were not due to a possible cannabinoid-induced nutritional deficit during the perinatal life. These results have been previously published (Bonnin et al.. 1995; Fernandez-Ruiz et al., 1996) and are not included in the present report.

Males and females that had been exposed perinatally to THC or vehicle were used when adult ( > 10 weeks: females were in diestrus) for three different pharmacological challenges. In the three cases, animals were assigned randomly to adult treatment conditions, being always the four different perinatal groups (females+ oil: females + THC: males -+ oil; males + THC) obtained from at least three different oil- or THC-exposed litters. Experiments with these animals were always performed between lO:OO~- 13:00 h. In experiment I, one third of the animals of each of these four groups was i.p. injected with a specific D, antagonist, SCH 23390 (purchased from Research Riochemicals Inc.. USA), used at a dose of 0.1 “g/kg, another third was injected with a specitic Dz antagonist, sulpiride (purchased from Sigma, USA), used at a dose of 30 mg!kg, and the last third was treated with vehicle. 30 min later, animals were tested behaviorally (open-field test). All doses were chosen because they were effective in markedly diminishing motor behavior in adult rats (they produeed more than 70% of reduction in ambulatory activity in both sexes) in dose-response studies conducted previously in our laboratory (data not shown) and also in previous reports (McDougall et al., 1990: Garrett and Holtzman, 1994). In experiment II, half of each perinatal group was S.C. injected with AMPH (purchased from Research Biochemicals, USA), used at a dose of 10 mg/kg and the other half of each group was treated with vehicle. 30 min later, animals were sacrificed. The dose of AMPH was chosen because it was effective in markedly diminishing intraneuronal DA metabolism in adult rats in a previous report (Elverfors and Niss- brandt. 1992). and in dose-response studies conducted previously in our laboratory (data not shown). In experiment III. half of each perinatal group was i.p. injected with NSD 1015 (purchased from Research Biochemicals, USA), used at a dose of 100 mg/kg, and the other half of each group was injected with vehicle. 30 min after treatment, animals were sacrificed. The

158 L. Garcia et al. 1 Drug and Alcohol Dependence 42 (1996) 155-166

dose of NSD 1015 was chosen because it was effective in inhibiting L-DOPA decarboxylase in adult rats, as previously reported (Steger et al., 1989; Rowlett et al., 1993) and validated in our laboratory (data not shown). Drugs were dissolved in distilled water (experiment I), although sulpiride was dissolved initially in a drop of glacial acetic acid, or saline (experiments II and III), and injected in a volume of 2-5 ml/kg (the differences in the volumes of injection depended on the experiment). In order to control a potential effect of acetic acid used for solubilizing sulpiride in experiment I, some control animals were injected with acidified distilled water in the same proportion as for preparing sulpiride solution; as these animals did not exhibit differences as compared to animals injected with distilled water alone, both were combined for the statistical analysis and presentation.

2.4. Sampling

In experiments II and III, animals were sacrificed and their brains removed and dissected according to Glowinski and Iversen (1966). The striatum and lim- bit forebrain (this contains olfatory tubercles, rostra1 limbic nuclei, the nucleus accumbens, septal nuclei and parts of the anterior amygdaloid nuclei) were dissected, weighed and immediately frozen and stored at - 70°C until assayed for DA indices.

2.5. Behavioral studies

In experiment I, animals were subjected to an open-field test 30 minutes after treatment. This consisted of a circular arena composed of hardboard (di- ameter: 90 cm) with a surround (height: 30 cm), both made of transparent polyvinylchloride. The floor was divided into five inner and eight outer parts using circles and radial segments. On the day of the test, the experimental animal was placed in the centre of the arena and its spontaneous activity was recorded on a TV-video system. In the pilot dose-response experiments, the duration of the test was 10 min, divided into two periods of 5 min. It was observed that the motor inhibition caused by increasing doses of the DA antagonists injected in both sexes was, in terms of percentages, similarly distributed in both periods, suggesting that the influence of the stress derived from the novel environment was not a determinant. Thus, we decided to perform the behavioral testing of the main experiment only during the first 5 min. The advantage of this option is the increase in the number of animals per each daily testing session, reducing the influence of interassay variation without markedly increasing the total time of each session and, hence, avoiding the effects of natural fluctuations during the

photoperiod. The apparatus was washed out with an odoriferous solution after each rat had been tested. The following parameters were analyzed: (i) spontaneous locomotor activity (ambulation)-number of sector crossings (a single line-crossing was defined as the rat placing the four paws into an adjacent quad- rant); (ii) frequency (score) of spontaneous stereotypic behaviors (grooming, rearing and shaking); and (iii) time spent in inactivity. The scoring of the different behaviors was carried out by investigators who had no knowledge of the treatment of each rat.

2.6. Neurochemical analyses

DA, DOPAC and L-DOPA contents were analyzed using HPLC with electrochemical detection. Striata and limbic forebrains were homogenized in ice-cold 0.2 N perchloric acid containing 0.5 mM sodium bisulfite and 0.45 mM EDTA (30 vol for the striatum and 10 vol for the limbic forebrain). Dihydroxybenzy- lamine was added as an internal standard. The homogenates were then centrifuged and the supernatants injected into the HPLC system. The HPLC system consisted of a Spectra-Physics 8810 isocratic pump. The column was a RP-18 (Spherisorb ODS-2; 125 mm, 4.6 mm, 5 pm particle size). The mobile phase consisted of 100 mM citric acid, 100 mM sodium acetate, 1.2 mM octane sulphonate, 1 mM EDTA and 5% methanol (pH 3.9) and the flow rate was 0.8 ml/min. The effluent was monitored with a coulo- chemical detector (Coulochem II, ESA) using a proce- dure of oxidation/reduction (conditioning cell: + 360 mV; analytical cell # 1: + 50 mV; analytical cell # 2: - 340 mV). The signal was recorded from the analytical cell # 2, with a sensitivity of 50 nA (10 pg per sample), on a Spectra-Physics 4290 integrator and the results were given as area under the peaks. Values were expressed as ng/mg of tissue.

2.7. Statistics

Data from the three experiments were mainly assessed by 3-way analysis of variance (variable 1: sex; variable 2: perinatal treatment; variable 3: adult challenge), using the computer package BMDP, followed by a post-hoc test (Student-Newman-Keuls test). Ad- ditionally, data were transformed to percentage of change over the mean value of the corresponding vehicle-injected group and, then, subjected to 2-way analysis of variance (variable 1: sex; variable 2: perinatal treatment) also followed by the Student-New- man-Keuls test. This was done to evaluate better the differences or to enhance some trends, as a function of sex and perinatal treatment, in the magnitude of changes caused by the different adult challenges.

L. Garcia et al. 1 Drug and Alcohol Dependence 42 (1996) 155 166

3. Results

3.1. Experiment I: challenge with DA antagonists

In this experiment, we analyzed the effects of challenges with DA antagonists on motor behavior in adult rats of both sexes perinatally exposed to THC or vehicle (Figs. 1 and 2). The 3-way analysis of variance (sex x perinatal treatment x adult challenge) used for the collective analysis of data of this experiment revealed that, when the three variables were analyzed individually, only two variables, adult challenge and sex, exhibited statistical differences. Thus, as expected, females collectively exhibited higher values of ambula-

Experiment I: Challenge with dopamine antagonists

60

0 fmwates + Oil femfw l THC m&4 l oil mater l THC

Experiment I: Challenge with dopamine antagonists

Freauencv of stereotvDY - I

- l vehicle &i%! l SCH 23390 = + rulplride

6

0 fsfn&s l Oil lam* * THC mat64 l oil molar + THC

Fig. 1. Ambulation and frequency of stereotypy, measured in the open-field test. 30 min after a challenge with SCH 23390, sulpiride or vehicle to adult males and females (in diestrus) that had been perinatally exposed to THC or oil (see text for details). Values are means k S.E.M. The number of animals used for each treatment group was 11 - 13 for SCH 23390, 6-7 for sulpiride and 1 l- 13 for vehicle. Data were assessed by 3-way (sex x perinatal treatment x adult challenge) analysis of variance followed by Student-Newman- Keuls test (*Pi 0.05, **P ~0.005, ***PC 0.0005 versus the corresponding vehicle-injected group; @ P < 0.005 versus the other sex for the same perinatal treatment).

Ex Challgnse wBh

inactivltv Is) 200

l- 160

120

60

40

0

m *w$irid4 + SCH 29890

m + which

li n

Fig. 2. Time spent in inactivity. measured in the open-field test. 30 min after a challenge with SCH 23390, sulpiride or vehicle to adult males and females (in diestrus) that had been perinatally exposed to THC or oil (see text for details). Values are means f S.E.M. The number of animals used for each treatment group was II-13 for SCH 23390. 6 7 for sulpiride and 1 l- 13 for vehicle. Data were assessed by 3-way (sex x perinatal treatment x adult challenge) analysis of variance followed by Student-Newman-Keuls test (*P < 0.05. **P < 0.005, ***P i 0.0005 versus the corresponding vehicle-injected group: ($ P < 0.05 versus the other sex for the same perinatal treatment).

tion (Fig. 1) (F(l, 110) = 13.01, P<O.O005) and lesser time spent in inactivity (Fig. 2) (F(1, I 10) = 10.72, P < 0.005) than males, but both sexes exhibited similar frequencies of stereotypy (Fig. 1) (F(1~ 109) = 0.5 1, ns). The analysis of variance also revealed that the administration of either SCH 23390, a D, antagonist, or sulpiride, a D, antagonist, to adult rats produced the expected motor inhibition (McDougall et al., 1990: Meyer, 1993; Garrett and Holtzman, 1994) in all groups examined, showing decreased ambulation (Fig. 1) (F(2, 110) = 55.79, P < 0.00005) and stereotypic movements (Fig. 1) (F(2, 109) = 12.81, P < 0.0005) and increased time spent in inactivity (Fig. 2) (F(2, 110) = 34.16. P < 0.00005). Finally, there was no effect of perinatal treatment with THC, individually analyzed,

adult parameters of ambulation (Fig. 1) Fl(l, 110) = 1.15, ns), frequency of stereotypy (Fig. I) (F(1, 109) = 0.79, ns) or inactivity (Fig. 2) (F( 1, 110) = 0.01, ns) in both males and females. This supports previous results (Navarro et al., 1994), which indicated the disappearance in adulthood of behavioral changes observed during the course of postnatal development, as suggested by the hypothesis of the present study.

However, when the different interactions among these three variables were analyzed, we observed some interesting results. Thus, the interaction between perinatal treatment and adult challenge was statistically significant for ambulation (F(2, 110) =r 3.62, P < 0.05), but not for stereotypy (F(2, 109) = 0.11, ns) or inactivity (F(2, 110) = 0.07, ns), indicating that animals ex-

160 L. Garcia et al. / Drug and Alcohol Dependence 42 (1996) 155-166

posed perinatally to THC exhibited a different degree of response, in terms of decreased ambulatory activity, to the administration of dopaminergic antagonists than oil-exposed animals. This difference was similar in both sexes since the 3-way interaction was not statistically significant (F(2, 110) = 0.01, ns). The remaining 2-way and 3-way interactions were not statistically significant, except for sex and adult challenge in inactivity data (F(2, 110) = 2.96, P < 0.05), showing higher increases after challenging with both SCH 23390 and sulpiride in both THC- and oil-exposed adult males than females.

To appreciate better the differences between THC- and oil-exposed animals in the magnitude of the decrease in ambulation after a challenge with DA antagonists, data were transformed to percentage of change over the correponding vehicle-injected group and the transformed data subjected to 2-way analysis of variance. This revealed that the decrease in ambulation caused by the challenge with SCH 23390 was more marked if the animals have been perinatally exposed to THC (F( 1,45) = 4.44, P < 0.05; oil-exposed females: - 59.8 + 11.4, versus THC-exposed females: - 79.9 + 7.4, P < 0.05; oil-exposed males: - 58.6 f 12.8, versus THC-exposed males: - 85.3 + 5.7, P < 0.05); this effect was similar in both sexes since the 2-way interaction between sex and perinatal treatment for the transformed data was not significant (F(1,45) = 0.09, ns). This did not occur after challenging with sulpiride.

3.2. Experiment II: challenge with AMPH

In this experiment, we tested the possible existence of differences, as a consequence of perinatal exposure to THC, in the magnitude of the response to a challenge with AMPH, by testing its effects on the contents of DA and DOPAC in the areas containing the terminals of midbrain dopaminergic neurons (Figs. 3 and 4). The 3-way analysis of variance revealed that the adult challenge with AMPH altered DA metabolism in the stria- turn (Fig. 3) (DA content: F(1,63) = 2.32, P = 0.133; DOPAC content: F(1, 63) = 44.27, P < 0.00005) and, in particular, in the limbic forebrain (Fig. 4) (DA content: F(1,63) = 4.27, P < 0.05; DOPAC content: F(1,63) = 27.13, P < 0.00005). As expected, no changes in the other variables analyzed, sex and perinatal treatment, were seen (Figs. 3 and 4). The interaction of this variable, adult challenge, with the others, sex and perinatal treatment, did not elicit any statistical differences in the striatum, whereas, in the limbic forebrain, the 2-way interaction, perinatal treatment x adult challenge, exhibited some non-significant trends (DA content: F(1, 63) = 2.66, P = 0.108; DOPAC content: F(1, 63) = 1.17, P = 0.283). These trends were consoli- dated when data were transformed to percentage of change over the corresponding vehicle-injected group and the transformed data subjected to 2-way analysis of

variance. Thus, we observed that the animals perinatally exposed to THC exhibited less marked reductions in DOPAC production (P(1, 31) = 3.78, P = 0.061), in parallel with more marked increases in DA stores (F(1, 31) = 4.39, P < 0.05), in the limbic forebrain. Al- though this was a priori valid for both sexes, as revealed the fact that the 2-way interaction between sex and perinatal treatment for the transformed data was not statistically significant for both parameters (DA: F(1,31) = 0.29, ns; DOPAC: F(1, 31) = 1.30, ns), the post-hoc analysis demonstrated the existence of statistically significant differences only in the case of females for both DOPAC (oil-exposed: - 66.7 _+ 6.4, versus THC-exposed: - 39.3 IfI 12.5, P < 0.05) and DA (oil-

t exposed: - 5.2 f 7.8, versus THC-exposed: + 32.2

Experiment II: challenge with AMPH

,6 DA content hg/mg tissue)

I / STRIATUM m * vehicle m l AMPH

12 -


2 DOPAC content (ng/mg tissue) I

~ STRIATUM / I+ vehicle RW l AMPH

105 1

male0 + THC

Fig. 3. Contents of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum 30 min after a challenge with amphetamine (AMPH) or vehicle to adult males and females (in di- es&us) that had been perinatally exposed to THC or oil (see text for details). Values are means + S.E.M. of more than eight determina- tions per group. Data were assessed by 3-way (sex x perinatal treatment x adult challenge) analysis of variance followed by Student-Newman-Keuls test (*P < 0.005 versus the corresponding vehicle-injected group).

L. Garcia et al. / Drug and Alcohol Dependence 42 (1996) 1.5.5 166 IhI


5 PA content hg/mg tissue) --__--_ ---7

ri l vahicte I

LlMBiC FOREBRAIN Em.AMPH /

47 L----L /

* *


DOPAC content (ng/mg tissue) 1.5 r __--____ ---

‘~ LIMBIC FOREBRAIN -___

I I* vehicle m + AMPH _____

Fig. 4. Contents of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) in the limbic forebrain 30 min after a challenge with amphetamine (AMPH) or vehicle to adult males and females (in diestrus) that had been perinatally exposed to THC or oil (see text for details). Values are means + S.E.M. of more than eight determina- tions per group. Data were assessed by 3-way (sex x perinatal treatment x adult chahenge) analysis of variance followed by Student-Newman-Keuls test (*P < 0.05, **P < 0.005 versus the corresponding vehicle-injected group).

10.8, P < 0.05). As expected, no differences were seen when transformed data for both DA and DOPAC contents in the striatum were subjected to 2-way analysis of variance.

3.3. Experiment III: challenge with NSD 1015

In this experiment, we tested the possible existence of differences, arising as a consequence of perinatal THC exposure, in the magnitude of NSD 1015-induced L- DOPA accumulation and DOPAC decrease in midbrain dopaminergic neurons (Figs. 5 and 6). The 3-way analysis of variance revealed that, of the three different variables individuahy analyzed, only the adult challenge exhibited statistically significant differences for both L-DOPA accumulation in the striatum (Fig. 5)

(F(l,43) = 139.44, P < 0.00005) and the limbic forebrain (Fig. 6) (F(1,43) = 75.56, P < 0.00005) and DO- PAC reduction also in both regions (Figs. 5 and 6) (striatum: F(1,42) = 38.24, P < 0.00005; limbic forebrain: F(1,43) = 46.32, P < 0.00005). These effects were seen independently of sex and/or perinatal treatment with THC, because none of the 2-way or 3-way interactions showed statistically sigmficant differences. How- ever, when data were transformed to percentage of change over the corresponding vehicle-injected group and the transformed data subjected to 2-way analysis of variance, we observed that both males and females perinatally exposed to THC exhibited similar NSD 1015-induced decreases in DOPAC contents in the lim- bit forebrain, whereas the magnitude of NSD 1015-in-

Experiment III: challenge with NSD 1015

L-DOPA accumulation (ng/mg tissue) ,*s --- -.--.---- ~--~-__; ._... ~~.- ..~

Experiment Ill: challenge with NSD 1015

DOPAC content (ng/mg tissue) ,,s -.----~~-...- -~-.---~----. __-.---__ ------. - _--_

STRIATUM I * v&id* m * NSD 30% ~

1.27 I

Fig. 5. Contents of r.-3,4-dihydroxyphenylalanine (L-DOPA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) in the striarum 30 min after a challenge with NSD 1015 or vehicle to adult males and females (in diestrus) that had been perinatally exposed to THC or oil (see text for details). Values are means + S.E.M. The number of animals used for each treatment group was 6--8~ Data were assessed by 3-way (sex x perinatal treatment x adult challenge) analysis of variance followed by Student-Newman-Ret& test (*P < 0.01, **P < 0.005. ***P < 0.0005 versus the corresponding vehicle-injected group).

162 L. Garcia et al. /Drug and Alcohol Dependence 42 (1996) 1.55-166

Experiment III: challenge with NSD 1015

2 L-DOPA accumulation (ng/mg tissue)

Experiment Ill: challenge with NSD 1015

1.6- DOPAC content (ng/mg tissue)

LIMBIC FOREBRAIN I I. vehic~s W. NSD mm

Fig. 6. Contents of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) in the limbic forebrain 30 min after a challenge with NSD 1015 or vehicle to adult males and females (in diestrus) that had been perinatally exposed to THC or oil (see text for details). Values are means + S.E.M. The number of animals used for each treatment group was 6-8. Data were assessed by 3-way (sex x perinatal treatment x adult challenge) analysis of variance followed by Student-Newman-Keuls test (*P < 0.01, **P < 0.005, ***P < 0.0005 versus the corresponding vehicle-injected grow).

duced L-DOPA accumulation in this region was signifi- cantly higher in females when perinatally exposed to THC (sex x perinatal treatment: F(1,21) = 5.53, P < 0.05; oil-exposed: 1860 f 180, versus THC-exposed: 5160 + 1660, P < 0.05); this effect did not exist in males, as revealed by the analysis of the response to NSD 1015 independently of sex (P(1,21) = 2.37, ns).

4. Discussion

The present study examined whether the response of midbrain dopaminergic neurons in adulthood to a pharmacological challenge with DA-acting drugs could be different in animals perinatally exposed to THC

from that of controls, despite the fact that the basal activity of these neurons appeared unaltered in adult animals perinatally exposed to THC as compared with controls (Navarro et al., 1994). This kind of experimental paradigm has been succesfully used for revealing the existence of silent behavioral disturbances in adulthood caused by the perinatal exposure to other drugs of abuse (Becker et al., 1995; Snyder-Keller and Keller, 1995) or to dopaminergic drugs (Scalzo et al., 1989). We have recently demonstrated for the first time that the response of midbrain dopaminergic neurons of THC-exposed animals to the administration in adulthood of drugs, which deplete DA stores and induce TH gene expression, differs from that observed in oil-exposed animals (Garcia et al., 1996). The results obtained in the present study by using three different DA-acting drugs, are also consistent with the initial hypothesis, because we could show the existence of subtle differences in the magnitude of the effects caused by these drugs in THC-exposed animals as compared with the response in oil-exposed animals.

4.1. Challenges with DA antagonists

The results obtained after challenging with DA antagonists proved that the administration of a D, antagonist, SCH 23390, to adult animals that had been perinatally exposed to THC induces patterns of re- sponding, in particular a more marked decrease in ambulatory activity indicating a higher motor inhibition, which differ from the typically observed normal pattern in controls. This was seen in both sexes and seems to be specifically related to D, receptors, because the pattern of response to sulpiride, a D, antagonist, was similar in both THC- and oil- exposed animals. In this sense, it is interesting to note that D, receptors, more than D, receptors, have been proposed recently as critical for the normal expression of motor activity (Xu et al., 1994).

Hence, these results are consistent with the notion that the perinatal exposure to THC alters the magnitude of the D, antagonist-induced motor inhibition. This finding is quite similar to that obtained in animals perinatally exposed to haloperidol, a D,/D, antagonist, which also exhibited differences in the sensitivity to a variety of CNS depressants, including haloperidol (Scalzo et al., 1989). It is unclear, at the moment, whether the changes in responsiveness to D, antago- nism were due to differences in drug metabolism, motor capabilities, receptor binding parameters, signal trans- duction mechanisms or other possible reasons. A priori, it would appear reasonable, based on the fact that perinatal cannabinoid exposure altered the development of D, and D, receptors (Rodriguez de Fonseca et al., 199 1; Fernandez-Ruiz et al., 1994), to accept that these differences should be related to changes in receptor

L. Garcia et al. I Drug and Alcohol Dependence 42 (1996) 155-I 66 163

binding parameters in areas recipient of midbrain dopaminergic outputs. However, we have recently reported that D, receptor binding parameters were unchanged in the striatum and the limbic forebrain of adult males perinatally exposed to THC (Navarro et al., 1994; Fernandez-Ruiz et al., 1994), despite the increase shown during the immature period (Rodriguez de Fonseca et al., 1991; Fernandez-Ruiz et al., 1992). In adult females exposed perinatally to THC, we observed a statistically significant, but very small, increase in the density of D, receptors in the striatum but not in the limbic forebrain (Navarro et al., 1994; Fernandez-Ruiz et al., 1994), with no changes in affinity. Taking into account these previous data, it appears unlikely that the SCH 23390~induced higher decrease in the ambulatory activity of adult males, when exposed perinatally to THC, might be related to changes in affinity or number of D, receptors, since there were no relevant changes in the binding characteristics of D, receptors in these animals (Navarro et al., 1994). This can be also applied to adult THC-exposed females, because, although we could observe changes in the receptor binding parameters. such as an increase in the density of D, in the striatum (Navarro et al., 1994) it is unlikely that this might explain a higher degree of motor inhibition. The higher density of these receptors in the striatum of THC-exposed females should lead to a higher proportion of unblocked D, receptors after SCH 23390 administration than in control females, thereby allowing a higher receptor activation by DA and more ‘normal’ motor behavior, rather than a more marked motor inhibition.

Hence, the higher decrease in the ambulatory activity caused by SCH 23390 in THC-exposed adult males and females seems to be unrelated to changes in binding parameters for this receptor. This would be similar to that seen with the behavioral sensitization to drugs that act by directly or indirectly stimulating dopaminergic sensitivity, which require the stimulation of DA receptors. although unaccompanied by changes in DA receptor affinity or density (Rowlett et al., 1993). Another possibilities could be the existence of changes in the availability of D, receptors or in the molecular mechanisms coupled to these receptors. Regarding the first of these two possibilities, there could be differences in the proportion of receptors that might be incorporated into the postsynaptic membrane, and, thus, easily available for neurotransmitter binding, and the proportion of receptors that might be incorporated into the intracellular membranes, presumably in recycling processes, and unable to bind the neurotransmitter in vivo. However, the in vitro binding analysis is not able to discriminate between both receptor locations (both are marked with the radioactive probe) because of the use of crude membrane fractions. The possible existence of differences at the postreceptor level, i.e., coupling to their

intracellular signal mechanisms, leading to differences in the magnitude of their biological action, could be also considered. This might be supported by the recent studies from Herkenham et al. (1991), who have reported that DA receptors, mainly D, receptors, are present in the same striatal neurons where cannabinoid receptors seem to be located, and by the fact that both cannabinoid and DA receptors act at the intracellular level by modifying the CAMP contents (for review, see Fernandez-Ruiz et al., 1992; Schwartz et al., 1992).

4.2. Challenges with AMPH

The administration of AMPH to adult rodents produced a well-characterized reduction in DA metabolism in midbrain dopaminergic terminals, which originates in a variety of potential sites of action for this drug (see Section 1). It is possible to evaluate the degree of AMPH action by examining the attenuation induced by this drug in DOPAC production, since this metabolite is formed intraneuronally from the reuptaken or newly synthesized DA (Elverfors and Nissbrandt, 1992). In the present work, we have observed that AMPH administration produced a marked attenuation in the DOPAC production, ranging around 60%. similar to that observed by Elverfors and Nissbrandt (1992). It can be asserted that the magnitude of the effects of AMPH, in terms of attenuation of DOPAC production, was similar in oil- and THC-exposed animals of both sexes, in view of the absence of statistical significance in the interaction between perinatal treatment and adult challenge, as well as in the 3-way interaction (sex x perinatal treatment x adult challenge). However, when data were transformed to percentages of change, we could appreciate that the effect of AMPH was slightly less marked when this drug was administered to animals that had been exposed perinatally to THC. The fact that this effect was seen only after transformation of the data. might indicate that the effect is subtle rather than pronounced. To determine its physiological relevance would require further experiments. Anyway, this subtle effect was seen only in the midbrain dopaminergic terminals reaching the Iimbic forebrain area, but not in those terminals reaching the caudate- putamen area, indicating that it is specific for mesolim- bit neurons. Moreover, it appears to occur only in adult females, but not in adult males. Our group has reported the existence of marked sexual differences in the response to cannabinoids when administered perinatally in rats (for review, see Fernandez-Ruiz et al.. 1992, 1994, 1996), and others have obtained similar findings in animals exposed perinataliy to other drugs of abuse such as cocaine, even when challenged pharmacologically in adulthood as in the present study (Snyder-Keller and Keller, 1995; for review, see Fernan- dez-Ruiz et al.. 1992, 1994). However an interesting

164 L. Garcia et al. 1 Drug and Alcohol Dependence 42 (1996) 155-166

aspect derived from our results after the AMPH challenge is that females were more responsive in adulthood, whereas previous reports demonstrated that males were more affected during development and that the effects in developing females were slight and tran- sient (for review, see Fernandez-Ruiz et al., 1992, 1994, 1996). In this sense, it is important to note that the maturation of midbrain dopaminergic neurons, in terms of increasing DOPAC production, during the first days after birth was accelerated profoundly in THC-exposed females but not in THC-exposed males (Bonnin et al., unpublished results). We interpreted this effect as arising from an early maturation of DA reuptake process induced by THC in females, which allowed a higher DOPAC production. It is possible that this accelerated maturation during the postnatal period might also result in a higher number of DA reuptake sites in adulthood, leading to an expected reduced effect of DA reuptake blockers as AMPH.

The above finding, a potential increase in the number of DA reuptake sites in THC-exposed females, might explain at the molecular level the differences in the responsiveness to AMPH, but other possibilities should be also considered. For example AMPH at the dose used in the present study presumably might inhibit monoamine oxidase activity leading to reduction of DOPAC production. To our knowledge, there is no evidence for possible changes in the activity of this enzyme as a function of perinatal THC exposure that might explain the subtle differences in the magnitude of AMPH action. Another possibillity could be changes at the level of DA synthesis; we have seen that animals perinatally exposed to THC exhibited AMPH-induced increases in the DA content in the limbic forebrain, but not in the striatum, an effect which is difficult a priori to explain. These changes did not exist in oil-exposed animals. In this respect, Elversfors and Nissbrandt (1992) have suggested the existence of an indirect AMPH stimulatory effect on DA synthesis. This could be produced as a consequence of the decrease in the concentrations of extravesicular intraneuronal DA caused by AMPH, which, in turn, may cause an increase of synthesis due to reduced end-product inhibition. It would be expected that THC-exposed animals-in particular females, in which the DA metabolism was reduced to a lesser extent than in oil-exposed animals-might secondarily develop an increased DA synthesis leading to higher DA contents. This finding seems to be strengthened by the data obtained in experiment III of the present study, which suggested that mesolimbic dopaminergic terminals of THC-exposed females had a subtlely higher rate of DA synthesis than oil-exposed animals, as will be discussed below.

4.3. Challenges with NSD 1015

The administration of NSD 1015, an inhibitor of L-DOPA decarboxylase, to adult rats produced the well-known stimulatory effect on L-DOPA concentrations, that has been used as an index of in vivo DA synthesis (Rowlett et al., 1993). As ocurred in the above experiment, it can be asserted that the magnitude of L-DOPA accumulation was similar in oil- and THC-exposed animals of both sexes, in view of the absence of statistical significance in the 2-way and 3-way interactions. However, the transformation of data to percentage of change allowed to appreciate that the magnitude of L-DOPA accumulation might be subtly higher in the midbrain dopaminergic terminals reaching the limbic forebrain area of THC-exposed females as compared with oil-exposed females. This did not exist in THC-exposed males and in the other constituent of midbrain dopaminergic terminals, the nigrostriatal system. Fur- ther experiments must determine the physiological relevance of this observation.

It is important to note that we have recently reported that the activity of TH in the limbic forebrain of THC-exposed females was similar to that measured in oil-exposed animals (Fernandez-Ruiz et al., 1994). However, it is important to consider that the analysis of TH activity was performed in vitro by measuring the amounts of L-DOPA formed when brain homogenates were incubated with saturating concentrations of L-tyrosine. It would be possible that, although the in vitro analysis leads to no changes, the in vivo analysis might show the existence of an altered synthesis capability, presumably due to circumstances that do not exist in the in vitro assay: substrate availability, intracellular location and others. Certainly, this might be occurring in THC-exposed females, which subtly exhibited a higher capability to accumulate L-DOPA when the activity of L-DOPA-decarboxylase had been blocked with NSD 1015.

5. Conclusions

In summary, our results are consistent with the possible existence of subtle changes in the sensitivity of midbrain dopaminergic neurons in adulthood caused by the exposure to THC during perinatal development. These silent changes can be revealed after the administration of drugs which specifically act on key processes of dopaminergic neurotransmission, such as the metabolism and reuptake of DA and its binding to receptors. A particular feature was the existence of a possible sexual dimorphism in these silent changes in the sensitivity of midbrain dopaminergic neurons. The neurochemical basis for these differences remains to be determined, but it is important to note that these might

L. Garcia et al.’ / Drug and Alcohol Dependence 42 (1996) 155166 165

presumably underly a variety of potential long-term disturbances, observed in adult animals perinatally exposed to cannabinoids, in the adult functionality of dopamine-related processes, such as stress, motor behavior and reward (Dalterio, 1980; Mokler et al., 1987; Navarro et al., 1994; for review see Dalterio, 1986; Fernrindez-Ruiz et al., 1992). In this sense, we have recently observed that both adult male and female rats became more intensely opiate-dependent in studies of self-administration, when exposed perinatally to THC (Ambrosio, FernBndez-Ruiz and Ramos, unpublished results), an effect that might be tentatively related to the above mentioned changes in mesolimbic dopaminergic neurons, in view of the well-known involvement of these neurons in reward- ing effects of habit-forming drugs (Herz and Shippen- berg, 1989).

Acknowledgements

This work has been supported by grants from CI- CYT (SAF-94-0540). The authors are indebted to the National Institute on Drug Abuse (USA) for kindly supplying THC.

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