Cheryl L. Kirstein

Cocaine Effects on the Developing Central Nervous System: Behavioral, Psychopharmacological, and Neurochemical Studies

IMPLICATIONS and FUTURE DIRECTIONS. The data we have collected thus far support the following conclusions: 1. Subcutaneous administration of cocaine results in dose-dependent increases in brain and plasma cocaine in both dams and fetuses, and maternal plasma levels in the range of or above those observed in human cocaine users. Fetal levels are lower than those of the dam, suggesting that the placenta may partially restrict cocaine entry into the fetus. Concentrations of the active cocaine metabolite benzoylecgonine, however, are greater in fetal than in maternal brain; this may have important implications for brain development given the calcium-binding properties of this metabolite. 2. Chronic subcutaneous administration of 10, 20 or 40 mg/kg cocaine from E8-E20 does not alter litter size, body weights at birth or weaning, or development of reflexes or physical landmarks in the offspring. 3. Offspring exposed gestationally to cocaine exhibit learning and/or retention deficits in some but not all conditioning situations. 4. Behaviorally and psychopharmacologically, there is evidence for a potential attenuation in DA activity in preweanling pups exposed gestationally to cocaine. There is, however, no sign of any alteration in DA turnover in treated offspring sacrificed at weaning, although preliminary data suggest that DA levels may be increased in exposed pups during the neonatal period. Possible alterations in DA receptor function are currently being assessed. We are still at the initial stages of our work, and the data we have collected thus far have raised as many questions as they have answered. We plan to assess further the cognitive deficits observed in cocaine-exposed offspring. Under what contingencies are these learning and/or retention deficits observed, and are they permanent deficits or does recovery eventually occur? Is there actually an attenuation in DA activity in treated offspring? If so, is this attenuation related to compensations at the presynaptic and/or postsynaptic level, and what is the time course for this effect on the DA system? What are the critical periods for the production of these alterations; is cocaine exposure during the second or third trimester alone sufficient? How do our results compare with those of other laboratories that are using other methods for administering cocaine during gestation? At some point in the future we also hope to examine potential therapeutic approaches to reduce the cognitive deficits observed in exposed offspring.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of prenatal cocaine exposure on behavior during the early postnatal period.

Offspring of Sprague-Dawley dams injected SC with 40 mg/kg/3 cc cocaine HCl daily from gestational days 8-20, pair-fed dams injected with the vehicle alone and nontreated control dams were examined behaviorally during the early postnatal period. No significant differences were observed among the treatment conditions in maternal weight gain during pregnancy, duration of pregnancy, or number of live male and female pups/litter. Offspring body weights at birth and weaning, physical maturation and reflex development were not significantly affected by prenatal cocaine exposure. In contrast, neonates exposed prenatally to cocaine were observed to exhibit significant deficits in learning of an odor/milk association that nontreated offspring learned and retained for a 24 hr period. On postnatal day 12, cocaine offspring exhibited an increase in locomotor activity and attenuated wall climbing precipitated by footshock, in the absence of any alteration in sensitivity to footshock. Given that wall climbing has been previously shown to be strongly related to levels of catecholamine activity at this age, these data suggest the possibility that there may be some attenuation in catecholaminergic function in pups exposed gestationally to cocaine. The results of this study provide evidence that prenatal cocaine exposure may have an impact upon behavioral and cognitive function even during the early postnatal period. More work is needed to fully characterize the range of alterations observed and the neural mechanisms underlying these early exposure effects.

Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats

The distribution of cocaine and the cocaine metabolite benzoylecgonine (BE) in brain and plasma of Sprague-Dawley rat dams and their near-term fetuses was assessed 0.5 and 2 h post-injection on gestational day 20 following chronic daily subcutaneous injections of 10, 20, or 40 mg/kg/3 ml cocaine hydrochloride beginning on gestational day 8. Plasma concentrations of cocaine reached in the dams were found to be in the range of, or to exceed, those reported in human cocaine users. Dose-related increases in plasma and brain levels of cocaine in the dams and the fetuses were observed, particularly at 2 h post-injection. Fetal concentrations of cocaine in brain and plasma were approximately 2–3-fold less than those of the dams, suggesting that the placenta may somewhat restrict cocaine entry into fetal circulation. Brain/plasma cocaine ratios, however, were generally equivalent in the dams and fetuses, suggesting that once cocaine enters the circulation, its affinity for brain tissue is similar in the fetus and dam. Whereas plasma levels of BE, like cocaine levels per se, were greater in the dams than fetuses, BE concentrations in fetal brain were greater than those observed in maternal brain. These high levels of BE may contribute to the production of neurobehavioral alterations in cocaine-exposed offspring, given that this active cocaine metabolite has been shown to form molecular complexes with calcium ions (Misra and Mule 1975), thereby having the potential to influence a multiplicity of calcium-regulated developmental events. Taken together, the results of the present study suggest that the subcutaneous route may prove to be an appropriate means in rats for administering cocaine prenatally in investigations designed to assess potential neurobehavioral ramifications of gestational cocaine exposure.

Cocaine reward and MPTP toxicity: alteration by regional variant dopamine transporter overexpression.

Polygenic factors play important roles in animal models of substance abuse and susceptibility to dopaminergic neurodegeneration. Genetic factors are also likely to contribute to the etiology of human drug abuse disorders, and may alter human vulnerabilities to Parkinsonian neurodegeneration. The dopamine transporter (DAT; SLC6A3) is densely expressed by the dopaminergic midbrain neurons that play central roles in drug reward and is believed to be a primary site of action for cocaine reward. This transporter is necessary for the action of selective dopaminergic neurotoxins, and is uniquely expressed on neurons that are the primary targets of Parkinsonian neurodegeneration. To study possible influences of variant DAT expression on these processes, we have constructed transgenic mice (THDAT) in which tyrosine hydroxylase (TH) promoter sequences drive expression of a rat DAT cDNA variant, increase striatal DAT expression by 20-30%, and provide modest alterations in striatal levels of dopamine and its metabolites. THDAT mice habituate more rapidly to a novel environment than wildtype littermates. These animals display enhanced reward conferred by cocaine, as measured by conditioned place preference. However, locomotor responses to cocaine administration are similar to those of wildtype mice, except at high cocaine doses. THDAT mice display more than 50% greater losses of dopaminergic neurons following a course of MPTP treatment than do wildtype control mice. These results document a model for allelic variation at a gene locus that can exert significant effects in murine models of human substance abuse vulnerability and dopaminergic neurodegeneration.

Extracellular dopamine increases in the neonatal olfactory bulb during odor preference training.

Young rats learn to approach an odor that has been paired with tactile stimulation. This attraction is accompanied by changes in the metabolism and anatomy within the olfactory bulb glomerular layer. In this study, we examined the changes that occur in the olfactory bulb during early olfactory learning, rather than after such pairings have occurred. Specifically, we determined whether the pairing of an odor with tactile stimulation would produce a modified response by olfactory bulb glomerular-layer neurons. To monitor one large subgroup of these neurons during early learning, we used in vivo microdialysis to assess the activity of dopaminergic neurons in the olfactory bulb of postnatal day (PND) 3 rats during simultaneous presentation of odor and tactile stimulation, tactile stimulation alone, odor alone, or clean air alone. Clean air evokes no change in extracellular dopamine (DA), while both odor alone and stroking alone induce prolonged increases in DA peaking at about 200% of baseline. The combination of odor and tactile stimulation, which allows an olfactory preference to be formed, induces a prolonged increase in DA which peaks at about 400% of baseline. The level of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) increases only in pups receiving both odor and tactile stimulation and peaks at about 200% of baseline. With the exception of the pups exposed to clean air, all groups show an increase in homovanillic acid (HVA) of between 150-200% following stimulation. The large and prolonged increase in DA may be linked to the longer term anatomical and physiological changes in the glomerular layer of the bulb that form as a consequence of early olfactory preference training.

Alcohol during adolescence selectively alters immediate and long-term behavior and neurochemistry

Alcohol use increases across adolescence and is a concern in the United States. In humans, males and females consume different amounts of alcohol depending on the age of initiation, and the long-term consequences of early ethanol consumption are not readily understood. The purpose of our work was to better understand the immediate and long-term impact of ethanol exposure during adolescence and the effects it can have on behavior and dopaminergic responsivity. We have assessed sex differences in voluntary ethanol consumption during adolescence and adulthood and the influence of binge ethanol exposure during adolescence. We have observed that males are sensitive to passive social influences that mediate voluntary ethanol consumption, and early ethanol exposure induces long-term changes in responsivity to ethanol in adulthood. Exposure to moderate doses of ethanol during adolescence produced alterations in dopamine in the nucleus accumbens septi during adolescence and later in adulthood. Taken together, all of these data indicate that the adolescent brain is sensitive to the impact of early ethanol exposure during this critical developmental period.

Repeated ethanol exposure during adolescence alters the developmental trajectory of dopaminergic output from the nucleus accumbens septi

Individuals who begin using alcohol prior to 14 years of age are 4 times more likely to progress to addiction than those who do not initiate use until 21 years of age. The nucleus accumbens septi undergoes dramatic developmental transitions during the adolescent period, and dopaminergic activity within this region has been identified as a central neurochemical mediator of drug reward, addiction and dependence. Thus, alcohol‐induced neurochemical alterations in dopaminergic activity within this brain region likely mediate the heightened vulnerability to addiction observed in adolescent alcohol users. To investigate this idea, Sprague–Dawley rats were exposed to intraperitoneal injections of either saline or ethanol (0.5, 1.0 or 2.0 g/kg) twice daily over four days beginning on postnatal day 21, 31, 41 or 56. Cannulas were implanted toward the nucleus accumbens septi, subsequent in vivo microdialysis was used to collect samples, and both basal and ethanol‐stimulated dopamine overflow was measured using high performance liquid chromatography with electrochemical detection. A developmental transition in basal levels of dopamine in the nucleus accumbens septi was apparent with peak levels at postnatal day 45. An ethanol challenge produced unique responses across ages, with greater peak effects relative to baseline in younger animals (postnatal day 25 and 35). Following repeated exposure to ethanol, a significant increase in basal dopamine was apparent for all ages, and when these animals were challenged with ethanol, peak effects relative to baseline were decreased in younger animals, but unchanged in older animals (postnatal day 45 and 60). Results indicate that there is a key developmental transition in the ability of rats to adapt to the effects of repeated ethanol exposure, which occurs between postnatal day 35 and 45. This alteration may explain the increased addiction vulnerability observed in individuals who initiate alcohol use during early adolescence.

Gestational cocaine exposure increases opiate receptor binding in weanling offspring

The use of cocaine during pregnancy produces a variety of adverse effects in offspring. Gestational cocaine exposure is known to affect developing dopamine systems, but other neurochemical systems may also be at risk. Regional density of opiate receptors labeled with [3H]naloxone was examined in the brains of 21-day-old male rats exposed to cocaine (0, 10, 20, or 40 mg/kg/day s.c.) between gestation days 8 and 20. Gestational cocaine exposure significantly increased labeling in a dose-dependent fashion in dopaminergic terminal (e.g. the nucleus accumbens, medial prefrontal cortex, olfactory tubercle, and caudatoputamen), limbic (e.g. basolateral amygdaloid nucleus, lateral habenula, hippocampus, dentate gyrus, entorhinal and cingulate cortices) and neocortical (e.g. somatosensory and motor cortices) regions, but had little effect in diencephalic or brainstem regions. The results suggest a functional linkage whereby drug-induced alteration of dopamine systems can regulate developing opioid systems in the brain. Moreover, gestational cocaine exposure produced long-lasting changes of opiate receptor labeling in certain brain regions. The implications of these results are uncertain. However, such effects on endogenous opioid systems could contribute to a developmental delay, cognitive or motor dysfunction.

Developmental Differences in Nicotine Place Conditioning

Abstract: To understand the motivations and implications of the prevalence of smoking, studies have compared the behavioral effects of nicotine, the psychoactive drug in tobacco, in adolescent and adult animals. The present study used a biased three‐chambered conditioned‐place preference procedure without prior habituation to examine the potential rewarding and anxiolytic effects of nicotine across adolescence and adulthood to assess the presence of age‐dependent differences in response to nicotine.

Effects of psilocybin on hippocampal neurogenesis and extinction of trace fear conditioning.

Drugs that modulate serotonin (5-HT) synaptic concentrations impact neurogenesis and hippocampal (HPC)-dependent learning. The primary objective is to determine the extent to which psilocybin (PSOP) modulates neurogenesis and thereby affects acquisition and extinction of HPC-dependent trace fear conditioning. PSOP, the 5-HT2A agonist 25I-NBMeO and the 5-HT2A/C antagonist ketanserin were administered via an acute intraperitoneal injection to mice. Trace fear conditioning was measured as the amount of time spent immobile in the presence of the conditioned stimulus (CS, auditory tone), trace (silent interval) and post-trace interval over 10 trials. Extinction was determined by the number of trials required to resume mobility during CS, trace and post-trace when the shock was not delivered. Neurogenesis was determined by unbiased counts of cells in the dentate gyrus of the HPC birth-dated with BrdU co-expressing a neuronal marker. Mice treated with a range of doses of PSOP acquired a robust conditioned fear response. Mice injected with low doses of PSOP extinguished cued fear conditioning significantly more rapidly than high-dose PSOP or saline-treated mice. Injection of PSOP, 25I-NBMeO or ketanserin resulted in significant dose-dependent decreases in number of newborn neurons in hippocampus. At the low doses of PSOP that enhanced extinction, neurogenesis was not decreased, but rather tended toward an increase. Extinction of “fear conditioning” may be mediated by actions of the drugs at sites other than hippocampus such as the amygdala, which is known to mediate the perception of fear. Another caveat is that PSOP is not purely selective for 5-HT2A receptors. PSOP facilitates extinction of the classically conditioned fear response, and this, and similar agents, should be explored as potential treatments for post-traumatic stress disorder and related conditions.