Spatiotemporal analysis of Fos expression associated with cocaine- and PTZ-induced seizures in prenatally cocaine-treated rats.

We previously reported that prenatal cocaine exposure (40 mg/kg s.c., E10-E20) increased susceptibility to convulsant-induced seizures later in life, with female rats becoming more sensitive to seizures induced by cocaine and pentylenetetrazol (PTZ), and males more sensitive to PTZ-induced seizures (Snyder-Keller and Keller, 1995, 2000). In order to determine the locus of enhanced seizure susceptibility in the brains of prenatally cocaine-treated rats, we examined the distribution and density of Fos-immunoreactive cells after cocaine- and PTZ-induced seizures in mature rats. Subconvulsive cocaine doses induced c-fos in cortical areas as well as densely dopamine-innervated regions such as striatum and nucleus accumbens. Following cocaine-induced seizures, intense c-fos induction was observed in piriform cortex, amygdala, and hippocampus. Quantification of the number of Fos-immunoreactive cells in the brains of prenatally cocaine-treated versus prenatally saline-treated rats revealed differences in piriform cortex and amygdala that were indicative of a lower threshold in prenatally cocaine-treated female rats. Following PTZ-induced seizures, the same pattern of limbic structures were recruited with increasing seizure severity. Only females exhibited changes in the number of Fos-immunoreactive cells as a result of prenatal cocaine treatment. Pretreatment with the noncompetitive NMDA antagonist MK-801 blocked both cocaine- and PTZ-induced seizures, and Fos expression in limbic areas was also blocked. The dopamine D1 antagonist SCH 23390 blocked cocaine-induced seizures and associated c-fos induction, but not PTZ-induced seizures or Fos. Examination of the pattern of Fos expression at 15-20 min postseizure revealed that the initial site of c-fos induction associated with PTZ-induced seizures appeared to be the piriform cortex, whereas cocaine-induced seizures induced early expression in both piriform cortex and lateral amygdala. These findings suggest that neural alterations residing in the piriform cortex and amygdala are likely to account for the increased seizure susceptibility of prenatally cocaine-treated rats.

Positive torsional strain causes the formation of a four-way junction at replication forks.

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-DeltaLk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-DeltaLk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (+)-DeltaLk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.

Acute treatment with tamoxifen reduces ischemic damage following middle cerebral artery occlusion.

Inhibitors of cell-swelling-activated anion channels, including the antiestrogenic compound tamoxifen (TAM), have been shown to attenuate the increase in excitatory amino acids (EAA) during ischemia. Since TAM enters the CNS we tested whether it provides protection from damage due to reversible middle cerebral artery occlusion (rMCAo) in rats. TAM (5 mg/kg, i.v.) infused 25 min before ischemia, potently reduced the total volume of the infarct from 328 +/- 34 mm3 to 41 +/- 21 mm3, a reduction of 87%, as measured by TTC staining. It was equally effective when infused starting at 1 h after reperfusion, i.e. 3 h after initiation of rMCAo. Protection of neurons was also found histologically. TAM had no effect on CBF as measured by hydrogen clearance. This appears to be the first report of a marked neuroprotective effect of TAM. Further studies are needed to determine whether its effects are due to inhibition of EAA release and/or other potential neuroprotective sites of action.

Prenatal cocaine exposure.

Cocaine abuse is a significant problem not only in the general population but also among pregnant women. Since cocaine readily crosses the placenta and is metabolized slowly in fetuses, they can be exposed to significant levels of cocaine for long periods. In humans the most common consequences of cocaine abuse during pregnancy include premature birth, lower birth weight, respiratory distress, bowel infarctions, cerebral infarctions, reduced head circumference, and increased risk of seizures. Behaviorally these newborns show an increased degree of "tremulousness," crying and irritability, and are over-reactive to environmental stimuli. Within a month these behaviors have recovered dramatically, but not to normal levels. Thus while there are a number of abnormalities associated with cocaine-exposed neonates, they are not imminently debilitating or life-threatening. However, the long-term consequences of this prenatal cocaine exposure remain to be elucidated. We have examined a rat model for neurochemical, neuroanatomical and behavioral changes resulting from prenatal cocaine exposure. Since cocaine is known to act by blocking the inactivation of the neurotransmitters dopamine, serotonin and norepinephrine, our studies have focused on brain dopamine (DA) and serotonin (5-HT) pathways. In this model system we have found neurochemical changes that are present at birth and that return to normal as the rat ages--similar to the recovery observed in infants. However, there are other neurochemical, anatomical and behavioral changes that persist after birth which may provide insights into the long-term consequences. It is hoped that by understanding the changes occurring in this rat model we will be better prepared to devise pharmacological interventions to circumvent the secondary consequences of prenatal cocaine exposure. These consequences might include increased susceptibility to drug addiction, seizures, depression, schizophrenia, Parkinson's disease, etc.

Enhanced susceptibility to cocaine- and pentylenetetrazol-induced seizures in prenatally cocaine-treated rats.

We previously reported that prenatal cocaine exposure increased susceptibility to cocaine-induced seizures later in life. Here we examine whether this enhanced susceptibility to seizures generalizes to other chemoconvulsants, and whether postnatal cocaine treatment similarly increases susceptibility. Following prenatal cocaine treatment (40 mg/kg; E10-20), both male and female rats were more likely to seize to a dose of 30 mg/kg pentylenetetrazol (PTZ) at 2 months of age, although the severity of the seizures observed was increased only in females. Daily cocaine injections (10-20 mg/kg SC) during the first 10 days after birth also produced effects that were dependent on the sex of the animal. Postnatally cocaine-treated female rats showed no greater incidence of seizures in response to an acute high dose of cocaine, but did exhibit an increased susceptibility to cocaine-kindled seizures. Male, but not female, postnatally cocaine-treated rats were more susceptible to PTZ-induced seizures. The increased susceptibility to seizures induced by two different chemoconvulsants after prenatal cocaine treatment suggests that developmental cocaine exposure, particularly during the second trimester equivalent, alters the balance between excitation and inhibition in the brain.

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail.

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.

Left and right 6-hydroxydopamine lesions of the medial prefrontal cortex differentially affect voluntary ethanol consumption.

Dopaminergic projections to the medial prefrontal cortex (mPFC) were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) to examine how dopamine (DA) asymmetry in the mPFC influences voluntary ethanol consumption. Differences in nucleus accumbens (NAS) DA neurotransmission have been related to individual differences in locomotor activity and in the rewarding efficacy of ethanol. Therefore, differences in locomotor activity were used to further characterize the effects of unilateral mPFC 6-OHDA lesions on ethanol consumption. Male Long Evans rats were assessed for high versus low levels of spontaneous locomotor activity. DA terminals in the left or right mPFC were unilaterally lesioned with 6-OHDA, resulting in an average DA depletion of 54% and 50%, respectively. After a minimum seven-day recovery period, preference for a 10% ethanol solution vs. water was determined in a 24-h 2-bottle home-cage free-choice paradigm. Left mPFC 6-OHDA lesions increased and right lesions decreased ethanol consumption. These differential effects of left and right lesions were primarily attributable to rats exhibiting low locomotor activity prior to surgery. The present data suggest that right greater than left cortical DA asymmetry in combination with low endogenous NAS DA (predicted by low locomotor activity levels) may increase the vulnerability to abuse ethanol.

Rotation, locomotor activity and individual differences in voluntary ethanol consumption.

Spontaneous turning behavior and locomotor activity were evaluated for their ability to predict differences in the voluntary consumption of ethanol in male Long-Evans rats. Animals were assessed for their preferred direction of turning behavior and for high vs. low levels of spontaneous locomotor activity, as determined during nocturnal testing in a rotometer. Subsequently, preference for a 10% ethanol solution vs. water was determined in a 24-h two-bottle home-cage free-choice paradigm. Rats exhibiting a right-turning preference consumed more ethanol than rats showing a left-turning preference. While locomotor activity alone did not predict differences in drinking, turning and locomotor activity together predicted differences in ethanol consumption. Low-activity right-turning rats consumed more ethanol than all the other groups of rats. Previous studies from this laboratory have shown that individual differences in turning behavior are accompanied by different asymmetries in dopamine (DA) function in the medial prefrontal cortex (mPFC). Individual differences in locomotor activity are associated with differences in nucleus accumbens (NAS) DA function. The present data suggest that variations in mPFC DA asymmetry and NAS DA function may underlie differences in the voluntary consumption of ethanol.

Ethanol induced differences in medial prefrontal cortex dopamine asymmetry and in nucleus accumbens dopamine metabolism in left- and right-turning rats.

Ethanol (0.5 g/kg i.p.) 15 min prior to sacrifice increased homovanillic acid (HVA) levels in the left medial prefrontal cortex (mPFC) of left-turning rats and in the right mPFC of right-turning rats. In the nucleus accumbens (NAS), ethanol decreased dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and HVA levels in rats that exhibited low levels of locomotor activity but not in rats that exhibited high levels of locomotor activity. This laboratory has previously shown that rats exhibiting differences in turning and locomotor activity behavior display different preferences for ethanol. The present results suggest that ethanol-induced differences in mPFC and NAS DA activity may be related to individual differences in the susceptibility to abuse ethanol.

Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker.

BACKGROUND AND PURPOSE: Increased activation of excitatory amino acid (EAA) receptors is considered a major cause of neuronal damage. Possible sources and mechanisms of ischemia-induced EAA release were investigated pharmacologically with microdialysis probes placed bilaterally in rat striatum. METHODS: Forebrain ischemia was induced by bilateral carotid artery occlusion and controlled hypotension in halothane-anesthetized rats. During 30 minutes of ischemia, microdialysate concentrations of glutamate and aspartate were measured in the presence of a nontransportable blocker of the astrocytic glutamate transporter GLT-1, dihydrokinate (DHK), or an anion channel blocker, 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS), administered separately or together through the dialysis probe. RESULTS: In control striata during ischemia, glutamate and aspartate concentrations increased 44+/-13 (mean+/-SEM) times and 19+/-5 times baseline, respectively, and returned to baseline values on reperfusion. DHK (1 mmol/L in perfusate; n=8) significantly attenuated EAA increases compared with control (glutamate peak, 9. 6+/-1.7 versus control, 15.4+/-2.6 pmol/ microL). EAA levels were similarly decreased by 10 mmol/L DHK. DNDS (1 mmol/L; n=5) also suppressed EAA peak increases (glutamate peak, 5.8+/-1.1 versus control, 10.1+/-0.7 pmol/ microL). At a higher concentration, DNDS (10 mmol/L; n=7) further reduced glutamate and aspartate release and also inhibited ischemia-induced taurine release. Together, 1 mmol/L DHK and 10 mmol/L DNDS (n=5) inhibited 83% of EAA release (glutamate peak, 2.7+/-0.7 versus control, 10.9+/-1.2 pmol/ microL). CONCLUSIONS: These findings support the hypothesis that both cell swelling-induced release of EAAs and reversal of the astrocytic glutamate transporter are contributors to the ischemia-induced increases of extracellular EAAs in the striatum as measured by microdialysis.

Stimulant-mediated c-fos induction in striatum as a function of age, sex, and prenatal cocaine exposure.

Induction of the immediate-early gene c-fos by the stimulants cocaine and amphetamine (AMPH) was analyzed by Fos immunocytochemistry at different ages in the brains of prenatally cocaine-treated and control rats. Cocaine and AMPH induced c-fos in patches of striatal neurons during the first postnatal week, and thereafter produced a progressively more homogeneous pattern that was more dense medially. Quantification of Fos-immunoreactive cells in older rats revealed differences related to sex and prenatal cocaine treatment. Both cocaine and AMPH produced dose-dependent increases in the number of Fos-immunoreactive cells in striatum. Prenatal cocaine exposure resulted in increased Fos in males in response to AMPH (2 mg/kg) at P18 and cocaine (10 mg/kg) at 1-2 months. In females, prenatal cocaine treatment resulted in a reduced response to cocaine at 1-2 months. Increased c-fos induction was observed in control females compared to control males in response to low doses of stimulants; no such sex difference was observed in prenatally cocaine-treated rats. The dopamine D1 antagonist SCH23390 blocked cocaine-mediated c-fos induction in all groups. The NMDA antagonist MK-801 blocked cocaine-mediated c-fos induction in the medial striatum. In females only, MK-801 pretreatment resulted in a dramatic increase in the number of Fos-immunoreactive cells in lateral striatum. These findings indicate differences in the neural basis of c-fos induction in males and females, and changes in stimulant-mediated c-fos induction resulting from prenatal cocaine exposure.

Paw preference, rotation, and dopamine function in Collins HI and LO mouse strains.

Mice have paw preferences that are consistent upon repeated measurement. The Collins HI and LO strains are two populations of mice that have been selectively bred to differ markedly in the degree of paw preference. They represent a unique genetic model of functional cerebral lateralization. Rotation (or circling) behavior in normal unlesioned animals reflects an endogenous lateralization of the functioning brain dopamine (DA) systems. In the present study, rotational behavior and lateralized brain DA neurochemistry were assessed in the Collins HI and LO strain mice. Confirming Collins findings, HI strain mice exhibited stronger paw preferences than LO strain mice. HI strain mice also showed stronger percent directional preferences during nocturnal tests of spontaneous rotation. Neurochemical differences were also apparent between the strains. DA and its metabolites were measured in the medial prefrontal cortex (PFC), nucleus accumbens (NAS), and striatum. Degrees of rotational and paw preference in HI, but not LO, mice were correlated with PFC asymmetries in DA and the DA metabolite dihydroxyphenyl acetic acid (DOPAC), respectively. Hemisphere, paw preference, turning preference, and strain interacted in a complex way to determine measures of DA utilization in the NAS and striatum. Even though the directions of paw preference and rotation were not correlated, HI and LO mice of differing paw and rotational directional preferences showed differences in DA neurochemistry in the NAS and striatum.

Effects of prenatal cocaine exposure on the mesocorticolimbic dopamine system: an in vivo microdialysis study in the rat.

Microdialysis studies were conducted on prenatally saline-treated and prenatally cocaine-treated rats, either as pups (10-30 days old) or young adults (40-190 days old), to study the effects of prenatal cocaine exposure on the mesolimbic dopamine (DA) system. In the n. accumbens of saline-treated rats, basal dialysate concentrations of DA were similar in pups and adults; however, the levels of DA metabolites, DOPAC, HVA, and the serotonin metabolite, 5-HIAA, were markedly lower in pups. In pups, prenatal cocaine exposure led to basal dialysate levels of DA in the n. accumbens that were twice control levels; however, there was no difference in response to a period of intermittent tail pinch or an acute injection of cocaine (20 mg/kg). In the adult, basal levels of DA, DOPAC, HVA and 5-HIAA in n. accumbens did not differ across prenatal treatments. However, in prenatally cocaine-treated adults a cocaine injection led to an enhanced rise in extracellular DA compared to controls. In frontal cortex of adult rats, basal levels of DA, DOPAC and HVA did not differ across prenatal treatments; however, basal levels of 5-HIAA in this region were significantly elevated in prenatal-cocaine rats. No group differences were observed in the frontal cortex in response to either tail pinch or cocaine. Thus prenatal cocaine exposure produces an increase in basal extracellular DA in the n. accumbens of pups which returns to normal with aging. While this initial difference normalizes, prenatal cocaine exposure induces other persistent changes in adulthood.

Left and right 6-hydroxydopamine lesions of the medial prefrontal cortex differentially alter subcortical dopamine utilization and the behavioral response to stress.

The effects of left and right prefrontal cortical dopamine (DA) depletion on circling behavior, stress-escape behavior and subcortical DA function were examined in rats exhibiting left or right turning biases. 6-Hydroxydopamine lesions of the medial prefrontal cortex (PFC) caused significant DA depletions when assessed in separate studies at 3 days and 3-4 weeks. However, depletions were smaller at 3-4 weeks and there was a significant increase in DA concentration on the left side following right lesions. Significant increases in striatal DA content were observed following lesions of either side at 3-4 weeks, but not at 3 days. No changes in DA concentration were observed in the nucleus accumbens septi (NAS). Left circling rats significantly increased their circling behavior following right sided lesions and showed disrupted footshock-escape behavior following left sided lesions. Performance of the footshock-escape task exerted an effect on striatal and NAS DA utilization as indicated by the ratio of 3,4-dihydroxyphenylacetic acid (DOPAC) to DA. The effects of footshock on NAS DA utilization were greater following left PFC lesions as compared to the right lesion and sham conditions. These lesion effects were also greater in left- than in right-turning animals. The data indicate that an intrinsic asymmetry in brain DA systems interacts with left and right PFC lesions to differentially determine subcortical DA function and behaviors that it subserves.

Enhanced cocaine self-administration in adult rats prenatally exposed to cocaine.

Rats that had been prenatally exposed to either cocaine or saline were examined as adults using continuous reinforcement (FR1) cocaine self-administration. Initially these rats were water-deprived and trained to bar-press for water; no differences across prenatal treatments were observed during this training phase. After complete rehydration and implantation of an intravenous cannula into the external jugular vein, animals were introduced to cocaine self-administration with a nocturnal and subsequent 3 h exposure. During daily test sessions rats were allowed to self-administer cocaine for 1 h/day. Prenatal cocaine exposure led to a marked and stable enhancement of the rates of self-administration for up to 13 days, the longest time point examined. These results suggest that prenatal cocaine exposure can alter cocaine reinforcement in adult animals.

Chronic antidepressant drug treatment reduces turning behavior and increases dopamine levels in the medial prefrontal cortex.

The effects of chronic administration of the antidepressant drugs desipramine, nortryptiline and paroxetine (PAR) (10 mg/kg/day, 21 days) on changes in turning (circling) behavior and on norepinephrine (NE), dopamine (DA) and serotonin and their metabolites 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid (5-HIAA) in the medial prefrontal cortex (PFC), nucleus accumbens and striatum were evaluated in rats. All three drugs eliminated turning biases in right turning rats. All drugs increased DA concentrations in the PFC while PAR increased NE in the PFC and reduced 5-HIAA in all three structures. The results are discussed with reference to previous findings involving brain asymmetry in depression.

Prenatal cocaine alters later sensitivity to cocaine-induced seizures.

Rats that had been prenatally exposed to cocaine were tested later in life for their sensitivity to cocaine-kindled seizures and acute cocaine-induced seizures. When treated daily with cocaine, beginning at one month of age, males prenatally exposed to 40 mg/kg cocaine developed seizures in a fewer number of days than those prenatally exposed to saline. Prenatally cocaine-treated females did not seize more rapidly than controls in the cocaine kindling paradigm; however, they were more susceptible to seizures in response to an acute high dose of cocaine. These results suggest that rats prenatally cocaine-treated are more sensitive to the seizure-producing effects of cocaine later in life, and this enhanced sensitivity is differentially expressed in males and females.

Effects of iboga alkaloids on morphine and cocaine self-administration in rats: relationship to tremorigenic effects and to effects on dopamine release in nucleus accumbens and striatum.

Ibogaine, a naturally occurring alkaloid, has been claimed to be effective in treating addiction to opioid and stimulant drugs and has been reported to decrease morphine and cocaine self-administration in rats. The present study sought to determine if other iboga alkaloids, as well as the chemically related harmala alkaloid harmaline, would also reduce the intravenous self-administration of morphine and cocaine in rats. Because both ibogaine and harmaline induce tremors, an effect that may be causally related to neurotoxicity in the cerebellar vermis, the temorigenic activities of the other iboga alkaloids were assessed. Lastly, in view of the involvement of the dopaminergic mesolimbic system in the actions of drugs of abuse, the effects of some of the iboga alkaloids on extracellular levels of dopamine and its metabolites in the nucleus accumbens and striatum were determined. All of the tested alkaloids (i.e., ibogaine, tabernanthine, R- and S-coronaridine, R- and S-ibogamine, desethylcoronaridine, and harmaline) dose-dependently (2.5-80 mg/kg) decreased morphine and cocaine intake in the hour after treatment; decreases in morphine and cocaine intake intake were also apparent the day after administration of some but not all of these alkaloids (i.e., ibogaine, tabernanthine, desethylcoronaridine, and the R-isomers of coronaridine and ibogamine). In some rats, there were persistent decreases in morphine or cocaine intake for several days after a single injection or after two or three weekly injections of one or another of these alkaloids; R-ibogamine produced such effects more consistently than any of the other alkaloids.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurochemical predisposition to self-administer cocaine in rats: individual differences in dopamine and its metabolites.

Using in vivo microdialysis, this study attempted to determine whether a neurochemical predisposition to self-administer cocaine could be identified. Estimated extracellular levels of dopamine and its metabolites were measured bilaterally in the mesocorticolimbic and nigrostriatal systems of naive rats that were subsequently trained to self-administer cocaine intravenously. There were several significant relationships between dopamine and dopamine metabolite (3,4-dihydroxyphenylacetic acid and homovanillic acid) levels and rates of cocaine self-administration during both acquisition and asymptotic phases of testing. Dopamine levels in the nucleus accumbens were non-monotonically related to rates of self-administration during both phases: low to moderate dopamine levels were positively correlated with self-administration rates whereas moderate to high dopamine levels were negative correlated with self-administration rates. Dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels in the striatum were inversely correlated with self-administration rates during the acquisition phase. DOPAC and HVA levels in the left and right sides of the medial prefrontal cortex were positively and negatively correlated, respectively, with self-administration rates during the asymptotic phase; left/right asymmetrics in cortical metabolite levels were also correlated with asymptotic rates. There were no significant relationships between any neurochemical indices and rates of bar-pressing for water. These results suggest that the normal variability in drug seeking behavior is at least in part attributable to individual differences in the activity of brain dopamine systems.(ABSTRACT TRUNCATED AT 250 WORDS)