Chronic haloperidol during development attenuates dopamine autoreceptor function in striatal and mesolimbic brain regions of young and older adult rats.

The effects of chronic haloperidol administration during the prenatal and preweanling periods on dopamine autoreceptor function were examined in striatum, olfactory tubercles, and nucleus accumbens of young (2-3 month) and older (12-13 month) adult rats. In striatum of young and older adult rats that had been chronically treated with haloperidol early in life, as well as in the nucleus accumbens of older adults receiving early chronic haloperidol, gamma-butyrolactone (GBL) did not induce significant increases in dopamine levels. In olfactory tubercles of young adults that had received early chronic treatment with haloperidol, apomorphine pretreatment failed to reverse the observed GBL-induced increase in dopamine levels. Thus, dopamine autoreceptor function appears to be attenuated in rats chronically treated with haloperidol during early development, in contrast to reports of autoreceptor supersensitivity following neuroleptic treatment in adulthood.

Ontogeny of the enhanced behavioral response to amphetamine in amphetamine-pretreated rats.

Repeated administration of amphetamine to adult rats results in enhanced behavioral responses to subsequent amphetamine exposure. These experiments were designed to determine the earliest age at which behavioral sensitization to amphetamine could be detected. Rats from both sexes (n = 6-8/group) at ages of 1, 7, 21 or 49 postnatal days (PNDs) were injected with either d-amphetamine sulfate (5 mg/kg) or saline, SC, twice daily for 5 consecutive days. Stereotyped behavior and locomotor activity responses to a challenge dose of d-amphetamine (2.5 mg/kg), or saline, IP, were assessed for a total of 90 min, 15 days after the last dose of pretreatment. Amphetamine-induced stereotyped behavior was significantly enhanced only when amphetamine pretreatment was initiated at PND 49, but not at the earlier ages of PND 1, 7 or 21. There was no apparent sex difference in this effect. Correspondingly, amphetamine-induced locomotor activity was reduced in both sexes of the same age group (PND 49), but not in groups pretreated earlier, when compared to the saline-pretreated rats. These results suggest that amphetamine sensitization may be a late-developing effect, one which occurs sometime after the 3rd week of postnatal life.

Ontogenetic alterations in the effects of food and/or maternal deprivation on 5-HT, 5-HIAA and 5-HIAA/5-HT ratios.

Sprague-Dawley rat pups were deprived of food and/or the dam in experiment 1 for 24 h prior to sacrifice at 4, 10, 16 and 22 days postnatally and analysis of 5-HT, 5-HIAA and 5-HIAA/5-HT ratios in cortex, forebrain minus cortex, and brainstem. Deprivation was observed to increase indoles and their ratio early in life, with the most pronounced effects being observed in early maturing caudal brain regions of neonatal rat pups. These neurochemical effects of deprivation gradually diminished during ontogeny, becoming virtually absent by the age of weaning. In neonates, both deprivation from the dam and food appeared to influence indoleamine levels, whereas absence of food appeared to be primarily influential in older preweanling animals. Ambient temperature during the treatment period did not influence indoles or their ratio in 4- and 10-day-old rat pups, but did begin to influence indoles at 16 days postnatally, an age when homeothermia begins to develop. In experiment 2, it was observed that significant increases in indoleamines were not seen until 16 h of deprivation in 4-day-old rat pups deprived of food, and the dam for 0, 2, 4, 16 or 24 h prior to sacrifice. Possible functional implications of these deprivation-related increases in indoleamines early in ontogeny are discussed.

Prenatal haloperidol exposure: effects on brain weights and caudate neurotransmitter levels in rats.

Monoamines may exert a trophic effect on early brain development. To assess the role of dopamine in prenatal neurological development of the rat, haloperidol (HAL) was given in daily 2.5 or 5 mg/kg SC doses to dams over gestational days 6 to 20. This treatment regime did not enhance fetal mortality, but did produce reliable, if modest, stunting of the body and brain weight of offspring. The 5 mg/kg HAL dose consistently reduced offspring brain weight to roughly 90% of controls. This effect was probably permanent, in that it was seen throughout maturation and in adults as late as 140 days of postnatal age. Appropriate controls showed that this effect was not due to drug-induced reductions in food intake, to the presence of HAL in maternal milk, or to behavioral abnormalities in HAL-exposed dams. These effects had, at best, modest regional specificity, in that most brain regions were affected, independently of degree of dopaminergic innervation. Closer investigation of HAL effects on the striatum suggested that this permanent weight reduction was not accompanied by alterations in striatal concentrations of monoamines, monoamine metabolites, amino acids, choline, acetylcholine, DNA, protein, or water. It is concluded that prenatal HAL does stunt growth, but that this effect may not involve a direct drug influence restricted to the fetal dopamine system in the brain.

The role of NMDA and sigma systems in the behavioral effects of phencyclidine in preweanling rats.

To determine the role of NMDA receptor blockade and sigma receptors in the behavioral effects of PCP during development, we assessed the behavioral effects of PCP, (+)-MK-801 and 1,3-Di(2-tolyl)guanidine (DTG) in preweanling rats. In the first experiment, rats were injected sc on postnatal day (PND) 19 with 0.5-4.5 mg/kg PCP, and locomotor activity and wall climbing behavior were scored. PCP induced high levels of locomotor activity on PND 19 in a dose dependent manner with the 2.0 mg/kg dose producing the greatest activity. In the second experiment, rats were injected on PND 12 or 19 with 1.0-4.0 mg/kg PCP or 0.1-0.4 mg/kg (+)-MK-801 and tested using the same procedures. Both PCP and (+)-MK-801 induced activity increases on PND 19 in a dose dependent manner, with 2.0 and 3.0 mg/kg PCP and 0.2 mg/kg (+)-MK-801 inducing the highest activity levels. Peak activity levels on PND 12 were approximately 30% of those observed on PND 19, with the lowest dose of PCP and (+)-MK-801 producing the greatest activity. Large amounts of wall climbing behavior were elicited by PCP on PND 12, whereas (+)-MK-801 induced only minor amounts of wall climbing. In the third experiment, the effects of 0, 1, 3, 6, or 12 mg/kg DTG were examined in PND 13-14 and 16-17 rats. DTG had little effect on locomotor activity on PND 13-14, although the highest dose did inhibit activity. On PND 16-17, all doses of DTG tended to increase locomotor activity. The results suggest (1) the robust locomotor effects of PCP on PND 19 are mediated in part by NMDA mechanisms (2) this period of increased sensitivity to both PCP and (+)-MK-801 might represent a critical period of development when systems mediating locomotor activity are vulnerable to neurotoxic insult (3) NMDA blockade alone does not mediate PCP-induced wall climbing behavior and (4) that at the doses of DTG and the ages tested, sigma receptors do not play a role in the locomotor-inducing effects of PCP.

Chronic prenatal haloperidol exposure: lack of effect on presynaptic dopamine autoreceptors.

Prenatal haloperidol exposure in rats is known to reduce striatal D2 receptor binding in postnatal day (PND) 30 offspring. The effects of prenatal haloperidol exposure on presynaptic dopamine (DA) autoreceptor function are not well described. Pregnant albino rats were given daily injections of haloperidol (2.5 or 5.0 mg/kg, sc) or vehicle over gestational days 6-20. Presynaptic DA autoreceptor function was assessed using the DOPA decarboxylase inhibitor NSD-1015 and gamma-butyrolactone in the caudate, nucleus accumbens and olfactory tubercles of male offspring sacrificed on PND 97. There appeared to be no impairment of autoreceptor function in any region studied. The data indicate that prenatal haloperidol exposure does not result in permanent alterations in presynaptic DA autoreceptor function.

Gestational exposure to cocaine or pharmacologically related compounds: effects on behavior and striatal dopamine receptors.

Gestational cocaine (COC) exposure has been reported to alter behavior and possibly dopamine (DA) receptors. In this paper, we further examined the effects of prenatal COC (40 mg/kg, s.c.) on DA receptor binding and the behavioral response to quinpirole, a DA D2 receptor agonist. In an attempt to elucidate possible mechanisms of such effects, we exposed pregnant dams to specific reuptake blockers; fluoxetine 12.5 mg/kg, a serotonin reuptake blocker; desipramine 10 mg/kg, a norepinephrine reuptake blocker; GBR-12909 10 mg/kg, a DA reuptake blocker; or to a local anesthetic, lidocaine 40 mg/kg. Drugs were administered once daily over gestational days 8-20. Control dams were injected with saline (SAL) or pair-fed to the COC group. Quinpirole challenge was performed in the offspring on post natal day 19. Two pups per litter were injected (s.c.) with 0.03 or 0.09 mg/kg quinpirole-HCl on post-natal day 19. The remaining pups in each litter were sacrificed for analysis of striatal DA receptors. Results showed that only COC exposure altered the behavioral response to the quinpirole challenge by increasing quinpirole-induced stereotypy and motor activity relative to SAL controls. DA receptor analysis showed no alteration in K(D) or B(MAX) for striatal D1 or D2 sites in any group. These results suggest that prenatal COC exposure produces alterations in function of the D2 receptor complex which are not reflected in K(D) or B(MAX) and that these effects are not fully mimicked by exposure to specific monoamine reuptake blockers or a local anesthetic.

Behavioral and neurochemical effects of orally administered MDMA in the rodent and nonhuman primate.

MDMA (methylenedioxymethamphetamine) is a recreational drug of abuse known as "Ecstasy" which markedly decreases regional brain serotonin (5-HT) content and produces 5-HT nerve terminal degeneration in forebrain areas of the rat. In order to determine the acute and chronic behavioral effects of MDMA, adult rats were given MDMA at 0, 5 or 10 mg/kg, po for 4 consecutive days. Alternatively, parachloroamphetamine (PCA) at 5 mg/kg was administered under the same regimen. Within 30 min after the first dose, the MDMA-treated rats exhibited the serotonin motor syndrome consisting of straub tail and splayed hindlimbs comparable to that seen in the PCA-treated rats. This serotonin motor syndrome, with a duration of about 2 hr, was less pronounced after subsequent doses. At 2-4 wk after the last dose, no significant differences between control and treated rats were seen in emergence, hot plate response, auditory startle response or complex maze behavior even though a significant dose-related decrease (50%) in 5-HT concentration was observed in the frontal cortex and hippocampus of these rats 4 wks after the last dose. Adult female monkeys dosed po with 5 or 10 mg/kg of MDMA twice/day for 4 consecutive days demonstrated no spontaneous behavioral changes or weight loss compared to controls, but forebrain 5-HT concentration was reduced by 80% 1 mon after dosing. These data indicate that at doses only 2-3 times the human dose, MDMA produces significant forebrain 5-HT decreases but does not produce detectable residual behavioral alterations as assessed by these behavioral paradigms.

Pharmacological blockade of blood pressure and heart rate increases following milk ingestion in 15-day-old SHR and WKY rat pups.

The effects of hexamethonium, a ganglionic blocker, on blood pressure (BP) and heart rate (HR) responses to milk ingestion were assessed in awake, 15-day-old spontaneously hypertensive rats (SHR) and their normotensive progenitor strain, Wistar-Kyoto rats (WKY) using two methods of milk delivery. SHRs had larger increases in BP compared to WKYs, but WKYs exhibited larger increases in HR following milk ingestion from an anesthetized dam. BP responses to milk ingestion from a tongue cannula were also larger in SHRs. Administration of hexamethonium prior to milk delivery resulted in a drop in BP following milk ingestion in both milk delivery situations for each strain. The results suggest that SHRs exhibit exaggerated sympathetic activation to milk ingestion compared to WKYs, and that in both strains, cardiovascular responses to feeding are modulated by the presence of the dam.

Blood pressure and heart rate responses of SHR and WKY rat pups during feeding.

These studies measured changes in blood pressure and heart rates of 15-day-old SHR and WKY rat pups immediately following milk ingestion using three different methods of milk delivery. In the first experiment milk ejections were induced, using IV oxytocin, from the pups' natural mothers, while in the second experiment ejections were elicited from Marland Farms Wistar foster mothers. In the last experiment cow's milk was delivered using tongue cannulas and no mother was present. In each case we found that the dramatic increases in blood pressure associated with milk ingestion were larger in the SHR pups, while heart rate changes tended to be larger in the WKY. We also found that the dynamics of these cardiovascular responses to feeding were significantly altered by the presence of the mother.

Interactions of inflammatory pain and morphine in infant rats: long-term behavioral effects.

Neonatal rat pups exposed to repetitive acute pain show decreases in pain threshold and altered behavior during adulthood. A model using prolonged inflammatory pain in neonatal rats may have greater clinical relevance for investigating the long-term behavioral effects of neonatal pain in ex-preterm neonates. Neonatal rat pups were exposed to repeated formalin injections on postnatal (P) days 1-7 (P1-P7), with or without morphine pretreatment, and were compared with untreated controls. Behavioral testing during adulthood assessed pain thresholds using hot-plate (HP) and tail-flick (TF) tests, alcohol preference, and locomotor activity (baseline and postamphetamine). Adult rats exposed to neonatal inflammatory pain exhibited longer HP latencies than controls and male rats had longer HP thresholds compared to females. Male rats exposed to neonatal morphine alone exhibited longer TF latencies than controls. Both neonatal morphine treatment and neonatal inflammatory pain decreased ethanol preference, but their effects were not additive. During adulthood, male rats exposed to neonatal inflammatory pain exhibited less locomotor activity than untreated controls. We conclude that neonatal formalin and morphine treatment have specific patterns of long-term behavioral effects in adulthood, some of which are attenuated when the two treatments are combined.

Behavioral effects of prenatal haloperidol exposure.

Pregnant albino rats were exposed to vehicle (CON), 2.5 mg/kg (LOW) or 5.0 mg/kg (HIGH) haloperidol (HAL) from the sixth through the twentieth day of gestation. The effect of prenatal HAL exposure on offspring was assessed with the following five behavioral measures: 1) milk-induced behavioral activation on the sixth postnatal day (PND 6), 2) shock-precipitated wall climbing (PNDs 9, 11, 13, 15 and 17), 3) amphetamine-induced stereotypies (PND 30), 4) apomorphine-induced stereotypies (PND 30) and 5) duration of barbiturate anesthesia (PNDs 34 and 62). Measures taken very early in life indicated that prenatal HAL reduced arousal. Inactivity scores were elevated in HAL-exposed pups on PND 6 during milk-induced behavioral activation. Shock-precipitated wall climbing was reduced in the HAL animals on PNDs 9 and 11, but not thereafter. At PND 30, no prenatal treatment effect was detectable on stimulant-induced stereotypies or on duration of barbiturate anesthesia. On PND 62, barbiturate anesthesia duration was significantly reduced in both sexes of HIGH HAL animals. These findings suggest that prenatal HAL effects follow a dynamic, changing course as the exposed rat pup matures. Early reductions in arousal (milk-induced behavior and shock-precipitated wall climbing) wane with age, perhaps to be replaced by an actual increase in arousal as HAL pups approach adulthood.

Weanling rats exposed prenatally to cocaine exhibit an increase in striatal D2 dopamine binding associated with an increase in ligand affinity.

Prenatal exposure to cocaine can result in abnormal neurobehavioral development. This study found an increase in D2 dopamine receptor binding, associated with an increase in ligand affinity, in striatum of weanling rats exposed prenatally to cocaine. There were no changes in D2 receptor binding in nucleus accumbens nor D1 receptor binding in either striatum or nucleus accumbens. Alterations in D2 dopamine receptors may be associated with neurobehavioral alterations following prenatal cocaine exposure.

Neurochemical effects of prenatal haloperidol exposure.

The neurochemical effects of prenatal exposure to dopamine receptor antagonists are as yet poorly characterized. To further examine this problem, pregnant rats were given daily subcutaneous injections of vehicle, 2.5 or 5.0 mg/kg haloperidol over gestational days 6 through 20. Membrane binding of [3H]SCH-23390 (D1-specific) and [3H]spiroperidol (D2-specific in most brain areas) was measured in four regions of the cerebral dopamine system at postnatal day (PND) 30. Dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were measured in caudate on PND 30 following a d-amphetamine challenge. Prenatal haloperidol exposure reduced [3H]SCH-23390 and [3H]spiroperidol binding in caudate in a dose-dependent manner. [3H]Spiroperidol binding was similarly reduced in nucleus accumbens, but only the low dose (2.5 mg/kg) group showed decreased [3H]SCH-23390 binding in this region. Binding of neither compound was significantly altered in amygdala or frontal cortex. Basal or drug-stimulated levels of caudate DA and DOPAC were unaltered. It is concluded that prenatal haloperidol exposure reduces D1 and D2 binding in some, but not all regions of the forebrain dopamine system.

The ontogeny of behavioral sensitization to phencyclidine.

Repeated exposure of adult rats to a variety of psychoactive compounds can result in altered behavioral responsiveness to later exposures depending on the dose, route, and frequency of administration and time of testing. The ontogeny and mechanism of this altered responsiveness are not well understood. To determine when behavioral sensitization to phencyclidine (PCP) occurs, neonatal and early developmental exposure effects of PCP were assessed on later behavioral responsiveness to a PCP challenge. Rat pups were injected daily for nine days beginning on either postnatal days (PNDs) 1 or 22 with 0.9% saline, 5.0 or 10.0 mg/kg PCP-HC1 (s.c.). Ten days following the last injection, rats were given one of the following drug challenges: 0.9% saline, 5.0 or 10.0 mg/kg PCP-HC1 (s.c.). Locomotor activity, ataxia, and several other behaviors were measured for 1 h beginning 2-3 min after the challenge injection. Two major findings emerged from these studies. First, pups treated subchronically with PCP on PNDs 1-9 did not exhibit any difference in behavioral sensitivity to a PCP challenge when tested on PND 19 compared to subchronically treated saline controls. In contrast, pups subchronically treated with PCP on PNDs 22-30 exhibited an increased sensitivity to the behavioral effects of a PCP challenge. These data suggest that PCP has age-dependent exposure effects that occur sometime after the first postnatal week and that result in an enhanced behavioral responsiveness to PCP later in life.

Retinoic acid exposure on gestational days 11 to 13 impairs swallowing in rat offspring.

We have previously reported that exposure to 10 mg/kg of all-trans-retinoic acid (RA) daily on the 11th, 12th, and 13th days of rat gestation is lethal to all fetuses so exposed, due to an inability to suckle [R.R. Holson et al., Neurotoxicol Teratol 19 (1997) 347-353]. Because this lethal RA effect could be due to any of a variety of causes, from olfactory problems in locating the nipple to a motor problem in sucking or swallowing, we performed the following experiment. Albino dams were exposed to 10-mg/kg RA or vehicle daily over gestational days (GDs) 11 to 13. On the afternoon of GD 21 all pups were delivered by c-section. Tongue cannulae were inserted into the oral cavity of these offspring, and used to infuse a solution of condensed milk directly into the mouth. During and after each of four infusions, the behavioral response to the infusion (typically rolling and curling) was recorded. Controls responded well to this procedure, typically swallowing all milk so infused. In contrast, almost no RA-exposed neonates were able to swallow milk infused into the oral cavity. In such cases the milk simply dribbled out of the mouth, while the stomach was found to be empty at autopsy. However, the RA-treated animals did seem aware that milk was entering their mouths, because they showed a normal behavioral response to milk infusion. We conclude that GD 11-13 retinoid lethality is due to motor not sensory problems in the control of swallowing.

Behavioral and neurochemical effects of prenatal methylenedioxymethamphetamine (MDMA) exposure in rats.

MDMA is a hallucinogenic drug that is used by the general public as a recreational drug of abuse. The neurobehavioral consequences of prenatal MDMA exposure are unknown. Groups of pregnant rats were gavaged with 0, 2.5, or 10 mg/kg MDMA during gestation on alternate gestational days 6-18. Gestational duration, litter size, neonatal birth weights and physical appearance at birth were unaffected by MDMA treatments. Pregnancy weight gain was significantly reduced by MDMA treatment. Progeny growth, maturational parameters (eye opening and incisor eruption times), surface righting reflex, swimming performance, forelimb grip strength, milk-induced behaviors, passive avoidance behavior, figure-8 maze activity over 48 hours, the density of brain serotonin (5-HT) uptake sites, and brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were unaffected by MDMA treatments. Olfactory discrimination on postnatal days (PND) 9-11 was enhanced in both male and female MDMA-treated progeny, while negative geotaxis (PND 7-10) was delayed in female pups. In contrast to progeny, MDMA caused dose-dependent decreases in 5-HT and 5-HIAA levels in discrete brain areas of the dam. It is concluded that prenatal exposure to MDMA at the levels used here produces only subtle behavioral alterations in developing rats. The dam is more at risk for MDMA-induced 5-HT depletion than is the conceptus.

Haloperidol effects on the developing dopamine system: conflicting results and implications for neurobehavioral teratology research.

In an attempt to further develop basic principles to guide research in neurobehavioral teratology, six experiments were conducted to examine the effects of prenatal haloperidol (a D2 dopamine antagonist) exposure on striatal D1 and D2 binding sites. Another laboratory has repeatedly reported that prenatal exposure to this dopamine antagonist reduces striatal dopamine binding sites in exposed offspring. Our initial studies were successful in replicating and extending these previously reported reductions in D2 dopamine binding sites in caudate of rats exposed prenatally to haloperidol. However, additional experiments in our laboratory, in which pups were exposed to a range of haloperidol doses over gestational periods when the dopamine system has been reported to be most vulnerable to prenatal haloperidol exposure effects, have repeatedly failed to replicate our initial findings. Three other laboratories have also failed to duplicate this effect. The results of these studies suggest that beyond "standard" confounding variables, neurobehavioral teratologists are faced with as yet poorly understood factors that influence replication of findings within and between laboratories. These findings also emphasize the importance of within- and between-laboratory replication of experimental findings.

Cardiovascular responses to feeding in newborn piglets.

Cardiovascular responses to feeding have been observed in several species during various periods of development and have been implicated in the development of cardiovascular regulation. In the rat, these responses are characterized by short-lasting, large increases in blood pressure (BP) and moderate increases in heart rate. These responses appear to be sympathetically mediated because pretreatment with ganglionic blockers eliminates the increase in BP associated with milk ingestion. The present study was designed to determine if similar cardiovascular responses occur during feeding in the newborn piglet. Piglets were obtained on postnatal d 2 and fed a milk diet via automatic feeder 6 times a day. On postnatal d 6, the piglets were instrumented with an external carotid artery catheter and an internal jugular vein catheter. On postnatal d 8 and 9, direct arterial BP and heart rate were recorded during feeding. BP responses to milk ingestion were immediate, and they reached a maximum increase of 50% above baseline on both test days and followed a response profile similar to that previously described in the 15-d-old rat. An increase in heart rate was also observed, reaching a maximum of 42% above baseline. The results show that early in life piglets have large cardiovascular responses to milk ingestion similar to those observed in young rats and human infants. These responses appear to model the cardiovascular responses to feeding observed in human infants and might be useful as a noninvasive method for assessing neonatal autonomic reactivity. These responses also have the potential to cause adverse effects in newborns already at risk for cardiovascular and cerebrovascular disease.

Can adverse neonatal experiences alter brain development and subsequent behavior?

Self-destructive behavior in current society promotes a search for psychobiological factors underlying this epidemic. Perinatal brain plasticity increases the vulnerability to early adverse experiences, thus leading to abnormal development and behavior. Although several epidemiological investigations have correlated perinatal and neonatal complications with abnormal adult behavior, our understanding of the underlying mechanisms remains rudimentary. Models of early experience, such as repetitive pain, sepsis, or maternal separation in rodents and other species have noted multiple alterations in the adult brain, correlated with specific behavioral phenotypes depending on the timing and nature of the insult. The mechanisms mediating such changes in the neonatal brain have remained largely unexplored. We propose that lack of N-methyl-D-aspartate (NMDA) receptor activity from maternal separation and sensory isolation leads to increased apoptosis in multiple areas of the immature brain. On the other hand, exposure to repetitive pain may cause excessive NMDA/excitatory amino acid activation resulting in excitotoxic damage to developing neurons. These changes promote two distinct behavioral phenotypes characterized by increased anxiety, altered pain sensitivity, stress disorders, hyperactivity/attention deficit disorder, leading to impaired social skills and patterns of self-destructive behavior. The clinical important of these mechanisms lies in the prevention of early insults, effective treatment of neonatal pain and stress, and perhaps the discovery of novel therapeutic approaches that limit neuronal excitotoxicity or apoptosis.