Opiate withdrawal in utero increases neonatal morbidity in the rat.

Long-term oral administration of the long-acting opiate 1-alpha-acetylmethadol (LAAM) to female rats beginning on the day of conception interfered with the dams' ability to carry litters to term. When treatment was initiated 3 weeks prior to mating this effect was not observed. Daily administration of the opiate antagonist naloxone from day 14 of gestation through term, to precipitate withdrawal in utero, resulted in increased stillbirths, decreased pup weight and size, and weight loss 24 hours after birth. These data question the validity of animal experiments which purport to be models for methadone maintenance programs but in which treatment is started immediately prior to or soon after conception. They also suggest that withdrawal in utero may be responsible for many of the adverse effects of opiates on human and animal development.

Prenatal withdrawal from opiates interferes with hatching of otherwise viable chick fetuses.

Fetal chicks were made opiate-dependent by injections of N-desmethyl-1-alpha-acetylmethadol into the chorioallantois on day 3 of embryogenesis. The injections had no effect on subsequent hatchability; however, spontaneous fetal motility was significantly depressed. Injection of naloxone caused a significant increase in the motility of the opiate-exposed fetuses but had no effect on control fetuses. That naloxone's effect was an expression of opiate withdrawal and not due to antagonism of depressed motility is also supported by the observation that naloxone significantly reduced the hatchability of opiate-exposed chicks and not of control chicks. Thus the withdrawal of a developing organism from a narcotic may be more deleterious to its survival than continued exposure.

Subtle consequences of methylmercury exposure: behavioral deviations in offspring of treated mothers.

Overt neurological impairment is the endpoint currently used to document a case of methylmercury poisoning. No consideration is given to possible subtle consequences. Offspring from mice exposed to methylmercury on day 7 or 9 of pregnancy were apparently unaffected during postnatal development. However, subtle behavioral differences between treated and control offspring were found when the overtly normal animals were tested in an open field and evaluated in a swimming apparatus at 1 month of age. Brain weight, protein, choline acetyltransferase, and cholinesterase were not significantly altered.

OCD-Like behaviors caused by a neuropotentiating transgene targeted to cortical and limbic D1+ neurons.

To study the behavioral role of neurons containing the D1 dopamine receptor (D1+), we have used a genetic neurostimulatory approach. We generated transgenic mice that express an intracellular form of cholera toxin (CT), a neuropotentiating enzyme that chronically activates stimulatory G-protein (Gs) signal transduction and cAMP synthesis, under the control of the D1 promoter. Because the D1 promoter, like other CNS-expressed promoters, confers transgene expression that is regionally restricted to different D1+ CNS subsets in different transgenic lines, we observed distinct but related psychomotor disorders in different D1CT-expressing founders. In a D1CT line in which transgene expression was restricted to the following D1+ CNS regions-the piriform cortex layer II, layers II-III of somatosensory cortical areas, and the intercalated nucleus of the amygdala-D1CT mice showed normal CNS and D1+ neural architecture but increased cAMP content in whole extracts of the piriform and somatosensory cortex. These mice also exhibited a constellation of compulsive behavioral abnormalities that strongly resembled human cortical-limbic-induced compulsive disorders such as obsessive-compulsive disorder (OCD). These compulsive behaviors included episodes of perseverance or repetition of any and all normal behaviors, repetitive nonaggressive biting of siblings during grooming, and repetitive leaping. These results suggest that chronic potentiation of cortical and limbic D1+ neurons thought to induce glutamatergic output to the striatum causes behaviors reminiscent of those in human cortical-limbic-induced compulsive disorders.

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.

Salicylate and cocaine: interactive toxicity during chicken mid-embryogenesis.

Increased free radical production, due to ischemia and reperfusion, has been postulated as a cause of cocaine's (COC) developmental toxicity. Salicylate reacts with hydroxyl free radicals (*OH) to form stable, quantifiable reaction products, which can be measured with high-pressure liquid chromatography (HPLC). To determine if chicken embryos' brains and hearts were exposed to increased *OH concentrations after injection of COC, an injection of a nontoxic dose of sodium salicylate (NaSAL, 100 mg/kg egg, or 5 mg/egg), followed by 5 injections of COC (13.5 mg/kg or 0.675 mg/egg, every 1.5 h), was administered to eggs containing embryos on the 12th day of embryogenesis (E12). In addition to finding increased *OH concentrations in E12 embryonic hearts and brains, we observed that the developmental toxicity of COC, manifest as vascular disruption (hemorrhage) and lethality, was enhanced by NaSAL injection. These results confirm and extend results of similar experiments performed upon older embryos (E18), and indicate that increased &z.rad;OH concentration in embryonic tissues after COC exposure and toxic interactions of COC and NaSAL can also occur at an earlier stage of development. The results are discussed in light of possible exposure of human fetuses to both COC and salicylates, since COC-abusing pregnant women can be misdiagnosed with pre-eclampsia and aspirin is used to treat this syndrome.

Distribution of methionine-enkephalin immunoreactivity in the chick brain: an immunohistochemical study.

The distribution of Met-enkephalin-like immunoreactivity in the brains of 2-week-old domestic chicks was studied with immunohistofluorescence and the unlabeled antibody peroxidase-antiperoxidase technique, using antibodies to Met-enkephalin generated in rabbits. Immunoreactive cell bodies were found in the telencephalon, diencephalon, and mesencephalon in areas as yet uncharacterized as discrete nuclei in birds (E-1, E-2, E-3, E-4, E-5); further cells were located in the diencephalic nucleus spiriformis lateralis, the midbrain medial intercollicular nucleus (E-6), the nucleus mesencephalicus lateralis, pars dorsalis, the Edinger-Westphal nucleus and dorsal occulomotor nucleus; and in the nucleus of cranial nerve X and an uncharacterized area in the dorsolateral medulla (E-7). Immunoreactive fibers and/or terminals were located around the immunoreactive cell bodies and, in addition in the lateral septal area of the telencephalon; in the preoptic and hypothalamic areas of the diencephalon; in the anterior intercollicular area, periaqueductal central gray, area C, and the midventral tegmentum of the mesencephalon; in the nucleus solitarius, nucleus IX-X, nucleus intercalatus, nucleus intermedius, and ventrolateral areas of the rhombencephalon. The pattern of distribution of met-enkephalin in the chick is compared with that in the rat. A possible functional role for Met-enkephalin in neural mechanisms mediating some behaviors of the chick is suggested.

Ontogeny of glucocorticoid receptors in the hyperstriatum-hippocampus-parahippocampal area and optic tectum of the embryonic chicken (Gallus domesticus) brain.

The importance of glucocorticoids and their perturbation during development is an active research area. Developmental insults, including direct and indirect consequences of exposure to drugs of abuse or withdrawal from them, may act upon or via the neuroendocrine axis of the pregnant experimental subject (e.g. rat) and/or directly upon the neuroendocrine axis of the embryo or fetus. The use of the domestic chicken embryo may constitute a good experimental subject for studying these effects in the absence of maternal influences. Thus, the pattern of brain glucocorticoid cytosolic receptors were characterized in an early developing brain region, the optic tectum (OT) and a later developing region with a different function, the hyperstriatum-hippocampus-parahippocampal (HHP) area, on embryonic days (E) 11, 15, 18 and on the day of hatching (HD). The influence of the glucocorticoid synthesis inhibitor metyrapone, injected into eggs on E14 and on E17, upon glucocorticoid receptors (on E15 and E18) was also studied to determine effects of a 'chemical adrenalectomy'. Receptors for this steroid are high on E11 and E15, decreasing as they approach the time of hatching, with the HHP generally showing greater numbers of specific binding sites for [3H]-corticosterone (CORT). Although metyrapone treatment did not alter the apparent number of receptors on E15, on E18 it unmasked receptors otherwise occupied by endogenous ligand(s) and/or induced their synthesis, resulting in significantly more receptors identified with [3H]-CORT. Nevertheless, the HHP continued to display more of these receptors than the OT on E15 and E18 after injection of metyrapone. These observations are consistent with the hypotheses that the HHP of embryos of this species contains a higher density of glucocorticoid receptors than does the OT; that glucocorticoid receptor quantification is related to steroid synthesis inhibition in late embryonic development; and that neuroendocrine feedback control of serum glucocorticoids may become functional between E15 and E18. The results also suggest the use of this experimental approach for assessing the effects of developmental insults with drugs, other than metyrapone, as a marker for altered neuroendocrine development and/or function.

Greater task difficulty amplifies the facilitatory effect of des-glycinamide arginine vasopressin on appetitively motivated learning.

Rats were trained in a discrete-trial forward autoshaping paradigm to touch an extended lever to earn food pellets. Reinforcement was delivered either simultaneously with or 6 s after lever retraction which occurred either non-contingently after 15 s or when the animal touched the lever. Treatment with des-glycinamide arginine vasopressin (DGAVP; 15 micrograms/kg, sc) 1 hr before sessions increased the rate of acquisition of the extended-lever-touch response and also facilitated development of intertrial (adjunctive) nose poking. Effects of the peptide were more robust in the more difficult, delayed reinforcement task. DGAVP lacks the classical peripheral activity of vasopressin. In both experiments, peptide treatment was terminated before asymptotic levels of performance were attained; the continued facilitation of acquisition in treated groups suggests a specific enhancement of learning and/or enhanced memory retrieval.

The serotonin2 antagonist ritanserin blocks quasi-morphine withdrawal at a time when mianserin is no longer effective.

A quasi-morphine withdrawal syndrome (QMWS), produced in opiate-naive rats with an injection of isobutylmethylxanthine (IBMX) and the opioid antagonist naloxone, allows one to study the expression of opiate withdrawal in the absence of the acute or chronic effects of opiates and the adaptive processes termed dependence. The allegedly selective and long-acting serotonin2 (5-HT2) antagonist ritanserin attenuated the QMWS-induced suppression of fixed ratio (FR) operant responding, which is a sensitive measure of the expression of a QMWS. When administered 30 min prior to precipitation of the QMWS, the lowest dose of ritanserin tested (0.158 mg/kg) was the most effective in blocking the expression of withdrawal; however, there was not complete reversal of the behavioral suppression. Acutely, the two higher doses of ritanserin tested (2.5 and 10 mg/kg) suppressed responding when given alone. This may have masked their ability to attenuate a QMWS. At a dose of 2.5 mg/kg, ritanserin completely blocked the QMWS-induced suppression of responding 24 h post-administration, at a time when its actions at other receptors (e.g., alpha 2) have dissipated. At an equivalent dose, the shorter-acting 5-HT2 antagonist mianserin was unable to attenuate the QMWS-induced suppression of FR operant responding 24 h post-administration. The 5-HT2 antagonists reportedly produce a paradoxical down-regulation of 5-HT2 binding sites upon chronic treatment, rather than the expected supersensitivity. Chronic treatment with ritanserin (2.5 mg/kg/day for 7 days), but not mianserin (same regimen), attenuated a QMWS 24 h after the final injection, thus supporting with a functional measure, the down-regulation of such binding sites by ritanserin.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects of neonatal exposure to isobutylmethylxanthine. I. Retardation of learning with antagonism by mianserin.

Pregnant women regularly ingest the methylxanthines, caffeine and theophylline, during pregnancy and lactation. Also, theophylline is used to treat apnea in premature infants. In this study, rat pups were treated with 3-isobutyl-1-methylxanthine (IBMX), on days 7-10 of life. Transient IBMX treatment during infancy caused a retardation of acquisition of a delayed reinforced autoshaped lever touch response in adulthood. Treated rats required more trials to learn the task, but did not show altered exploratory activity in the operant chambers. Coadministration of the serotonin (5-HT) antagonist mianserin with IBMX was able to attenuate significantly the effects of IBMX in both males and females, even though mianserin treatment alone caused an apparent learning deficit in the males. The results indicate that 5-HT and 5-HT receptors are important during development for normal expression of a specific cognitive function later in life. Furthermore, a 5-HT system appears to play a role in the mechanism whereby perinatal methylxanthine exposure could lead to learning impairments or other undesirable behavioral consequences. The use of IBMX in developing rats may also offer a model for studying the long-term consequences of the expression of opioid withdrawal during the neonatal period, since this agent induces a quasi-morphine withdrawal syndrome (QMWS) in mature rats. It is of interest that mianserin can block or attenuate effects of both quasi- and true morphine withdrawal.

Modification of quasi-morphine withdrawal with serotonin agonists and antagonists: evidence for a role of serotonin in the expression of opiate withdrawal.

Methylxanthines produce a quasi-morphine withdrawal syndrome (QMWS) in opiate naive rats. Additionally, methylxanthine-induced suppression of conditioned behavior in rats is reversed by the alpha 2 adrenergic agonist clonidine which also attenuates true opiate withdrawal and the QMWS. Therefore, the operant behavioral effects of 3-isobutyl-1-methylxanthine (IBMX) provide a model with which to study mechanisms involved in the expression of opiate withdrawal. In order to examine the role of serotonin (5-HT) in the rate-decreasing effects of IBMX on operant behavior, the 5-HT precursor 5-hydroxytryptophan, and 5-HT reuptake blocker fluoxetine were administered in combination with IBMX to rats performing a fixed-ratio 30 operant for food reinforcement. Both drugs failed to reverse the behavioral suppression caused by relatively low doses of IBMX, suggesting that elevated 5-HT neurotransmission contributes to, rather than attenuates, the QMWS. The relatively selective 5-HT2 antagonists mianserin and pirenperone blocked the IBMX-induced suppression, whereas the classic 5-HT antagonist methysergide had no effect. The results indicate that the operant behavioral effects of IBMX and possibly the QMWS may be mediated by serotonergic mechanisms.

Cocaine and salicylate: documentation of hydroxyl radical formation in hearts and brains of 18-day-old chick embryos and unexpected interactive toxicity.

RATIONALE: Multiple low doses of cocaine (COC) may cause intermittent vasoconstriction and reperfusion, leading to elevations in damaging reactive oxygen species, such as hydroxyl free radicals (*OH). Salicylate may offer protection because it reacts with *OH and/or because of its anti-inflammatory actions. OBJECTIVE: To measure *OH concentrations in hearts and brains of chicken embryos exposed to multiple, small doses of COC, and to determine if otherwise non-toxic doses of sodium salicylate (NaSal) protected against the marginal but significant reduction in hatchability caused by a model of "binge" COC exposure. METHODS: Three experiments were carried out. In the first, 67.5 mg COC/kg egg was administered as five doses of 13.5 mg/kg egg or 0.675 mg/egg every 1.5 h, injected just beneath the shell, on day 18 of development (E18), 1 h after NaSal (25 or 100 mg/kg egg) was injected as a bolus. Hearts and brains taken shortly afterward were analyzed for *OH. In experiment 2, the dose of COC was reduced to 56.5 mg/kg egg so as to achieve a small but significant reduction in hatchability in order to determine if NaSal protected against or enhanced COC's toxicity, manifest as an increase or decrease in hatchability. The doses of NaSal for this experiment were 50, 100 or 200 mg/kg egg, all devoid of effects upon hatchability when injected alone. Experiment 3 was done to confirm the presence of vascular disruptions/hemorrhages observed on COC-exposed embryos while harvesting hearts and brains for chemical analyses and to quantify what appeared to be enhanced COC-related vascular accidents associated with NaSal pretreatment. The dose of NaSal used in experiment 3 was 200 mg/kg egg and COC was injected again at 5x13.5 mg/kg egg. RESULTS: COC increased *OH in hearts and brains of chicken embryos on E18, and non-toxic doses of NaSal (i.e. 100 or 200 mg/kg egg) enhanced COC's toxicity in a dose-related manner. The lowest NaSal dose (50 mg/kg egg) may have offered some protection against the effects of COC, as the reduction in hatchability caused by 56.5 mg COC/kg egg was no longer significant. Vascular disruptions/hemorrhages were associated with and most likely responsible for the interactive toxicity. CONCLUSIONS: Our unexpected findings may be of clinical relevance because of the use of aspirin for treatment of misdiagnosed "preeclamptic" COC-abusing pregnant women and its possible use for COC abusers at risk for reduced cerebral blood flow and stroke.

Long-term effects of neonatal exposure to isobutylmethylxanthine. II. Attenuation of acute morphine withdrawal in mature rats.

An acute model of morphine withdrawal was used to determine if neonatal exposure to 3-isobutyl-1-methylxanthine (IBMX) would cause alterations in the expression of withdrawal in the adult rat. IBMX induces a quasi-morphine withdrawal syndrome (QMWS), which is almost identical to true morphine withdrawal both behaviorally and neurochemically. Transient IBMX treatment during infancy (on days 7-10 of life) caused an attenuated suppression of fixed ratio (FR) responding during acute morphine withdrawal in adulthood; however, there appeared to be no attenuation of withdrawal-induced hypothermia. The attenuated behavioral response was not due to an altered ability to express withdrawal, as these rats did not react differently to various doses of IBMX plus naloxone (i.e., varying severities of quasi-morphine withdrawal) in adulthood. Coadministration of the serotonin (5-HT) antagonist mianserin with IBMX in the neonate prevented the effects of IBMX. Both the mianserin-treated and the IBMX plus mianserin-treated groups had increased levels of [3H]naloxone binding in brainstem, while IBMX treatment alone apparently had no significant effect. None of the neonatal drug treatments affected [3H]naloxone binding in frontal cortex. Thus, the long-term effects of IBMX on the opioid withdrawal response cannot be explained by changes in the number of opioid binding sites (labelled with [3H]naloxone) within the brain. The results indicate that exposure to a methylxanthine, and thus quasi-morphine withdrawal, during development results in long-lasting alterations of a system which is involved in opioid withdrawal. Because coadministration of mianserin prevented the effects of IBMX, 5-HT and 5-HT2 receptors are implicated in these effects.

The effects of morphine and delta-9-tetrahydrocannabinol on motor activity in rats.

Acute treatment of rats either by high doses of morphine or delta 9-tetrahydrocannabinol (THC) decreased locomotor activity. Naloxone reversed morphine-induced depression completely and reversed THC-induced depression only partially. On day 3 of treatment, tolerance developed to the locomotor inhibitory action of THC or morphine and partial cross-tolerance was observed to the depressant action of THC. Naloxone slightly depressed locomotor activity in THC-tolerant rats, but increased motor activity in morphine-tolerant rats.

Selective learning impairment of delayed reinforcement autoshaped behavior caused by low doses of trimethyltin.

The organometal neurotoxin trimethyltin (TMT), induces impaired learning and memory for various tasks. However, administration is also associated with other "non-specific" behavioral changes which may be responsible for effects on conditioned behaviors. To determine if TMT treatment causes a specific learning impairment, three experiments were done using variations of a delay of reinforcement autoshaping task in which rats learn to associate the presentation and retraction of a lever with the delivery of a food pellet reinforcer. No significant effects of TMT treatment were found with a short (4 s) delay of reinforcement, indicating that rats were motivated and had the sensorimotor capacity for learning. When the delay was increased to 6 s, 3.0 or 6.0 mg TMT/kg produced dose-related reductions in behaviors directed towards the lever. Performance of a group given 7.5 mg TMT/kg, while still impaired relative to controls, appeared to be better than the performance of groups given lower doses. This paradoxical effect was investigated with a latent inhibition paradigm, in which rats were pre-exposed to the Skinner boxes for several sessions without delivery of food reinforcement. Control rats showed retardation of autoshaping when food reinforcement was subsequently introduced. Rats given 7.5 mg TMT/kg exhibited elevated levels of lever responding during pre-exposure and autoshaping sessions. The results indicate that 7.5 mg TMT/kg produces learning impairments which are confounded by hyperreactivity to the environment and an inability to suppress behavior toward irrelevant stimuli. In contrast, low doses of TMT cause learning impairments which are not confounded by hyperreactivity, and may prove to be useful models for studying specific associational dysfunctions.

Modulation of the behavioural effects of interleukin-1 in mice by nitric oxide.

Interleukin-1 is a cytokine which mediates the host response to infection and inflammation and is responsible for sickness behaviour. Inhibition of nitric oxide synthase activity by N omega nitro-L-Arginine-Methyl-ester (30 mg kg-1, i.p.) potentiated the depressive effects of interleukin-1 (375 ng, i.p.) on social investigation in mice. This effect was attenuated by L-arginine (180 mg kg-1, i.p.) but not by D-arginine. The same treatment did not alter the body weight loss induced by interleukin-1. These results suggest that nitric oxide plays a protective role in the neural effects of interleukin-1.

Parallel changes in operant behavioral adaptation and hippocampal corticosterone binding in rats treated with trimethyltin.

Rats were given water vehicle or trimethyltin (TMT; 3.0, 6.0 or 7.5 mg/kg, p.o.). Lever responding for food was measured 3 months later, in a test in which the fixed ratio requirement was doubled daily (FR1-128). Response rates for all groups were inverted U-shaped functions of FR values. However, the effect of increasing ratio values was attenuated in the 6.0 mg/kg group, which responded less than controls when control rates were maximal (at FR16 and FR32). In contrast, rats given the high dose responded at higher rates (at FR4 and FR64). [3H]Corticosterone binding to hippocampal cytosolic protein was maximally reduced for the group given 6.0 mg TMT/kg. The greatest reduction in hippocampal weight resulted from injection of 7.5 mg TMT/kg, but a smaller reduction in [3H]corticosterone binding (i.e. 22%) was observed for this group. In the absence of an effect of 3.0 mg TMT/kg upon weight of hippocampus, there also was a reduction in steroid binding, indicating the sensitivity of this parameter for TMT toxicity. The results support the notion that hippocampal corticosteroid receptors are important for behavioral adaptation, and rats given moderate doses of TMT may be useful for studying functions of corticosterone receptors.

Des-Gly-vasopressin improves acquisition and slows extinction of autoshaped behavior.

The effects of a vasopressin analog (DGAVP) with minimal endocrinological activity, were assayed on acquisition and extinction of a discrete trial, food reinforced, autoshaped lever touch response. Magazine-trained rats, maintained at 80-85% of free-feeding body weights, were injected s.c. with saline, 5 or 10 micrograms/kg of DGAVP 1 h before each of two sessions in which they learned to touch a retractable lever, presented on a 45 s random interval (RI 45) schedule. Retracted lever contacts (nose-pokes) and unconditioned rearing activity were simultaneously monitored. After acquisition of the extended lever touch response, rats were reassigned to treatment groups, and again injected with saline, 5 or 10 micrograms/kg of DGAVP 1 h before each of two extinction sessions. DGAVP facilitated acquisition (5 micrograms/kg) and slowed extinction (5 and 10 micrograms/kg) of conditioned behavior, while having no effects on the other behaviors, thus demonstrating the specificity of the effect of a vasopressin-like compound on both tasks (enhanced acquisition and retarded extinction) used to study learning.