Embryonic "binge" cocaine exposure alters neural-immune and neural-endocrine interactions in young chickens: involvement of serotonin(2) receptors.

As part of our characterization of the developmental consequences of prenatal cocaine exposure, cocaine was injected into eggs containing viable chicken embryos on embryonic day (E) 18 and the fever response to the endotoxin lipopolysaccharide (LPS) and a delayed-type hypersensitivity response to phytohemagglutinin (PHA) were assessed postnatally. E18 cocaine exposure did not affect basal body temperature. LPS induced a fever in the chicks at 4 h post-injection on post-hatch day (D) 4 and 2 h post-injection on D24. E18 cocaine exposure suppressed the peak LPS-induced fever by 50% at both ages. E18 cocaine exposure also suppressed the hypersensitivity reaction to an intradermal injection of PHA on D17, while having no effect on the response to a saline injection. To determine the importance of serotonin(2) (5-HT(2)) receptors in the developmental toxicity of cocaine, varying doses of the 5-HT(2) antagonist ritanserin were injected on E17 followed by cocaine on E18. Ritanserin, like cocaine, did not alter basal temperature, but it dose-relatedly attenuated or blocked cocaine's effect on LPS-induced fever on both D4 and D24. Ritanserin pretreatment was also able to block the blunted isolation stress response seen in D16 chicks following E18 cocaine exposure. Thus, late prenatal cocaine exposure significantly alters adaptive fever and hypersensitivity responses, and embryonic 5-HT(2) receptors played a mediating role in the fever effect.

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.

Embryonic cocaine exposure and corticosterone: serotonin(2) receptor mediation.

Cocaine activates the mature hypothalamic-pituitary-adrenal (HPA) axis, increasing corticosterone concentrations in animals and humans and serotonin(2) receptors (5-HT(2)) are involved in this effect. Although prenatal cocaine exposure is associated with altered responsiveness of the HPA axis to "stress" and serotonergic compounds postnatally, it is unknown whether cocaine directly activates the embryonic HPA axis or if 5-HT(2) receptors are involved. Domestic chicken eggs with viable embryos were exposed to either the 5-HT(2) receptor agonist dimethoxyiodophenylaminopropane (DOI: 0.4, 0.8, or 1.2 mg/kg egg) or saline on embryonic day 18 (E18). In a second study, the 5-HT(2) antagonist ritanserin (0.3 mg/kg egg, a dose found effective against other effects of DOI or cocaine) or vehicle was administered on E17, prior to treatment on E18 with either saline or cocaine (5 injections of 12 mg/kg egg, equivalent to a total dose of 3.5 mg/egg). Radioimmunoassay was used to measure serum corticosterone from blood samples taken approximately 1-2 h after drug injections. DOI significantly raised corticosterone in a dose-related fashion. Cocaine-induced corticosterone elevations were blocked by pretreatment with ritanserin, whereas ritanserin by itself did not affect corticosterone concentrations. These data indicate that 5-HT(2) receptors are involved in cocaine's effect on the HPA axis during late chicken embryogenesis.

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.

Late embryonic ritanserin exposure fails to alter normal responses to immune system stimulation in young chicks.

Prior studies in our laboratory have demonstrated that prenatal treatment with the serotonin2 (5-HT2) antagonist ritanserin is effective in blocking some of the lethal, dysmorphic, cardiovascular, and behavioral consequences of excessive direct or indirect stimulation of 5-HT2 receptors in the developing chicken. The efficacious dose range for ritanserin in these studies had very little or no effect on the above measures of toxicity when administered alone. In the present study, we extend our characterization of ritanserin's potential toxicity, or lack thereof, to include the normal behavioral and endocrine responses to immune system stimulation by the endotoxin lipopolysaccharide (LPS). LPS administration induces a syndrome collectively known as sickness behavior, manifest as altered thermoregulatory processes leading to fever, and increased serum concentrations of neuroendocrine hormones, including corticosterone. These survival-promoting responses to LPS were assessed in young chickens that had been treated with doses of ritanserin ranging from 0 to 2.7 mg/kg on embryonic day 17 (E17). When sickness behavior was assessed in 5-7-day-old chicks 1 h post-LPS injection, E17 ritanserin-treated subjects did not differ from controls. At 4-6 h post-LPS, 4-day-old chicks displayed a robust fever, and E17 ritanserin did not affect the magnitude of this response. Similarly, E17 ritanserin treatment failed to affect corticosterone concentrations 2 h post-LPS in 14-day-old chicks. Thus, ritanserin treatment during late embryogenesis, a time when it is effective against direct and indirect acting 5-HT2 agonists, failed to modify the survival promoting and beneficial interactions between the nervous, endocrine, and immune systems that are elicited following immunostimulation.

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.

Voltage associated with spontaneous embryonic motility in the developing chicken: an automated characterization during mid-late embryogenesis.

Movement of developing chicken embryos and their associated membranes generates voltage detectable with electrodes inserted just beneath the eggshell. Use of such voltages as a motility indicator offers an embryonic behavioral assessment method less subjective and invasive than observational methods using windows that disrupt substantial portions of the eggshell. We used a computerized signal recording and processing procedure to compare voltages from embryonic Day 12 (E12), E15, and E18 chicken eggs with embryos, assessed on the same day. Larger voltages were recorded from E18 subjects than from E12 or E15 subjects. Because this could have been due to embryonic size (mass) and/or proximity to the electrodes, making age comparisons uninterpretable, we used standard deviation-normalized and Z-score-based data transformations, comparing groups for relative deviations from basal voltages. E18 subjects still appeared more active than E12 subjects, with E15 a transitional age, in contrast to results from earlier window-based studies. The automated assessment method we used could enhance behavioral teratology studies of avian species.

Open-field and LPS-induced sickness behavior in young chickens: effects of embryonic cocaine and/or ritanserin.

Exposure to drugs of abuse during embryogenesis may adversely affect nervous, immune, and endocrine systems development. We compared exposure on embryonic day 18 (E18) by single or multiple cocaine (COC) injections (56.25 mg/kg total dose for both) or saline on hatching and activity measures. In saline-exposed controls, repeated testing, age, and gender affected activity levels. A single or multiple COC injections increased the median latency to explore and multiple COC injections decreased the median number of lines crossed by female chicks in the open field. We also determined if pretreatment with the serotonin2 (5-HT2) receptor antagonist ritanserin could attenuate COC's effects on open-field behavior as well as behaviors sensitive to immune system stimulation (lipopolysaccharide (LPS)-induced sickness behavior). Eggs containing embryos were pretreated on E17 with 0.4 mg ritanserin/kg or its vehicle followed by multiple COC injections or saline on E18. E18 COC treatment decreased the median number of lines crossed and distress vocalizations in females. Ritanserin pretreatment mitigated the COC induced effects. E18 COC exposure also suppressed LPS-induced sickness behaviors in both males and females, increasing food consumption and the time spent awake and active, as well as decreasing the time spent sleeping. Ritanserin alone had no effect on the food consumed or time spent active, nor did this dose affect COC-induced alterations in sickness behavior. Ritanserin alone decreased time spent sleeping and also failed to affect the COC-induced suppression. Thus, embryonic COC exposure can suppress open field and LPS-induced sickness behavior in the young chick, and ritanserin pretreatment can block the former, but not the latter effects at the dose chosen for these experiments.

Acute heat stress model of seizures in weanling rats: influence of prototypic anti-seizure compounds.

The present study tested the therapeutic potential for prototype anti-epilepsy drugs using an animal model of infantile febrile seizures. The model consisted of immersion of weanling rats (21 days old) in a 45 degrees C water bath for a maximum of 4 min (four exposures over a 2 week period) and observing for the progression to stage-5 seizures. All compounds were administered orally at the respective ED50 for prevention of seizures in the maximal electroshock (MES) test. Clonazepam effectively lowered the score for seizure grade, shortened the duration of seizures, as well as reduced the number of animals experiencing seizures during three of the four testing periods. MK801 reduced both the maximum seizure grade, and the number of animals experiencing seizures during sessions two and three. However, the dose of MK801 caused behavioral side effects. Valproate actively decreased seizure grade, while it modestly acted to attenuate seizure duration, extended the time to seizure onset, and reduced the number of animals experiencing seizures on testing day 1. Remacemide hydrochloride and phenobarbital were not effective. The method appears useful for evaluating the potential of agents to prevent acute febrile seizures.

Relationships between midembryonic 5-HT2 agonist and/or antagonist exposure and detour learning by chickens.

The importance of serotonin (5-HT) as both a transmitter and a regulatory signal during development of many species is well established. The availability of 5-HT receptor subtype agonists and antagonists will enable pharmacological dissection of the importance of one or more of the 5-HT receptors for their involvement in the mediation of developmental insults by drugs that are less selective but include actions upon serotonergic function. Such insults include exposure to cocaine or opiate withdrawal, both of which are blocked or attenuated by 5-HT2 antagonists. The 5-HT2 receptor agonist dimethoxyiodophenylaminopropane (DOI), like cocaine, causes vasoconstriction during embryogenesis, herniated umbilici in hatchlings, and altered detour learning by young chickens after injection into eggs at late stages of embryogenesis. The 5-HT2 antagonist ritanserin (RIT) blocks or significantly attenuates these effects. This study describes an effect of DOI on posthatch detour learning when injected earlier during embryogenesis (i.e., on embryonic day 12, E12) which is opposite its effect when injected later (i.e., on E15). Both effects are blocked by an inactive dose of RIT (0.3 mg/kg egg) and by a higher dose of RIT (0.9 mg/kg egg), which itself retards posthatch detour learning following E12 injection. Thus, excessive stimulation or blockade of 5-HT2 receptors around midembryogenesis can cause a similar behavioral teratogenic outcome. The data are discussed in relation to the likelihood that potential use of 5-HT2 antagonists for treating pregnant women and their fetuses who are not at risk is nil.

Behavioral and neuroendocrine assessment of ritanserin exposure in the developing chicken: lack of toxicity at effective doses.

The 5-HT2 antagonist ritanserin (RIT) is undergoing Phase III clinical trials for the treatment of substance abuse disorders. RIT has also shown preclinical therapeutic potential for attenuating or blocking lethal and/or toxic effects of exposure to cocaine or the selective 5-HT2 agonist dimethoxyiodophenyl-aminopropane (DOI) in the developing chicken. To assess the potential toxicity ("side effects") of RIT itself during development, we exposed chicken embryos to 0, 0.1, 0.3, 0.9, or 2.7 mg RIT/kg egg by injecting the drug into eggs with 14-day-old embryos (E14). Voltage generated by spontaneous embryonic activity (motility) was measured on E15 to assess short-term effects of RIT; none were observed. There was no overall effect of these RIT doses on hatchability, though sample sizes were small (n = 13-15 per group). One to 2 weeks after hatching, chicks' acquisition of a detour learning response was tested. There were no observable effects of any RIT dose on detour learning. To assess potential effects of RIT on responsiveness to stress, some chicks were exposed to isolation stress approximately 3 weeks after hatching and killed 15 min later. Blood was assayed for serum corticosterone. There was no effect of any embryonic RIT dose on corticosterone concentrations in nonstressed subjects. Although corticosterone was elevated in all stressed groups, the group exposed to the highest embryonic RIT dose (2.7 mg/kg egg) showed a stress-induced elevation greater than other groups. Thus, except for the highest RIT dose (six to seven times greater than a therapeutically effective dose used in earlier work), embryonic RIT exposure on E14 had no effect on embryonic behavior, hatchability, posthatch learned behavior, and basal serum corticosterone concentrations. At a supraefficacious dose it appears to have modified the responsiveness of the neuroendocrine axis to mild stress.

Evidence for a serotonin-mediated effect of cocaine causing vasoconstriction and herniated umbilici in chicken embryos.

Some of cocaine (COC)'s pathophysiological effects on exposed embryos likely result from its vasoconstrictive action, and serotonin2 (5-HT2) agonists such as dimethoxyiodophenylaminopropane (DOI) can mimic these effects. Infusions of COC (5 mg/kg/min) or DOI (0.5 mg/kg/min) for 15 min into chicken eggs with embryos on E15 caused a significant reduction in blood vessel diameters (14 and 30%, respectively). Pretreatment with the 5-HT2 antagonist ritanserin (RIT, 0.9 mg/kg) 18-22 h earlier blocked the effect of COC and blocked or attenuated the effect of DOI. In separate groups of chicken embryos exposed to multiple injections of low doses of COC on E18, herniated umbilici were prominent in hatchlings. A single bolus injection of the same absolute amount of COC did not cause herniated umbilici. An additional experiment replicated the induction of herniated umbilici by multiple injections of COC and demonstrated the probable involvement of 5-HT2 receptors because RIT blocked COC's ability to induce this anomaly. These data suggest that COC's vasoconstrictive effect, via 5-HT2 receptors, may play a mechanistic role in some adverse outcomes in embryos exposed to COC.

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.

Ritanserin blocks DOI-altered embryonic motility and posthatch learning in the developing chicken.

Developing chicken embryos exposed to cocaine show altered motility, hatchability, and posthatch detour learning. Pretreating such subjects with the serotonin2 (5-HT2) antagonist ritanserin (RIT) can block the motility suppression and reduced hatchability, indicating 5-HT2 receptor involvement in these cocaine effects. To study behavioral consequences of more selective 5-HT2 receptor stimulation and its blockade during development and to compare such exposure with that of cocaine, we injected eggs with 15-day-old chicken embryos with the 5-HT2 agonist dimethoxyiodophenylaminopropane (DOI, 1.0 mg/kg egg) and 1 h later, with RIT (0.3 and 0.9 mg/kg egg). Motility was recorded 2.5 or 24 h after DOI. This DOI dose suppressed motility 2.5 h but not 24 h after administration. Both RIT doses blocked DOI's motility suppression. No treatment affected hatchability. Subjects were tested on posthatch days 6-9 for detour learning acquisition. DOI "enhanced" learning (i.e., reduced latency), a cocaine-like effect observed in prior work, which was also blocked by both RIT doses. Thus, some consequences of DOI exposure late during embryonic development resemble cocaine's and are blocked by RIT, suggesting a therapeutic role for RIT-like drugs against cocaine's potential developmental toxicity.

Excessive stimulation of serotonin2 (5-HT2) receptors during late development of chicken embryos causes decreased embryonic motility, interferes with hatching, and induces herniated umbilici.

The existence and functional significance of 5-HT2 receptors in chicken embryos was studied by injecting the selective agonist dimethoxyiodophenylaminopropane (DOI), alone or in conjunction with the selective 5-HT2 antagonist ritanserin (RIT), into domestic chicken eggs with embryos of varying ages. DOI caused dose-dependent reductions in hatchability and herniated umbilici in hatchlings. These effects were observed after injection early, mid, or late during embryonic development, with evidence of the toxic effects of DOI being greater in older embryos, probably due to 5-HT2 receptor activation late in development, even after injecting DOI as early as on day 3 of embryogenesis. This is based upon the fact that embryos in eggs injected with DOI early continued to develop apparently normally, failing to hatch, often after pipping their shells. Additionally, those that hatched often did so with herniated umbilici, as did late-exposed embryos, indicating that DOI's effects upon this organ were most likely mediated during the prehatching period (i.e., days 18-20). The agonist's selectivity was confirmed by the capacity of RIT to dose dependently block both of these toxic effects of DOI. Reduced embryonic motility monitored on day 19, after injection of DOI on the evening of day 18, suggests that excessive activation of 5-HT2 receptors late during development of this species interferes with some normal embryonic behaviors and physiological changes necessary for inducing and/or maintaining the hatching process.

Potential efficacy and toxicity of GM1 ganglioside against trimethyltin-induced brain lesions in rats: comparison with protracted food restriction.

GM1 ganglioside (one week each at 10, 5, and 2.5 mg GM1/kg per day, ip) or gradual food restriction leading to a reduction in body weight to 75% of control were tested for their ability to block or reverse histopathologic and behavioral effects of trimethyltin (TMT) poisoning in rats. TMT (a single oral gavage of 6.0 mg TMT HCI/kg body weight) reduced hippocampal weight, decreased hippocampal cell counts, decreased autoshaped learning measures, and suppressed progressive fixed ratio (PFR) lever pressing without affecting stable lever pressing. Neither GM1 nor greater food restriction affected hippocampal weight. Greater food restriction prevented TMT's effects on autoshaping but not on PFR behavior, was without behavioral effects in animals not treated with TMT, and did not affect hippocampal histology. GM1 prevented certain TMT-induced decrements in autoshaping and PFR behavior but also suppressed autoshaping and stimulated stable fixed ratio behavior in animals not treated with TMT. GM1 also reduced hippocampal serotonin concentration, another "lesion-like" change. GM1 blocked TMT-induced hippocampal CA3b cell loss, but did not protect CA3c cells, the main locus of TMT hippocampal damage. The results support the idea that exogenous GM1 is a potent neuroactive agent with complex actions in intact organisms, potentially beneficial and potentially toxic. Like GM1, food restriction induces complex and potentially beneficial effects, but it lacked GM1's biochemical and behavioral "side effects" (i.e. toxicity) in these experiments.

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.

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.

Ganglioside treatment partially counteracts neurotoxic effects of trimethyltin but may itself cause neurotoxicity in rats: experimental results and a critical review.

We have demonstrated a deficit in working memory and/or consolidation of information in working memory into reference memory by a single oral dose of the neurotoxin trimethyltin(TMT). Moreover, TMT causes loss of hippocampal corticosterone receptors and increases brain glial fibrillary acidic protein(GFAP), an index of the astrocytic reaction to diverse types of CNS lesions. We tried to block the TMT-induced cognitive deficit and these biochemical markers by treating rats with purified mixed gangliosides (GS) for 21 days, starting 2 days before the TMT treatment. As expected, TMT decreased the number of corticosterone receptors in hippocampi and increased the GFAP concentration in hippocampi and to a lesser extent, in frontal cortices, measured more than 8 mon after treatment. The small increase in GFAP in frontal cortices was attenuated by GS but not in hippocampi. The pronounced learning deficits caused by TMT were attenuated to a small extent by GS in the TMT-GS group, when a learning criterion was used for the last session's performance of acquired lever-directed behavior. GS also delayed the appearance of significant performance differences between Controls and TMT-treated rats, when probed with a progressive fixed ratio schedule of reinforcement. However, most measures of learning and performance indicated that GS did not block the dysfunctional consequences of TMT treatment but instead caused similar functional decrements in rats treated with water instead of TMT. Corticosterone receptors in hippocampi were reduced to about 65% of Controls in the TMT-Water, TMT-GS, and Water-GS groups. A reduction in corticosterone receptors in hippocampi after TMT treatment probably reflects the loss of one or more cell types (e.g., pyramidal cells), which is supported by the increase in GFAP in this region. However, we did not observe a reciprocal relation between steroid receptors and GFAP after GS alone, indicating that GS did not cause detectable cell loss or cell damage, measured in this manner. Thus, reactive gliosis probably was not a pre-condition for the cognitive dysfunction. The fact that the cognitive deficits are probably related to hippocampal dysfunction supports the notion of a causal relationship between corticosterone receptor reduction and/or their altered function and cognitive impairment of this special type. The possibility that our results demonstrate potential neurobehavioral toxicity of GS is discussed in light of many reports which present data that can be similarly interpreted.

Acute opiate withdrawal in rats undernourished during infancy: impact of the undernutrition method.

Acute morphine withdrawal was assessed in adult rats following early postnatal undernutrition produced by two different methods (Large Litter procedure-20 pups/litter and Modified Slob procedure-rats cross-fostered on days 2, 4, and 6 to nonlactating dams for 24-hour periods). Response rates were first stabilized on a FR16 operant schedule. A single dose of morphine (20 mg/kg) was then administered, followed 4 h later by a single injection of naloxone (2.5 mg/kg). Males reared in large litters showed little behavioral disruption after morphine, suggesting either insensitivity to the opiate or the rapid development of tolerance. After naloxone. Modified Slob males displayed milder withdrawal than those in the well-nourished control or large litter groups. Thus the method of undernutrition influenced morphine's action and expression of withdrawal. A clear sex difference was also evident, females appearing to be generally less sensitive to the opiate- and naloxone-induced withdrawal than males. Body temperature underwent a characteristic elevation following morphine and a depression following naloxone across all groups, but undernutrition did not affect these responses. Hence, behavior proved to be the more sensitive measure for revealing differences in opiate dependence and withdrawal following early life undernutrition, under the test conditions employed.