Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum.

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.

Glyceryl triacetate for Canavan disease: a low-dose trial in infants and evaluation of a higher dose for toxicity in the tremor rat model.

Canavan disease (CD) is a fatal dysmyelinating genetic disorder associated with aspartoacylase deficiency, resulting in decreased brain acetate levels and reduced myelin lipid synthesis in the developing brain. Here we tested tolerability of a potent acetate precursor, glyceryl triacetate (GTA), at low doses in two infants diagnosed with CD, aged 8 and 13 months. Much higher doses of GTA were evaluated for toxicity in the tremor rat model of CD. GTA was given orally to the infants for up to 4.5 and 6 months, starting at 25 mg/kg twice daily, doubling the dose weekly until a maximum of 250 mg/kg reached. Wild-type and tremor rat pups were given GTA orally twice daily, initially at a dose of 4.2 g/kg from postnatal days 7 through 14, and at 5.8 g/kg from day 15 through 23, and thereafter in food (7.5%) and water (5%). At the end of the trial (approximately 90 to 120 days) sera and tissues from rats were analysed for changes in blood chemistry and histopathology. GTA treatment caused no detectable toxicity and the patients showed no deterioration in clinical status. In the high-dose animal studies, no significant differences in the mean blood chemistry values occurred between treated and untreated groups, and no lesions indicating toxicity were detectable in any of the tissues examined. Lack of GTA toxicity in two CD patients in low-dose trials, as well as in high-dose animal studies, suggests that higher, effective dose studies in human CD patients are warranted.

Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.

Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.

Adult vs. adolescent rats differ in biobehavioral responses to chronic nicotine administration.

More than 90% of smokers begin smoking during adolescence, suggesting that nicotine's actions may differ in adults vs. adolescents in ways that render adolescents vulnerable to smoking initiation. This experiment tested the hypothesis that nicotine's biobehavioral actions differ in adult and adolescent rats. Forty-two male (21 adolescents, 21 adults) and 41 female (21 adolescents, 20 adults) Sprague-Dawley rats were administered saline or 12 mg/kg/day nicotine via osmotic minipump for 21 days. Body weight, feeding, and locomotion (horizontal activity, vertical activity, center time) were measured before, during, and after saline or nicotine administration. Nicotine's effects depended on age and sex. Nicotine reduced body weight and feeding of adult males and females, and of adolescent males, but not of adolescent females. In addition, adolescent males were more sensitive than adults or adolescent females to nicotine's activity-enhancing effects. In cessation, nicotine-exposed adolescent males continued to exhibit greater activity than saline-exposed animals. Results indicate that nicotine's biobehavioral actions differ depending on age and sex.

Nicotine administration interacts with housing conditions to alter social and non-social behaviors in male and female Long-Evans rats.

Despite the fact that the anxiety-relieving effects of smoking are widely reported by smokers, human laboratory studies have not found consistent evidence for this effect. In animals, contrasting results also have been reported, with Sprague-Dawley rats unaffected by nicotine in behavioral tests of anxiety but with other rat strains (e.g., Wistar, Fischer-344) displaying behaviors indicative of anxiolysis. The present experiment used the social interaction test to evaluate effects of nicotine (12 mg/kg/day) chronically administered via minipump on social and non-social behaviors in 48 male and 48 female Long-Evans rats--a strain that has not previously been evaluated and that is known to differ from Sprague-Dawleys in other behavioral responses to nicotine. Because environmental conditions can alter behavioral effects of drugs, animals lived in either individual or same-sex group housing. Social interactions were measured on day 10 of drug administration and social behaviors (touch, follow, sniff-other, wrestle) as well as non-social behaviors (anxiety-related behaviors: freezing, grooming-self; exploratory behavior: moving) were scored. Group housing decreased social behaviors and increased anxiety-related behaviors. Nicotine's effects depended upon housing condition, such that nicotine further decreased social behaviors and increased anxiety-related behaviors of group-housed animals, with weaker effects in single-housed animals. Overall, the results suggest that nicotine's effects are modified by environmental conditions, and that nicotine administration at this dosage and in this particular rat strain results in anxiogenic effects.

The importance of acclimation in acoustic startle amplitude and pre-pulse inhibition testing of male and female rats.

The acoustic startle reflex (ASR) and pre-pulse inhibition (PPI) of the ASR are used extensively to index drug effects in rodents. Important methodological issues exist, however, with regard to the specific procedures and equipment used. In particular, the effects of acclimation to the startle procedure on response stability and the effects of testing animals in groups vs. individually have not been examined but are relevant to data interpretation. The present experiment measured acoustic startle responses with and without a pre-pulse of 25 adult Sprague-Dawley rats (12 male, 13 female) tested individually and in same-sex groups at four time points. Individual testing increased startle responses and PPI of males at time 1 and altered PPI of females at times 1, 2, and 3 compared with group testing. Responses were indistinguishable in the two testing environments at time 4. Results indicate that testing environment may affect responses when subjects have not been acclimated to the testing situation and that there are sex differences in these effects. Because responses stabilized by the fourth testing point, repeated testing of subjects particularly females, may be an important methodological inclusion when evaluating effects of drugs and other manipulations on ASR and PPI.

Prenatal nicotine exposure: effects on locomotor activity and central [125I]alpha-BT binding in rats.

Maternal smoking during pregnancy or in utero exposure of the fetus to nicotine may result in learning difficulties and hyperactivity in the child. To elucidate possible involvement of the alpha(7) nicotinic receptor subtype in these behavioral impairments, pregnant dams were treated with nicotine (9 mg/kg/day) via osmotic minipumps throughout gestation. Male offspring were weaned at postnatal day 18, and were tested for locomotor activity at postnatal days 20-24. Pups were sacrificed on postnatal day 36-38 and 18 discrete brain areas were analyzed for [125I]alpha-bungarotoxin (alpha-BT) binding by quantitative autoradiography. Prenatal nicotine caused an elevation in locomotor activity (vertical movements) in offspring. [125I]alpha-BT binding was significantly reduced in the hippocampal CA1 region (29%), dentate gyrus (22%), and medial geniculate nucleus (29%). These findings suggest that some of the behavioral abnormalities induced by prenatal nicotine exposure may be due to a reduction of alpha(7) nicotinic receptors in discrete brain regions.

Effects of nicotine and stress on startle amplitude and sensory gating depend on rat strain and sex.

We recently reported that 14 days of nicotine administration (12 mg/kg/day) reduced acoustic startle reflex amplitude and impaired prepulse inhibition (PPI) of startle in male and female Long-Evans rats. These findings contrasted with reports of nicotine-induced enhancement of startle and PPI in Sprague-Dawley (a different strain) male rats. The present experiment administered 0, 6, or 12 mg/kg/day nicotine via osmotic minipump for 14 days to 120 Sprague-Dawley rats (male and female) and to 120 Long-Evans rats (male and female) and examined ASR and PPI. Half of the subjects also were stressed by immobilization once each day to examine nicotine-stress interactions. Nicotine enhanced ASR and PPI responses of Sprague-Dawley rats but impaired these responses in Long-Evans rats, regardless of sex. Effects of stress were complex and depended on strain, sex, and drug dose. These findings indicate that effects of nicotine on measures of reactivity (ASR) and sensory gating (PPI) depend on genotype and that nicotine stress interactions depend on genotype, sex, and nicotine dosage.

Effects of chronic nicotine administration on locomotion depend on rat sex and housing condition.

Nicotine's behavioral actions in the human smoker by self-report depend, in part, on the individual's gender and environment. The purpose of the present experiment was to determine whether effects of nicotine on unconditioned behaviors of rats also depend on sex and environmental conditions. Long-Evans rats (96 males and 96 females) living in individual or grouped housing were administered saline or 12 mg/kg/day nicotine via osmotic minipump for 14 days. Horizontal activity (a measure of overall activity and arousal), vertical activity (a measure of exploratory behavior), and center time (a possible index of anxiety) were measured on Day 10 of drug administration and on Day 2 of nicotine cessation. Group housing decreased horizontal and vertical activity and center time, with effects occurring sooner in females. Nicotine's effects depended on housing and sex. For males, nicotine altered indices of arousal and exploration, increasing these variables for group-housed males but decreasing them for individually housed males. For females, nicotine altered possible indices of anxiety, reducing anxiety for group-housed females. In cessation, housing effects continued in females and appeared more robustly in males. Results indicate that nicotine's chronic effects depend on subjects' sex and living environment.

Sex differences in effects of opioid blockade on stress-induced freezing behavior.

The present experiment examined the effects of naloxone on freezing behavior in male and female rats following stress and no-stress conditions. Twelve male and 12 female Wistar rats were exposed to 10 min of mild, unpredictable footshock stress and to a comparable no-stress condition. Immediately following stress or no-stress conditions, subjects were injected with naloxone or saline, and two independent observers measured freezing behavior. In male rats, naloxone potentiated freezing following stress but had no effect on freezing following no-stress. In females, naloxone did not affect freezing regardless of stress conditions. These results reveal a sex difference in effects of naloxone on freezing behavior and suggest that sex differences may exist with respect to the role of endogenous opioids under stress.

Nicotine administration impairs sensory gating in Long-Evans rats.

In rats, effects of nicotine administration on sensory gating as indexed by prepulse inhibition (PPI) of the acoustic startle reflex (ASR) are unclear. We have found that nicotine administration enhances ASR and PPI in Sprague-Dawley rats, but other investigators, using Long-Evans rats, have reported no effects or enhancement of PPI only. Numerous methodological differences exist among studies in addition to subject strain, however, making it unclear whether inconsistent behavioral responses are the result of different experimental procedures or indicate a true strain difference. To investigate the role of strain in nicotine's effects on ASR and PPI, 192 male and female Long-Evans rats were administered 12 mg/kg/day nicotine via osmotic minipump for 14 days using identical methodologies employed in studies with Sprague-Dawley subjects. Effects of grouped vs. individual housing on these responses also were examined. Nicotine administration impaired ASR and PPI in Long-Evans subjects. These effects occurred in female rats regardless of housing condition, and interacted with housing in male rats. Results indicate that sex and housing are important variables in nicotine's effects. Results suggest that subject strain may be an important variable in nicotine's effects on sensory gating, and that responses of Sprague-Dawley vs. Long-Evans rats may represent a true strain difference.

Effects of stress on circulating nicotine and cotinine levels and in vitro nicotine metabolism in the rat.

The effects of physical and psychological stress on circulating nicotine levels and conversion of nicotine to cotinine were examined in the rat. Animals received one of three dosages of nicotine (0, 6, and 12 mg/kg) via miniosmotic pumps. On day 14 of drug infusion, animals from each drug condition were randomly assigned to one of three stress conditions (noise, rubber ligature, or no stress). After 2.5 h of stress exposure, animals were killed and plasma nicotine and cotinine were measured in vivo and hepatic conversion of nicotine to cotinine was determined in vitro. Stress lowered blood nicotine levels. However, this difference was statistically significant only among animals receiving 12 mg/kg per day nicotine. In contrast, stress had no consistent effect on either measure of conversion of nicotine to cotinine in rats. Taken together, these results suggest that stress lowers circulating nicotine levels. However, the mechanism by which this occurs remains unclear.

Hyperactivity induced by prenatal nicotine exposure is associated with an increase in cortical nicotinic receptors.

Prenatal exposure to nicotine may lead to hyperactivity. To evaluate possible involvement of central nicotinic receptors in this condition, pregnant Sprague-Dawley rats were implanted with osmotic minipumps to receive nicotine (6 mg/kg/day) or saline throughout gestation. A total of 222 pups (118 males and 104 females) from 24 dams were measured for locomotor activity. Male and female hyperactive and nonhyperactive offspring from each treatment group were selected and analyzed for nicotinic receptor concentrations in various brain regions. Hyperactive male offspring that were prenatally exposed to nicotine exhibited a significant increase in the cortical receptor densities without a change in binding affinity. Hyperactive offspring of saline-treated dams did not show an increase in cortical nicotinic receptors. These results suggest that hyperactive male offspring of nicotine-exposed dams are also susceptible to neurochemical effects of intrauterine nicotine exposure.

Sex differences in effects of predictable and unpredictable footshock on fentanyl self-administration in rats.

An operant conditioning paradigm was used to examine effects of predictable and unpredictable footshock on oral fentanyl (50 micrograms/ml self-administration (SA) in 12 female and 12 male Wistar rats (Rattus norvegicus). Rats were tested for drug SA under a progressive ratio schedule with and without repeated predictable or unpredictable footshock over 8 weeks. Female rats consumed greater amounts of fentanyl than did male rats. Male rats exhibited greater withdrawal behaviors following naloxone challenge. Predictable footshock with repeated exposure (i.e., chronic stress) was accompanied by greater fentanyl SA than was unpredictable footshock, particularly for female rats. Corticosterone levels were positively correlated with fentanyl SA. Predictability of the stressor also had a greater effect on maintenance of fentanyl SA than it did on relapse to fentanyl SA. Results suggest that sex plays an important role in drug-taking behavior by rats.

Disulfiram causes sustained behavioral and biochemical effects in rats.

The present experiment examined effects of disulfiram (Antabuse) administration on behavioral measures of nociception (hot plate and tail flick), peripheral muscular performance (grip strength), motivated performance, balance, and coordination (rotorod) in 24 male Sprague-Dawley rats during and 2 wk after an eight-day administration of disulfiram. In addition, peptidylglycine 5(-hydroxylating monooxygenase (PHM) activity in several tissues and levels of alpha-amidated alpha-melanocyte stimulating hormone (alpha-MSH) in the neurointermediate lobe of the pituitary were assayed to evaluate biochemical effects of disulfiram. These particular assays were included because it has been reported that disulfiram affects alpha-amidated peptides via alteration of PHM activity. Decrements in all behavioral measures, except tail flick, occurred after one week of disulfiram administration. Decrements in grip strength continued for the 2 wk after cessation of disulfiram. Dose-related reductions in changes in PHM activity and levels of alpha-MSH were found 2 wk after cessation of disulfiram administration. The time course of the results suggest that changes in PHM activity may underlie decrements in grip strength. The present experiment provides a paradigm for further investigations of effects of alpha-amidated peptides on behavior.

Effects of ddC and AZT on locomotion and acoustic startle. I: Acute effects in female rats.

Several synthetic nucleoside analogues, including AZT(RETROVIR), ddC (HIVID), ddI (VIDEX), and d4T (ZERIT), are currently being used in the treatment of HIV infection. Unfortunately, in clinical use the appearance of severe and sometimes debilitating peripheral neuropathy and pain has been associated with the long-term use of several of these drugs (i.e., ddC, ddI and d4T), although not with AZT. To date, standard pre-clinical animal toxicity studies have failed to reveal any adverse neurologic effects of these compounds. However, previously reported preliminary findings suggest that ddC may alter several neuro-behavioral parameters (including locomotor activity, acoustic startle responding, and aggression) in rats and mice following presentation in the animals' drinking water for 7 days. The current series of experiments examined effects of acutely administered ddC and AZT on spontaneous locomotor activity and acoustic startle responses (with and without pre-pulse) in female Sprague-Dawley rats. Following intragastric administration, ddC reduced locomotion at all but the highest dose, whereas AZT had no significant effect on locomotor activity. Acutely administered ddC had no effect on ASR, whereas AZT increased ASR at the highest stimulus intensity. These data support the use of behavioral testing in the development of the antiviral nucleoside analogues, as behavioral testing may be more effective in identifying the neurologically active agents than is standard toxicity testing.

Prenatal exposure to nicotine: effects on prepulse inhibition and central nicotinic receptors.

The present experiment examined effects of prenatal nicotine exposure (6 mg/kg/day via osmotic minipump) throughout gestation on prepulse inhibition of the acoustic startle response (PPI) and on the density of nicotinic acetylcholine receptors (nAchRs) in the brains of 5-week-old Sprague-Dawley rats. A total of 117 male and 103 female offspring were used. Prenatal nicotine reduced subsequent percent PPI to a 98 dB stimulus in female but not in male offspring. There was an inverse correlation between the percent of PPI and nAchR density in the cortex of male rats and the striatum of female rats.

Effects of environmental conditions on food consumption in female and male rats.

The present experiment examined food and water consumption under different housing conditions in 20 female and 20 male Wistar rats. Food and water consumption were measured for 6 h a day following an 18-h same-sex crowded or individual housing period for each of 6 days. All subjects were individually housed during the 6-h measurement period and had access to food and water. Female rats consumed more food and water than did male rats during the 6-h period, regardless of their 18-h housing condition. In addition, previously crowded rats consumed more food and water during the 6-h period than did rats that were previously individually housed. During the 18-h period, when subjects were differentially housed, males consumed more food and water than did females; crowded rats ate less than did individually housed rats; and crowded rats drank more water than did individually housed rats. Based on plasma corticosterone data, the female and male rats were differentially affected by housing conditions. The present results are discussed with regard to housing conditions per se and sex differences in stress responses to housing.

Effects of housing on male and female rats: crowding stresses male but calm females.

Housing conditions affect behavioral and biological responses of animals. Effects of same-sex grouped, crowded, or individually housed conditions on plasma corticosterone levels of male and female Wistar rats were examined in two experiments. Experiment 1 examined the effects of individual vs. crowded housing conditions on corticosterone, a biochemical index of stress, in seven male and seven female rats. Experiment 2 extended the findings of Experiment 1 by separately manipulating spatial and population aspects of housing with 50 male and 50 female rats. Male rats had higher corticosterone levels under crowded conditions. In contrast, female rats had higher levels when individually housed. Spatial crowding was the key variable for males, whereas the number of other animals was more important for females. These results indicate that investigators must consider housing conditions as an intervening variable that is likely to differentially affect behaviors of male and female rats.