R.Robert Holson

Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species

Developmental studies often assess the effect of treatment of the pregnant mother on offspring. The use of multiparous species such as rats and mice in such studies creates a special set of design and analysis problems. These arise for two reasons. First, the availability of many offspring per litter tempts the experimenter to inflate sample size by treating scores from several pups per litter as independent observations. Second, large litter size seldom makes it practical to measure exposure effects in all offspring of an exposed dam. Such studies commonly involve two-stage sampling: Drawing a random sample of dams for treatment, then drawing a second sample of pups per dam for neurobehavioral measurements. In this article, such sampling was modeled by two different simulations. The first, a standard Monte-Carlo approach, sampled from random-normal distributions for litter mean and within-litter variability. The second simulation sampled without replacement from actual data on weight of all pups in a series of 39 nontreated rat litters. These mutually-supportive approaches demonstrate that litter effects, even over as few as three litters, are generally large and statistically meaningful. Consequently, statistical significance tests are sensitive to litter effects. Inflation of sample size by treating as few as 2 pups per litter as independent measurements can almost triple the nominal 0.05 alpha level. Furthermore, two-stage sampling increases the within-treatment error term and correspondingly reduces statistical power relative to one-stage sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Low environmental temperatures or pharmacologic agents that produce hypothermia decrease methamphetamine neurotoxicity in mice

Recently we have reported that methamphetamine (METH) neurotoxicity in rats depends on the environmental temperature. Here, we evaluate whether a cold environment (4 degrees C) or drugs which chloride and glutamate ion channel function block METH neurotoxicity in mice. Adult male CD mice received METH i.p. (4 x 10 mg/kg METH at 23 degrees C along with saline. 2.5 mg/kg (+)-MK-801, 40 mg/kg phenobarbital or 2.5 mg/kg diazepam and either 4 x 10 or 4 x 20 mg/kg METH at 4 degrees C). Multiple injections of METH (4 x 10 mg/kg i.p.) at room temperature (23 degrees C) produced a significant depletion of dopamine (DA) in striatum at 24, 72 h, 1 and 2 weeks. Three days post 4 x 10 mg/kg METH at 23 degrees C, an 80% decrease in striatal dopamine (DA) occurred while the same dose at 4 degrees C produced only a 20% DA decrease, and 4 x 20 mg/kg METH at 4 degrees C produced a 54% DA decrease. At 23 degrees C (+)MK-801 completely blocked while phenobarbital (40% decrease) and diazepam (65% decrease) partially blocked decreases in striatal DA produced by 4 x 10 mg/kg METH. Decreases in DOPAC and HVA were similar to the decreases in DA after METH and antagonists. Multiple injections of METH (4 x 10 mg/kg, i.p.) at room temperature also produced a significant depletion of serotonin (5-HT) in striatum at 24, 72 h, 1 and 2 weeks. This depletion of 5-HT at room temperature was blocked either by changing the environmental temperature to 4 degrees C, or by pretreatment with MK-801, diazepam and phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamines in rat caudate

Extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) were assayed in the caudate of freely moving rats using microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-EC) to detect changes in their release. Dialysates were assayed at 20-minute intervals for four hours after an intraperitoneal (IP) injection of MDMA (10 mg/kg). In a separate study to determine MDMA effects on total caudate levels of the above neurochemicals, animals were injected IP with MDMA (10 mg/kg) and then sacrificed at 20, 60, 120 and 180 minutes after treatment. Brains were quickly removed, and caudate nuclei were dissected for neurochemical analysis using HPLC-EC. MDMA elicited an amphetamine-like increase in DA release, followed by an increase in DA content. DOPAC and HVA were both reduced in homogenate. 5-HT release was also increased, followed by a drop in caudate homogenate levels by three hours. DA extracellular content was 686% of control at 80 minutes; caudate homogenate levels were 122% at 120 minutes. 5-HT extracellular release was 123% at 20 minutes, then decreased thereafter. It is concluded that the acute effect of MDMA on caudate is at least as great on the DA as it is on the 5-HT system.

Effects of a cold environment or age on methamphetamine-induced dopamine release in the caudate putamen of female rats

Extracellular levels of dopamine (DA) and metabolites as well as serotonin [5-hydroxytryptamine (5-HT)] and 5-hydroxyindoleacetic acid (5-HIAA) were determined in the caudate putamen (CPU) of either 6- or 12-month-old female rats using microdialysis and high-performance liquid chromatography with electrochemical detection (HPLC-ED) before, during, and after four consecutive injections (given at 2-h intervals) of methamphetamine (METH). In 6-month-old rats administered 4 x 5 mg/kg METH at an environmental temperature (ET) of 23 degrees C, peak extracellular DA levels (between 50 and 150 rho g/10 microliters) were attained 30-45 min after each dose of METH while dihydroxyphenylacetic acid (DOPAC) decreased steadily after the first doses of METH until it reached a plateau at 50% of control (550-700 pg/10 microliters) levels. Increases in 5-HT levels during METH administrations paralleled DA increases while 5-HIAA decreases paralleled DOPAC decreases. The total CPU DA and 5-HT content of these rats was about 65% of control at 3 days post-METH. Reducing the ET to 4 degrees C during dosing decreased the peak and average DA levels attained during the 4 x 5 mg/kg METH administration to about 50% of that observed at a 23 degrees C ET. Increasing the dose to 4 x 10 mg/kg METH (4 degrees C ET) increased peak and average CPU DA levels to 200% that observed during 4 x 5 mg/kg METH at a 23 degrees C ET. However, no significant decreases in total CPU DA content of any rats dosed with METH at a 4 degrees C ET were observed 3 days post-METH. In 12-month-old rats dosed with 4 x 5 mg/kg METH (23 degrees C ET), the peak and average extracellular DA levels were only 30-60% that of 6-month-old rats. However, the CPU DA content of older rats was significantly decreased both 3 (30% control) and 14 (60% control) days post-METH. In summary, METH toxicity may not be predicted solely by the extracellular levels of DA attained during METH administration; age and ET also greatly influence METH neurotoxicity.

Prenatal dexamethasone or stress but not ACTH or corticosterone alter sexual behavior in male rats

Prenatal maternal stress in rats and mice can demasculinize and feminize the sexual behavior of adult male offspring. Causal mechanisms are unknown, but one attractive hypothesis is that stress activation of maternal adrenal glucocorticoid secretion is the responsible agent. To test this hypothesis, pregnant rats were exposed to a variety of substances which enhance glucocorticoid actions. These included ACTH (20 IU of a gel preparation, SC once daily), corticosterone (CORT; 7 mg/kg SC in oil, three times daily), or dexamethasone (DEX; 0.1 mg/kg, SC once daily). Controls included noninjected dams and a positive stress control group (restraint under bright lights three times daily). All treatments reduced maternal weight gain, DEX most potently. No treatment altered litter size, stillbirths, or sex ratio, but DEX reduced weight at birth, an effect still seen at postnatal day 85. DEX, CORT, and stress reduced male adrenal weight at birth, while DEX and CORT altered sexual differentiation as measured by anogenital distance. Stress impaired adult male sexual performance but not the lordosis quotient following exposure of animals to stud males. DEX affected both measures. No other treatment had any significant effect on sexual behavior. No treatment altered plasma LH levels, either basal or in response to an estrogen challenge in adult gonadectomized males. In adulthood there was no treatment effect on stress reactivity, measured behaviorally or by plasma glucocorticoids. Correlational analysis revealed that weight gain during pregnancy was the single best predictor of subsequent sexual performance. It is concluded that prenatal dexamethasone exposure demasculinizes and feminizes male offspring.(ABSTRACT TRUNCATED AT 250 WORDS)

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 eitherd-amphetamine sulfate (5 mg/kg) or saline, SC, twice daily for 5 consecutive days. Stereotyped behavior and locomotor activity responses to a challenge dose ofd-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 gropus pretreated earlier, when compared to the saline-pretreated rats. These results sugges that amphetamine sensitization may be a late-developing effect, one which occurs sometime after the 3rd week of postnatal life.

Nitric oxide regulation of methamphetamine-induced dopamine release in caudate/putamen

A possible role for NO modulation of dopamine (DA) release in the caudate/putamen (CPU) during methamphetamine (METH) exposure was investigated using in vivo microdialysis in rats. Inclusion of the nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine (NOARG), NG-nitro-L-arginine methyl ester (L-NAME) or D-NAME (less potent inhibitor) in the microdialysis buffer prior to METH minimally affected basal levels of DA, DOPAC or HVA in CPU microdialysate. However, L-NAME and NOARG produced concentration-dependent decreases of up to 64% (100 microM) in CPU DA levels in microdialysate during exposure to four doses of METH (5 mg/kg i.p./2 h), with lesser effects on DOPAC or HVA. Reversal of the NOARG inhibition was produced by inclusion of 500 microM of either L-arginine or L-citrulline in the microdialysate. D-NAME (100 microM) minimally affected levels of DA or metabolites. Paradoxically, inclusion of from 20 to 2 microM of the NOx generators isosorbide dinitrate (ISON) or sodium nitroprusside (SNP) in the microdialysis buffer decreased DA and DOPAC levels in microdialysate during METH exposure. This paradox might result from the concentrations of NOx produced by SNP or ISON being great and not regionally specific resulting in inhibition of DA release and/or synthesis while the NO generated endogenously during METH exposure may have localized and site-specific actions. Alternatively, NOx may inhibit NOS or other enzymes in the NO synthesis pathway, thereby reducing levels of an intermediate (other than NO) which potentiates DA release. In their entirety, our results indicate that NO generation in the CPU may augment the release of DA during METH exposure.

Neonatal dexamethasone on day 7 causes mild hyperactivity and cerebellar stunting.

To investigate the effects of glucocorticoid treatment on central nervous system development, rats were injected with dexamethasone (DEX) (1-3 mg/kg) on postnatal day (PND) 3 or 7. DNA and protein content and concentration were measured in the cerebellum and hippocampus on PND 28 and 112. Whole and regional brain weights were measured at PND 28, 84, and 112. Nest odor preference (PND 10-11), open field activity (PND 18-21), running wheel activity (PND 50-56), and complex maze performance (PND 60-63) were measured in rats treated twice with 1.5 mg/kg DEX on PND 7. DEX treatment on PND 7 resulted in reductions in PND 28 whole brain and regional weights (frontal cortex, cerebellum, and brain stem) and, by PND 112, all except whole brain and cerebellar weights had recovered. A mild syndrome of hyperactivity (increased open field rearing and activity) was apparent in rats treated with DEX on PND 7. These results are discussed in terms of the developmental stage specificity in production of brain, and, specifically, cerebellar insults and their resulting effects.

Neonatal dexamethasone on day 7 in rats causes behavioral alterations reflective of hippocampal, but not cerebellar, deficits.

Developmental glucocorticoid treatment in rats has been shown to cause body and brain weight decrements concurrent with behavioral alterations. Here, Sprague-Dawley rats were treated with the synthetic glucocorticoid, dexamethasone (DEX), on postnatal day (PND) 7 (1.5 mg/kg, sc, injected in the morning and afternoon). Behavioral assessments of negative geotaxis, locomotor activity (open field, maze exploration, residential running wheel, residential figure 8 maze), open-field activity response to amphetamine, acoustic startle, prepulse inhibition (PPI) of acoustic startle, juvenile play behavior, anxiety (emergence tests), motor coordination (rotarod performance), spatial learning (Morris water maze and food-reinforced complex maze), and operant performance (time estimation and response inhibition) were assessed in male rats. Body weight was decreased beginning at PND 43 until sacrifice on PND 127. Whole and regional brain weights were less, especially hippocampus, cerebellum, brainstem, and cortical remnant. Indications of delayed development were apparent; specifically, DEX-treated rats took significantly longer to turn on PND 8, but not PND 9, in the negative geotaxis test. DEX treatment induced deficits in the Morris water maze that were similar to hippocampal deficits. Open-field activity changes were inconsistent; however, DEX-treated rats were hyperactive during the dark period in running wheel tests. There were no indications of changes in reactivity or emotionality.

Euthanasia by decapitation: Evidence that this technique produces prompt, painless unconsciousness in laboratory rodents

Rapid euthanasia of laboratory rodents without the use of anesthesia is a necessary research technique whenever there is the likelihood of anesthesia or stress interfering with the chemistry of the tissues under investigation. Decapitation has long been the procedure of choice under such circumstances. Recently, however, the American Veterinary Medical Association (AVMA) panel on euthanasia recommended that decapitation be avoided on the grounds that the decapitated head may be conscious and suffering for as much as 15 seconds. The panel further recommended that if decapitation was scientifically necessary, the decapitated head be immediately immersed in liquid nitrogen. These AVMA guidelines now enjoy regulatory status; the recommendation that decapitation be avoided has thus caused considerable difficulty for all research requiring rapid, anesthesia-free collection of tissues. The scientific validity of these recommendations is consequently a matter of great practical as well as theoretical importance. The decision to discourage decapitation appears to have been based on a single literature report claiming that the EEG of the decapitated head revealed conscious suffering for more than 10 seconds (Mikeska and Klemm 1976). This review carefully examines the scientific literature on this subject. It is concluded that the report by Mikeska and Klemm of EEG activation in the decapitated head is correct, but that this phenomenon is also seen when the decapitated head is under deep anesthesia, and in normal brains under ether anesthesia or during REM sleep. Hence these findings do not demonstrate either consciousness or the perception of pain.(ABSTRACT TRUNCATED AT 250 WORDS)