Regional Brain Weights Research Articles

Insulin-like growth factor-1 supplementation promotes brain maturation in preterm pigs.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurological outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and regional and cellular brain development. Pigs were treated with 2.25 mg/kg/day of recombinant human IGF-1/IGF binding protein 3 complex from birth until Day 5 or 9 before collection of brain samples for quantitative immunohistochemistry (IHC), RNA-seq and qPCR analyses. Brain protein synthesis was measured using in vivo labeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region- and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at Day 5 and 14% at Day 9 after IGF-1 treatment. Treatment had no effect on Iba1-positive microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.SIGNIFICANCE STATEMENTDeficiency of systemic insulin-like growth factor-1 (IGF-1) is associated with delayed neurological development in preterm infants. Here, we show that the IGF-1 receptor is primarily expressed in immature neurons in the developing brain of the translational preterm pig model. Supplementation with IGF-1 accelerates neuron differentiation in the hippocampus and promotes myelination in subcortical white matter regions in a time-dependent way. Furthermore, systemic IGF-1 supplementation stimulates cerebral protein synthesis. Our study suggests that IGF-1 therapy in the early postnatal period might be supportive for neurodevelopment in preterm infants.

Sex-Specific Survival, Growth, Immunity and Organ Development in Preterm Pigs as Models for Immature Newborns.

Background: After very preterm birth, male infants show higher mortality than females, with higher incidence of lung immaturity, neurological deficits, infections, and growth failure. In modern pig production, piglets dying in the perinatal period (up to 20%) often show signs of immature organs, but sex-specific effects are not clear. Using preterm pigs as model for immature infants and piglets, we hypothesized that neonatal survival and initial growth and immune development depend on sex.Methods: Using data from a series of previous intervention trials with similar delivery and rearing procedures, we established three cohorts of preterm pigs (90% gestation), reared for 5, 9, or 19 days before sample collection (total n = 1,938 piglets from 109 litters). Partly overlapping endpoints among experiments allowed for multiple comparisons between males and females for data on mortality, body and organ growth, gut, immunity, and brain function.Results: Within the first 2 days, males showed higher mortality than females (18 vs. 8%, P < 0.001), but less severe immune response to gram-positive infection. No effect of sex was observed for thermoregulation or plasma cortisol. Later, infection resistance did not differ between sexes, but growth rate was reduced for body (up to −40%) and kidneys (−6%) in males, with higher leucocyte counts (+15%) and lower CD4 T cell fraction (−5%) on day 9 and lower monocyte counts (−18%, day 19, all P < 0.05). Gut structure, function and necrotizing enterocolitis (NEC) incidence were similar between groups, but intestinal weight (−3%) and brush-border enzyme activities were reduced at day 5 (lactase, DPP IV, −8%) in males. Remaining values for blood biochemistry, hematology, bone density, regional brain weights, and visual memory (tested in a T maze) were similar.Conclusion: Following preterm birth, male pigs show higher mortality and slower growth than females, despite limited differences in organ growth, gut, immune, and brain functions. Neonatal intensive care procedures may be particularly important for compromised newborns of the male sex. Preterm pigs can serve as good models to study the interactions of sex- and maturation-specific survival and physiological adaptation in mammals.

Developmental Treatment with Bisphenol A or Ethinyl Estradiol Causes Few Alterations on Early Preweaning Measures

Because bisphenol A (BPA) exposure is nearly ubiquitous, increased knowledge of its potential effects on development will enable better risk assessment and regulatory guidance. Here, Sprague-Dawley rats were reared in low exogenous estrogen environments. After breeding at adulthood, dams were gavaged on gestational days (GDs) 6-21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day BPA, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE₂). Offspring were orally treated on postnatal days (PNDs) 1-21 with the same dose the dam received. Relative to the VEH group, dams of both EE₂-treated groups weighed less throughout gestation and lactation. PND 1 absolute anogenital distance and anogenital index were unaltered by any treatment. Ages at fur development and eye and ear opening were unaffected by any treatment. Despite a significant treatment effect, no group was significantly different from VEH in PNDs 3-6 righting latencies; although males had shorter latencies and all latencies decreased with age. PNDs 8-11 slant board behavior was unaffected by any treatment; however, males had shorter turning latencies and latencies decreased with age. Preweaning body weights of BPA- and EE₂-treated groups as well as naive controls were less than VEH. No treatment affected PND 21 whole or regional brain weights or levels of estradiol, testosterone, corticosterone, T3, T4, luteinizing hormone, ghrelin, or leptin. These results add to the literature indicating that developmental BPA treatment at these doses has no effects on gestational or lactational body weight, offspring anogenital distance, preweaning behaviors or hormone levels, and whole and regional brain weights measured at weaning.

Altered adult locomotor activity in rats from phencyclidine treatment on postnatal days 7, 9 and 11, but not repeated ketamine treatment on postnatal day 7

Neonatal ketamine (KET) or phencyclidine (PCP) treatment can trigger apoptotic neurodegeneration in rodents. Previously, we described KET- and PCP-induced altered body weight and home cage, slant board and forelimb hang behaviors in preweaning rats (Boctor et al., 2008). In that study, l-carnitine (LC) attenuated the KET-induced behavioral alterations and body weight decrements. The four subcutaneous (sc) treatment groups were: (1) saline; (2) 10 mg/kg PCP on PNDs 7, 9 and 11; (3) 20 mg/kg KET (6 injections; one every 2 h on PND 7); or (4) a regimen of KET and 250 mg/kg LC (KLC) both administered on PND 7, with additional 250 mg/kg doses of LC on PNDs 8–11. A portion of each treatment group was evaluated for postweaning behaviors which included grip strength and motor coordination (postnatal days (PNDs) 22 or 71), locomotor sensitization (PND 42), spatial alternation (PNDs 22–70) and residential running wheel activity (PNDs 72–77). On PND 42 or 78, whole and regional brain weights were measured. Grip strength and motor coordination were unaffected at either age by neonatal treatment. On PND 42, neonatally treated KET- or KLC-treated rats responded to a challenge of 5 mg/kg KET with activity similar to controls that received the same challenge. Neonatal PCP treatment, however, induced significant sensitization to a challenge of 3 mg/kg PCP on PND 42 relative to controls that received the same challenge, causing increased activity which was especially profound in females. Performance on a continuous spatial alternation task requiring a “win-shift, lose-stay” strategy appeared unaffected by neonatal KET or KLC treatment. PCP treatment, however, caused significantly increased random responding and shorter choice latencies. In addition, neonatal PCP treatment elevated light and dark period running wheel activity and reduced PND 42 and 78 body and whole brain weights. These findings provide further evidence that PCP treatment on PNDs 7, 9, and 11 causes subtle cognitive deficits and long-term alterations in activity that are unrelated to deficits in grip strength or motor coordination. Further, repeated KET treatment on PND 7 does not appear to result in severe behavioral modifications.

Chemotherapeutic choice of ranimustine or nimustine on the basis of regional polyamine levels in rat brain

The aim of the present study was to improve the chemotherapeutic efficacy of anticancer drugs by choosing on the basis of the polyamine level induced by the drug in each host cancer-bearing tissue. We propose an "organ-specific therapy" in the article. The polyamines, putrescine, spermidine and spermine are strongly associated with tumor cell growth. The effects of ranimustine (MCNU) and nimustine (ACNU) on body weight, regional brain weights and concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, combined thalamus and hypothalamus and diencephalon of the brain were examined in rats. MCNU and ACNU reduced spermidine and spermine in the corpus striatum, and spermine in the diencephalon, but increased putrescine in the corpus striatum and combined thalamus and hypothalamus. These results indicate that both MCNU and ACNU are suitable for the treatment of cancers of the corpus striatum, but ACNU is not suitable for cancers of the corpus striatum, thalamus and hypothalamus.

A rat neurodevelopmental evaluation of offspring, including evaluation of adult and neonatal thyroid, from mothers treated with ammonium perchlorate in drinking water.

The purpose of this study was to evaluate the potential neurodevelopmental toxicity of perchlorate exposure during gestation and the first 10 days of lactation. Mated Sprague-Dawley rats (25/exposure group) were given continual access to 0, 0.1, 1.0, 3.0, or 10.0 mg/kg-day ammonium perchlorate (AP) in drinking water, starting gestation day 0 (mating) through lactation day 10 (DL 10). One pup/sex/litter/exposure group was assigned to (1) juvenile brain weights, morphometry, and neuropathology; (2) passive avoidance and watermaze testing; (3) motor activity and auditory startle habituation; and (4) adult regional brain weights, morphometry, and neuropathology. AP had no effect on body weights, feed consumption, clinical observations, or sexual maturation of pups at exposures as high as 10.0 mg/kg-day. There were no behavioral effects in the offspring exposed as high as 10.0 mg/kg-day as evaluated by passive avoidance, swimming watermaze, motor activity, and auditory startle. Increases in hypertrophy and hyperplasia of the thyroid follicular epithelium and a decrease in the thyroid follicle size were observed in culled male pups in the 10.0 mg/kg-day group on DL 5. The exposure level for effects on triiodothyroxine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels for pups were 0.1, 1.0, and 3.0 mg/kg-day, respectively. There was an apparent increase in the thickness of the corpus callosum of the 10 mg/kg-day group pups on DL 12. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was greater than 10.0 mg/kg-day. Based on the thyroid morphometric and histopathologic findings, the NOAEL for pup toxicity was 0.1 mg/kg-day.

Gestational retinoic acid exposure in the rat: effects of sex, strain and exposure period

Effects of gestational exposure to all- trans retinoic acid (RA) were assessed in the Long–Evans (hooded) and Sprague–Dawley (albino) rat strains. Two exposure periods were evaluated against vehicle controls. Both involved three consecutive daily per os doses of either 2.5 mg/kg RA on gestational days (GD) 11 through 13 or 10 mg/kg RA on GD 14 through 16. All assessments were conducted on at least one male and one female per litter. Substantial main effects of sex, strain and treatment were obtained, but with few significant interactions. Main effects of strain were found on surface righting, neonatal mortality, litter weight and postnatal day (PND) 35 regional brain weight. Among strain effects, the most interesting was the finding that weights of whole brain, frontal cortex, brainstem and cerebellar vermis were lower in hooded than in albino rats. These strain effects seldom interacted with treatment. Among the treatment effects was the finding that GD 11–13 but not GD 14–16 RA exposure impaired the righting reflex in both strains. Moreover, GD 11–13 exposure reduced weight of the cerebellar vermis more than did GD 14–16 RA exposure, while GD 14–16 RA exposure had greater impact on the weight of the cerebellar hemispheres than did GD 11–13 exposure. Covariate analysis suggested that these effects were independent of reductions in body weight. It is concluded that there are few strain or sex differences in the effects of gestational RA exposure, at least for the rat strains evaluated in this study.

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.

Minimal behavioral effects from developmental cerebellar stunting in young rats induced by postnatal treatment with α-difluoromethylornithine

Postnatal treatment with α-difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase, reduces polyamine levels in rats. Because polyamines are critically involved in growth and development, body and/or brain weights are often decreased by DFMO treatment. Here, rats were injected subcutaneously with 0, 250, 500, or 750 mg/kg of DFMO on postnatal days (PNDs) 5–10. Behavioral assessments included righting reflex, negative geotaxis, forelimb hanging, open field activity, and rotarod performance. Additionally, day of eye opening was recorded and on PND 28, whole and regional brain weights were measured. Cerebellar/whole-brain ratio was decreased in a dose-dependent manner whereas frontal cortex/whole-brain ratio was increased. Eye opening was delayed to a similar extent in all treated groups whereas body weight was unaffected. α-difluoromethylornithine treatment had no significant effects on the assessed behaviors. These results indicate that 6 days of DFMO treatment can substantially impact cerebellar development, but this appears to have few effects on these early assessed behaviors. However, potential behavioral alterations may not be apparent until adulthood. Published by Elsevier Science Inc.

Gestational stage-specific effects of retinoic acid exposure in the rat.

Although, or perhaps because, retinoids are among the earliest known behavioral teratogens, there is still little agreement about the behavioral effects of stage-specific exposure to these compounds. In these studies, pregnant albino rats were gavaged once daily with retinoic acid (RA) for 3 consecutive gestational days (GD), GD 8-10), GD 11-13, or GD 14-16. Dose levels were maximal levels compatible with survival (10, 2.5, or 12.5 mg/kg RA, over GD 8-10, 11-13, and 14-16, respectively). Two studies were conducted. The first assessed the effects of RA exposure on GD 8-10 or 14-16 on regional brain weight and on a large behavioral test battery. The second study assessed the effects of RA exposure on GD 11-13 or GD 14-16 on many of the same variables. Taken together with an earlier study of the behavioral effects of GD 11-13 RA exposure, these studies permit the following conclusions. 1) RA exposure at the above doses at any of the three exposure periods produced an apparent reduction in amphetamine-induced open field activity. 2) RA exposure on GD 14-16 but not earlier produced a robust, replicable rotarod deficit in exposed offspring. 3) RA exposure on GD 11-13, but not earlier or later, increased daytime activity in residential running wheels. 4) RA exposure on GD 11-13 or GD 14-16 but not GD 8-10 reduced weight of cerebellum. 5) No RA effect at any exposure period was seen on maze learning, activity in novel open fields, or on auditory startle.

Lack of developmental neurotoxicity of MN rgp 120/HIV-1 administered subcutaneously to neonatal rats.

The potential for neurotoxic effects was evaluated in rat off-spring after exposure in utero and/or during the neonatal period to a recombinant subunit vaccine of gp120 prepared from the MN strain of HIV-1 (MN rgp 120/HIV-1). Thirty pregnant female rats were given MN rgp120/HIV-1 with alum adjuvant, and 30 rats were given vehicle, once every 3 days from Day 1 of presumed gestation until parturition. One pup/sex/litter from treated and control group dams were given a daily subcutaneous injection, from Day 1 through Day 22 postpartum (PP) of vehicle, MN rgp120/HIV-1, MN rgp120/HIV-1 with alum, or MN rgp120/HIV-1 with QS-21 adjuvant. Neurobehavioral and physical development were evaluated (preweaning reflex and development, sexual maturation, motor activity, acoustic startle, passive avoidance, functional observational battery, and water M-maze testing), and tissues were processed for anatomical examination (whole and regional brain weights, and neuropathology). Administration of MN rgp120/HIV-1, with or without adjuvant, to pups did not cause any persistent effect on any parameter evaluated. Neurohistological examination did not reveal any pathological effects related to treatment. Thus, MN rgp120/HIV-1 alone or formulated as a vaccine does not cause neurotoxicity or developmental toxicity in neonatal rats after exposure in utero and/or during the neonatal period.

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.

Behavioral effects of low-dose gestational day 11-13 retinoic acid exposure.

In a comparison article we report that maternal PO exposure to 2.5 mg/kg all-trans retinoic acid (RA) daily for 3 consecutive days over gestational days (GD) 11-13 produces a 10% reduction in weight of cerebellum at 4 weeks of age, not accompanied by other malformations. Here we report the results of a preliminary behavioral analysis of offspring exposed gestationally to RA as above. Exposed dams were allowed to deliver normally, and litters were culled to eight pups (4 +/- 1 of each sex) at birth. Both male and female offspring were tested prior to weaning on GD 21. Thereafter females were killed on postnatal day (PND) 28 for verification of RA effects on regional brain weight, and all subsequent behavioral testing was conducted on males. Preweaning tests were restricted to negative geotaxis (PND 8-9) and open field activity (PND 22). Postweaning tests included open field activity (PND 43), auditory startle response (three times, on PNDs 22, 43, and 84), 2-week activity in residential running wheels (PNDs 62-76), complex maze performance for 5 consecutive days (PND 83-87), emergence latency (PND 106), and assessment of the behavioral response to an amphetamine challenge (PND 107). Males were then killed on PND 108 for verification of RA effects on regional brain weights. In this study, RA reduced weight of cerebellum but not striatum. Cerebellar weight was 92% of control values in PND 28 females, and this weight difference had diminished to 95% of control weight by PND 108 in males. There were no treatment effects on negative geotaxis, activity in a small open field, auditory startle amplitude, or latency to enter an illuminated alley from a dark chamber. Maze learning occurred at levels equal to or slightly better than controls. Running wheel activity was enhanced by RA exposure, whereas activity in response to an amphetamine challenge was reduced by such exposure. We conclude that RA doses low enough to produce mild weight reductions in cerebellum, without attendant malformations, can alter behavior. The precise nature of these alterations remains to be elucidated, but the findings reported here suggest that effects may be more pronounced on activity than on learning.

Gestational retinoic acid exposure: a sensitive period for effects on neonatal mortality and cerebellar development.

This is the first in a series of studies investigating the developmental stage-specific neurobehavioral effects of all-trans retinoic acid (RA) exposure. Because high doses of this compound are known to be lethal to the developing organism, we first conducted a dose-response study to identify RA doses that produce low enough levels of gestational/postnatal mortality to make a behavioral analysis possible in survivors. Secondarily, at doses found to produce sufficient survivors on PND 28, effects on body and regional brain weights were examined. Finally, at these doses, effects on somatic malformations were evaluated. Four separate exposure periods were analyzed: gestational days (GD) 8 through 10, 11 through 13, 14 through 16, or postnatal days (PND) 3 through 5. In the postnatal exposure period rat pups were injected (s.c.) with three consecutive daily doses of 0, 5, 10, or 20 mg/kg RA on PND 3 through 5. This postnatal exposure had no detectable effect on survival, body or brain weight. In contrast, there was a marked sensitivity to RA in the GD 11-13 group. Many pups from dams given 10 mg/kg RA PO on GD 11-13 were found dead in the cage on the day of birth, and all surviving pups died within 4 days of birth. Examination of milkbands revealed no evidence of effective suckling in these short-term survivors. The same 10 mg/kg dose at GD 8-10 or GD 14-16 produced much lower mortality and pups appeared to suckle normally. To produce adequate PND 28 survival in the GD 11-13 group, it was necessary to reduce dosage to 2.5 mg/kg daily. Even this lower exposure produced effects on PND 28 body and brain weight, significantly lowering weights of body (84% of control), whole brain (94%), and cerebellum (90%). Cerebellar weight was also depressed as percent of whole brain weight, suggesting an effect focused specifically on this region. RA at 10 or 12.5 mg/kg over GD 14-16 also reduced cerebellar weight (92% and 91% of control, respectively). Thus, exposure on GD 14-16 had effects similar to those seen at GD 11-13, but only at considerably higher doses. In contrast, exposure to RA on GD 8-10 did not affect whole body or brain weight, and of eight brain regions examined, only brain stem weight was reduced (91% of control). The GD 8-10 exposure also differed substantially from later exposures in that it was the only treatment to produce substantial malformations, including exencephaly, eye and skeletal defects. We conclude that gestational exposure to RA produces lethality and regional brain stunting that is dose and developmental stage specific, with a pronounced sensitive period on GD 11-13. In contrast, the GD 8-10 period is most sensitive for production of malformations, albeit at somewhat higher doses.

Prenatal retinoic acid exposure: I. Effects on neonatal lethality and regional brain weights.

Prenatal retinoic acid exposure: I. Effects on neonatal lethality and regional brain weights.

Prenatal retinoic acid exposure: I. Effects on neonatal lethality and regional brain weights

Prenatal retinoic acid exposure: I. Effects on neonatal lethality and regional brain weights

A single day of alcohol exposure during the brain growth spurt induces brain weight restriction and cerebellar Purkinje cell loss

The period of rapid brain growth that occurs relatively late in development has been shown to be vulnerable to alcohol-induced brain growth deficits and neuron loss in rats using repeated daily exposure to alcohol. This study examined whether a condensed exposure (binge) restricted to a single day during this period would be sufficient to restrict brain growth and produce neuron depletion. Using artificial rearing, alcohol was given to neonatal rats in two consecutive feedings on postnatal day 4, using a total dose of either 6.6 g/kg or 3.3 g/kg. The higher dose produced mean peak blood alcohol concentrations of 362 mg/dl, while the lower dose produced mean peak BACs of 153 mg/dl. The higher dose significantly restricted whole brain, forebrain, cerebellum and brain stem weights on postnatal day 10, with the cerebellum being most severely reduced. Cerebellar Purkinje cells, counted from a 2-μm section from the midsagittal vermis, were significantly reduced in the 6.6 g/kg group. Purkinje cell loss relative to controls was significant in most lobules (I–V, IX and X), but, notably, was not significant in the later maturing regions (lobules VI and VII). The 3.3 g/kg group had no significant reductions in regional brain weights. Although the overall number of Purkinje cells was nonsignificantly reduced relative to controls, the 3.3 g/kg dose did produce significant loss of Purkinje cells in lobule II. Exposure to high peak BACs, even for a relatively short period during the brain growth spurt, constitutes a substantial risk to the developing brain, and even a moderate exposure may result in loss of more vulnerable neurons. The pattern of cell death produced suggests that the maturational or metabolic state of Purkinje cells at the time of the alcohol insult may influence the vulnerability of these neurons to an alcohol insult.

Effect of Clonazepam on Brain Development of Mice

Twenty-seven Kun-Ming white mice were divided randomly into three groups of 9 animals. One of these groups served as a control group. Clonazepam (CZP), 0.1 or 0.5 mg/kg/day, was administered by oral gavage for 22 days. The water-maze test was performed before giving CZP and 10 and 22 days later. The step-down test was performed on the 1st and the 2nd day after discontinuing CZP treatment to determine toxic effects on brain development and learning-memory function. Body weights and brain weight were also recorded. Brain development and learning-memory function, evaluated by water-maze and step-down tests, were not impaired by CZP. Although there was a trend toward lower weights in the higher-dose group, CZP treatment did not significantly affect either body weight or total or regional brain weight. The results of this study indicate that 4 weeks of treatment with CZP does not impair brain development and learning-memory function in baby mice.

Implementation of a primary screen for development neurotoxicity

A battery of tests to evaluate physical growth/development and neurobehavioral function was conducted with 78 litters of control Sprague-Dawley rats given purified water by intubation. The objectives of this study were to optimize test methods and to document the range and variability of the experimental endpoints. Data are presented for maternal evaluations (body weight gain, food consumption), gestation length, litter size, and postnatal survival. Pup body weight was used to assess postnatal somatic growth rate from birth to 85 days of age, while whole and regional brain weight measurements at 7, 28, and 85 days provided a more specific measure of physical growth relevant to a neurobehavioral study. Physical landmarks of development evaluated were pinna unfolding, incisor eruption, and eye opening while reflex landmarks of development evaluated were the negative geotaxis and pupillary reflexes. The mean percentage of litters acquiring a physical trait or reflex increased sigmoidally with age, and the data suggest that the potential to detect developmental delays would be optimal when ca. 90% of control litters reach the test landmark. Functional evaluations were arranged according to four testing subsets so that each litter was evaluated in each test (1 pup/sex/litter), but repeated testing on pups was minimized. Auditory and tactile startle reflexes, as well as preoulse inhibition, were measured at ages 22 days and 60–64 days and found to increase with age. A passive avoidance paradigm (age 40–43 days) was used to assess exploratory behavior (approach) and memory (avoidance). Swimming performance in a water maze was used to evaluate learning. In this test, escape times and error rates improved to their highest level by five or six trials and showed acceptable degrees of variability. Spontaneous motor activity was monitored for 23 hr at age 54–61 days to evaluate exploratory activity, photoperiod entrainment, and catecholamine-induced locomotion (amphetamine challenge). Finally, landmarks of sexual maturation (balanopreputial separation evident at 45 days of age, vaginal perforation evident at 33 days) and estrous cyclicity (4.8 cycles per 21 days) were evaluated as measures of reproductive neuroendocrine function. In sum, the test battery provided an efficient yet comprehensive screen for evaluating effects on physical growth/development and neurobehavioral function which meets practical criteria for preclinical testing of pharmaceutical agents.

Preliminary findings of a reduction of otoconia in the inner ear of adult rats prenatally exposed to phenytoin

Pregnant Sprague-Dawley CD ® rats were administered phenytoin by gavage on days 7–18 of gestation in doses of 0 or 200 mg/kg. Following completion of a series of behavioral tests (20), progeny (18 months of age) were examined for otoconia in the vestibular labyrinth of the inner ear. None of the controls (N=22) had reduced otoconia, while 23.3% of phenytoin-treated offspring (N=43) had reductions. None of the controls, but 44.2% of phenytoin-treated offspring exhibited abnormal circling behavior during systematic examinations conducted in dry and swimming environments. Of the phenytoin-treated offspring exhibiting circling, 21.0% had reduced otoconia in either the utricle or saccule of one ear, while 25.0% of phenytoin-treated offspring not circling exhibited similar reductions. Conversely, 79% of phenytoin-treated offspring exhibiting circling did not exhibit any otoconial reductions. Thus, otoconial reduction cannot account for the majority of the cases of circling. The 21% vs. 25% otoconial reduction difference was not significant, however, when ratings of the magnitude of reduction were analyzed, circling offspring had significantly lower scores in their utricles than those not circling. More specifically, otoconial reduction in the right utricle and circling behavior were significantly related, although the number of concordant cases was small. Otoconial ratings did not differ for saccules. No differences in regional brain weights were found at the time of otoconial examination (560 days). The evidence provide preliminary support for the idea that prenatal exposure to phenytoin induces a reduction in otoconial crystals of the vestibular labyrinth in some of the exposed offspring, but it cannot account for most of the behavioral effects that have been observed in these offspring.