Alteration in the pattern of nerve terminal protein immunoreactivity in the perforant pathway in Alzheimer's disease and in rats after entorhinal lesions.

Neurons in layer II of the entorhinal cortex consistently develop neurofibrillary tangles in Alzheimer's disease (AD). Experimental neuroanatomical studies have shown that these neurons give rise to the perforant pathway, a major excitatory projection to the hippocampal formation, which terminates in a discrete pattern in the outer portion of the molecular layer of the dentate gyrus. The distribution of two nerve terminal associated proteins, synaptophysin and NT75, was studied in the molecular layer of the dentate gyrus in AD and control cases to determine whether Alzheimer neuronal pathology is associated with loss of synaptic markers. In parallel studies, the effect of ablation of the entorhinal cortex in rats was evaluated. In AD as compared to controls, a decrease in synaptophysin immunostaining was evident in the terminal zone of the perforant pathway. NT75 nerve terminal immunostaining was too weak to interpret in the human hippocampal formation. Both synaptophysin and NT75 immunoreactivity were found in association with some neuritic plaques. In rats, entorhinal lesions resulted in diminished immunoreactivity for both synaptophysin and NT75 in the perforant pathway terminal zone. These results suggest that nerve terminal protein loss is a concomitant feature of neuronal pathology in AD.

Attenuation of reversal deficits of mice with septal lesions by shifts in the motivational context.

Response perseveration following septal lesions, demonstrated on tasks that require change of a learned pattern of responding, depends in part on a relatively constant motivational context. The present experiment examined the effects of concurrent changes in the temperature of the water in a water-escape maze and the reversal of a spatial discrimination in mice that had received either septal lesions or control surgery. Control and experimental mice were trained on a spatial discrimination in either 17 or 34 degrees C water. Following acquisition, reversal training was conducted in the maze filled with the same-temperature water used during acquisition or water temperature as well as spatial reinforcement contingencies were reversed. When the water temperature remained constant across acquisition and reversal, the mice with septal lesions showed persistence during reversal training. Changing the water temperature concurrently with the institution of reversal training attenuated the perseverative deficits of mice with lesions, in contrast to the relative lack of effect in control animals. These results are discussed within the framework of the role that the septal region plays in attention.

Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice.

Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

Long-term deficits in water maze spatial conditional alternation performance following retrohippocampal lesions in rats.

The effects of large bilateral retrohippocampal lesions on long-term performance of conditional spatial alternation, incorporating a strong working memory component, were examined using a T-maze task motivated by swim-escape. The lesions, which included entorhinal cortex, subiculum, pre- and parasubiculum and invaded the molecular layer of the dentate gyrus, completely eliminated the previously acquired conditional alternation learning, and performance failed to recover with 40 days of testing. These findings support the contention that retrohippocampal structures are an important and necessary component of the neural circuitry mediating working memory.

Constraints on water maze spatial learning in rats: implications for behavioral studies of brain damage and recovery of function.

In an effort to develop spatial learning tasks not requiring food or water deprivation for use in studies of recovery of function after brain damage, T-maze spatial alternation learning was examined in intact rats using water maze swim-escape procedures. Consistent with previous studies, rewarded spatial alternation involving food or water deprivation was readily learned by intact rats. However, none of the groups of rats trained in the swim-escape tasks learned to alternate goal arm choices in the water maze at reliable rates. This was true regardless of whether non-correction or correction procedures were used, and regardless of intertrial delay intervals. Although average alternation rates over sessions did increase from chance levels, the majority of the rats did not reach criterion levels, even with as many as 38 consecutive days of testing. In contrast, a conditional spatial alternation task in the water maze, using a win-shift procedure, was readily learned. Surprisingly, a win-stay version of this conditional spatial task was not learned over 21 days of testing. These unexpected constraints on spatial learning and memory processes in rats cannot be attributed simply to failure of spatial information processing, nor to strict limitations on working memory in swim-escape tasks, since excellent spatial navigation abilities have been documented, and mastery of at least some working-memory tasks have now been demonstrated in swim-escape tasks.

Impaired spatial navigation in adult female but not adult male rats exposed to alcohol during the brain growth spurt.

Two groups of male and female rats were given the same dose of alcohol using an artificial rearing procedure on postnatal days 4-10. One group received the alcohol in a condensed manner each day which caused cyclic blood alcohol concentrations (BACs) with high peaks. A second group received the alcohol in a uniform manner over each day which resulted in moderate, stable BACs. Two control groups consisted of male and female rats artificially reared but not exposed to alcohol and rats reared normally by dams. All rats were raised to 90 days of age and then tested for spatial navigation ability in the Morris water maze, which involved locating a hidden underwater platform using distal extramaze cues. Neither the alcohol treatments nor the artificial rearing had any effects on performance of adult male rats relative to suckle controls in this task. In contrast, the condensed alcohol exposure but not the uniform alcohol exposure resulted in detrimental performance in the Morris water maze by adult female rats. When the ability to locate and escape onto a visible platform was examined, there were no differences between the female groups given condensed alcohol exposure or artificially reared on milk solution alone. Thus, exposure to high BACs during the brain growth spurt has a lasting and selective detrimental effect on spatial navigation learning in adult female but not adult male rats.

Therapeutic effects of complex motor training on motor performance deficits induced by neonatal binge-like alcohol exposure in rats . I. Behavioral results.

The effects of complex motor task learning on subsequent motor performance of adult rats exposed to alcohol on postnatal days 4 through 9 were studied. Male and female Long-Evans rats were assigned to one of three treatments: (1) alcohol exposure (AE) via artificial rearing to 4.5.g kg-1 day-1 of ethanol in a binge-like manner (two consecutive feedings), (2) gastrostomy control (GC) fed isocaloric milk formula via artificial rearing, and (3) suckling control (SC), where pups remained with lactating dams. After completion of the treatments, the pups were fostered back to lactating dams, and after weaning they were raised in standard cages (two-three animals per cage) until they were 6 months old. Rats from each of the postnatal treatments then spent 20 days in one of three conditions: (1) inactive condition (IC), (2) motor control condition (MC) (running on a flat oval track), or (3) rehabilitation condition (RC) (learning to traverse a set of 10 elevated obstacles). After that all the animals were tested on three tasks, sensitive to balance and coordination deficits (parallel bars, rope climbing and traversing a rotating rod). On parallel bars, both male and female rats demonstrated the same pattern of outcomes: AE-IC rats made significantly more mistakes (slips and falls) than IC rats from both control groups. After 20 days of training in the RC condition, there were no differences between AE and both SC and GC animals in their ability to perform on the parallel bars test. On rope climbing, female animals showed a similar pattern of abilities: AE-IC rats were the worst group; exercising did not significantly improve the AE rats' ability to climb, whereas the RC groups (SC, GC and AE) all performed near asymptote and there were no significant differences among three neonatal treatment groups. There was a substantial effect of the male rats' heavier body weight on climbing ability, and this may have prevented the deficits in AE rats behavior from being detected. Nevertheless, male animals from all three postnatal treatments (SC, GC and AE) were significantly better on this task after RC. Female and male rats from all three postnatal groups demonstrated significantly better performance on the rotarod task after 20 days of 'rehabilitation'. These results suggest that complex motor skill learning improves some of the motor performance deficits produced by postnatal exposure to alcohol and can potentially serve as a model for rehabilitative intervention.

Regional differences in the timing of dendritic outgrowth of Purkinje cells in the vermal cerebellum demonstrated by MAP2 immunocytochemistry.

Detailed, within-subjects Golgi analyses of regional differences in cerebellar Purkinje cell dendritic development are impractical due to the capriciousness of that technique. Immunocytochemical labeling of microtubule-associated protein 2 (MAP2) was used to reveal the dendritic development of Purkinje cells, and indicated marked differences in the timing of initial outgrowth of Purkinje cell dendrites for different lobules in the developing rat cerebellar vermis. In particular, an early maturing region of Purkinje cell dendritic outgrowth (lobules I, II, IX and X and along the primary fissure), and a late maturing region (distal lobule VI, lobule VII and dorsal lobule VIII) were documented.

Alcohol-induced Purkinje cell loss depends on developmental timing of alcohol exposure and correlates with motor performance.

Several reports indicate that neonatal ethanol exposure induces cerebellar Purkinje and granule cell loss if exposure occurs before postnatal day (PD) 7, and that cerebellar damage may underlie ethanol-induced motor deficits. The present study used an unbiased stereological method, the optical fractionator, to count total cerebellar Purkinje cell number in groups of Sprague-Dawley rats given binge-like ethanol exposure at different neonatal ages. Correlations between Purkinje cell number (of 55-day-old rats) and parallel bar motor performance (previously tested on PD 30-32) were also evaluated. One group was given binge-like exposure to 6.6 g/kg per day of ethanol via artificial rearing on PD 4 and 5 (PD 4/5); a second group on PD 8 and 9 (PD 8/9); and a third group on both PD 4 and 5 and 8 and 9 (Comb). Gastrostomy (CG) and suckle (SC) control groups were also included. Purkinje cells were significantly reduced in all three ethanol-treated groups compared to controls, but the severity of loss was significantly greater in the PD 4/5 and Comb groups (reduced by 42% and 45%, respectively, relative to GC) compared to the PD 8/9 group (reduced by 15%). Across treatment groups, the total cerebellar Purkinje cell number was significantly correlated with successful parallel bar traversal (r = +0.74), supporting the contention that ethanol-induced motor deficits may be associated with cerebellar cell loss. These data confirm the presence of windows of vulnerability of Purkinje cells to neurotoxic effects of binge ethanol treatment, and demonstrate that both the behavioral and neuroanatomical consequences of binge exposure depend on the developmental timing of the exposure.

Transient cortical astrogliosis induced by alcohol exposure during the neonatal brain growth spurt in rats.

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.

Prenatal alcohol exposure retards the migration and development of serotonin neurons in fetal C57BL mice.

Incomplete neural tube fusion (iNTF), induced by alcohol, in midline floor and roof plates was found in our recent study. In this study, serotonin (5-HT) neurons, known to be born entirely in the midline raphe at brainstem, were examined during their development with fetal alcohol exposure. Weight-matched C57BL mice pregnant dams were divided into three groups on E8: one received ethanol via a chocolate Sustacal liquid diet providing 20% ethanol-derived calories as the sole source of nutrients (ALC); the second received an isocaloric Sustacal liquid diet and was pair-fed to individual dams in the ethanol-fed group (PF); the third was fed ad lib rat chow (Chow). Fetal brains were obtained on E15 and were processed for immunostaining of 5-HT and its trophic factor, S100 beta. The ascending 5-HT neurons, in normal development, appear bilaterally near midline on E12, and by E15, as seen in chow and PF groups, migrate from the midline germinal zone laterally and dorsally to their final position with rich fibers. In contrast, in the E15 ALC group, many 5-HT-im neurons were found remaining in the midline germinal region or had migrated, but with under-differentiated, sparse fibers. There were 20--30% fewer 5-HT-im neurons in ALC as compared to PF and Chow. In addition, the number of S100 beta cells was less in ALC as compared with PF and Chow groups. No difference was found between PF and Chow in number of 5-HT-im or S100 beta-im cells. The 5-HT neurons found compromised in migration and differentiation may, in part, stem from failure of access to floor plate or midline tissue induction and the insufficient support by S100 beta. As 5-HT neurons have been implicated for signaling brain maturation, fewer 5-HT neurons may have lasting effects on the development of brain or, if persistent in the adult, profoundly affect adult brain function.

Alz-50 immunoreactivity in the neonatal rat: changes in development and co-distribution with MAP-2 immunoreactivity.

Alz-50 is a monoclonal antibody that recognizes pathological alterations in Alzheimer's disease. It has recently been noted also to mark some subplate neurons in human infants under the age of 2 years. We now report that Alz-50 recognizes many neurons in the normal neonatal rat in a pattern that changes with development. Immunoreactivity decreases substantially in intensity as the rat matures. This immunoreactivity co-distributes with microtubule-associated protein-2 (MAP-2) immunoreactivity in terms of topography, cellular localization and changes over the developmental time-course. This observation raises the possibility of exploring cytologic triggers that may lead to re-expression of Alz-50 immunoreactivity in aging and in pathological conditions.

Manipulating peak blood alcohol concentrations in neonatal rats: review of an animal model for alcohol-related developmental effects.

Fetal alcohol syndrome (FAS) is now well documented, but factors that affect the severity of the accompanying central nervous system damage are still not well understood. In a series of experiments, artificially reared neonatal rats were exposed to alcohol during postnatal days 4-10 (during the brain growth spurt of the rat) to evaluate the consequences of various patterns of alcohol consumption in contributing to the severity of alcohol-related brain damage. In the first experiment, groups of rat pups were given different doses of alcohol (6.6 to 9.8 g/kg) in a milk formula in eight 15-min feedings over each 24 hr. Measures of brain weight on day 10 indicated an inverse relationship between dose and brain weight. Re-evaluating the results with respect to blood alcohol concentration (BAC) revealed an even stronger correlation between BAC and microencephaly. A series of experiments followed in which various doses of alcohol were condensed into fewer and fewer hours each day. Condensing the dose produced higher BACs for a given dose and produced more severe microencephaly, greater neuronal loss, behavioral hyperactivity and impaired spatial navigation. Some of these effects were permanent with females more affected than males on some measures. These data suggest that patterns of alcohol consumption that produce high BACs, such as binge drinking, may be especially harmful to the brain of the developing fetus.

Genetic influences on brain growth restriction induced by development exposure to alcohol.

Genetic factors have been implicated as contributing to the considerable variation in the severity of alcohol-related birth defects in offspring of women who drink heavily during pregnancy. Two animal models of alcohol-related developmental effects incorporated different behavior genetic approaches to examine genetic influences on brain and body growth following alcohol exposure during development. The first, extensively developed in Sprague-Dawley rats, examined the effects of three doses of alcohol administered to two inbred rat strains (MR and M520) via artificial-rearing procedures during the early postnatal brain growth spurt. In both strains, alcohol produced a dose-dependent restriction of brain weight (but not body weight) on postnatal day 10, compared to artificially reared controls. The MR strain was more susceptible to cerebellar growth restriction than the M520 strain, an effect not attributable to strain differences in blood alcohol concentrations. In the second model, pregnant female Long-sleep and Short-sleep mice, selectively bred for differences in initial sensitivity to the hypnotic effects of acute alcohol administration, were intubated with ethanol from gestational days 7-18. Controls included either sucrose or maltose/dextrin intubation controls and non-intubated controls. The LS offspring showed growth deficits and brain weight reductions in adulthood, while the SS offspring were resistant to these detrimental effects of the prenatal alcohol exposure. Thus, differences in either maternal or fetal genotype may contribute to individual differences in the severity of the effects of alcohol exposure during development.

Effects of pre- and early postnatal protein malnutrition on carcass composition and lipoprotein lipase activity in male rats.

Little is known about the effects of pre- and early postnatal protein malnutrition on energy storage (e.g., carcass lipid) and expenditure (e.g., brown adipose tissue [BAT] thermogenesis) and their reversibility by nutritional rehabilitation. Therefore, the purpose of these experiments was to examine the permanence of prenatal and early postnatal protein malnutrition on energy balance in rats. Five weeks before mating and through gestation adult female rats were fed either a 25 or 8% casein diet (designated 25 or 8). Diet reversals were performed at birth and/or at weaning (designated B or W) and the pups were cross-fostered at birth. Thus, the groups were: 25-25B (controls), 8-25W (gestational and lactational protein malnutrition, 8-25B (gestational protein malnutrition), and 25-8B-25W (lactational protein malnutrition). Animals were weighed and sacrificed 200-250 days postpartum for carcass composition. Retroperitoneal white adipose tissue (RWAT) and interscapular BAT (IBAT) wet weights and lipoprotein lipase (LPL) activity were also measured in the 25-25B and 8-25W groups. Nutritional rehabilitation at birth (8-25B) resulted in normal body weights as adults. Lactational protein malnutrition (25-8B-25W) resulted in intermediate body weights to the controls (25-25W), which had the greatest weights, and the 8-25W group, which had the lowest weights. The 8-25W and 25-8B-25W rats also had significantly decreased carcass wet weight and total body water, fat and fat-free dry mass relative to the 25-25B and 8-25B groups, the latter two of which did not differ in their carcass composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of environmental rearing history and postsurgical environmental change on the septal rage syndrome in mice.

Reactivity to handling was examined in Binghamton Heterogeneous (HET) mice given septal lesions or control surgery following 5 weeks of postweaning rearing in enriched or restricted environments. Three housing conditions were employed: continuous enrichment, continuous restriction (both pre- and postsurgically), and a group switched from environmental enrichment to restriction 24 hours after surgery. The restricted mice were more reactive to handling than the enriched mice presurgically (60 days old), and interactions of housing conditions and surgery were found over the seven day postsurgical handling and testing period. The restricted mice given septal lesions greatly increased in reactivity postsurgically, while the intact controls declined in reactivity over the postsurgical week. The enriched septals were only moderately increased over the low presurgical reactivity baseline, and were much less reactive than the restricted septals. Perhaps the most remarkable finding was the dramatic and immediate increase in reactivity in the septals switched from enrichment to restriction, while intact controls showed no effect of the switch. These results emphasize the importance of experience in determining the effects of septal damage, and may be useful in examining other correlates of septal rage.

Differential effects of clonidine, B-HT 933 and B-HT 920 in immature rat pups.

The effects of the alpha-adrenergic agonist clonidine were compared with two experimental hypotensive drugs, B-HT 920 and B-HT 933, in 10-day-old rat pups. Clonidine induced the expected dose-dependent (0.1-1.0 mg/kg) motor activation and wall-climbing syndrome typical at this age. B-HT 933, thought to be a more selective alpha 2-agonist than clonidine, elicited locomotor activity and wall-climbing only at the highest dose used (50 mg/kg). The high dose of B-HT 933 necessary to begin to mimic the effects of clonidine, a finding consistent with some studies using B-HT 933 in adults, suggests that the wall-climbing syndrome is mediated by receptors which have a low affinity for B-HT 933. In striking contrast, B-HT 920, a presynaptic dopamine agonist in mature rats, produced a very different behavioral profile. B-HT 920 induced long periods of sniffing accompanied by locomotion at low doses (peak at 0.12 mg/kg) and ataxic locomotion and poorly coordinated wall-climbing at high doses (30-50 mg/kg). Experiment 2 demonstrated that the active sniffing evoked by low doses of B-HT 920 was dose-dependently blocked by haloperidol (0.035-1.0 mg/kg). These findings of behavioral effects in 10-day-old rats suggest that B-HT 920 stimulates dopaminergic receptors in immature rats, presumably located on postsynaptic neurons. We propose that B-HT 920 and B-HT 933 also may be differentiated in terms of the time of onset of functional development of dopaminergic and noradrenergic autoreceptors, respectively.

Temporal determinants of neonatal alcohol-induced cerebellar damage and motor performance deficits.

The timing and duration of alcohol exposure was manipulated in neonatal rats by using a "binge" model of alcohol exposure during the "third trimester equivalent." Groups of Sprague-Dawley rats were exposed to binges via artificial rearing on postnatal days (PD) 4-9, on PD 4-6 or on PD 7-9, which produced peak blood alcohol concentrations representative of human alcohol abusers (approximately 250 mg/dl). Motor performance was assessed using parallel bar traversal on PD 42-44, and total Purkinje cell numbers were determined by using the 3-dimensional stereological optical fractionator method. PD 4-9 binge exposure induced the most severe Purkinje cell loss (to 68% of controls) and PD 4-6 binge exposure also produced significant loss (to 86% of controls), whereas PD 7-9 binge exposure had no significant effect (98% of controls). Unexpectedly, all three alcohol treatments resulted in significant impairments on the parallel bar task. The time of exposure during the early neonatal period in rats strongly influences the degree of Purkinje cell loss, but Purkinje cell loss is not necessary for the alcohol-induced motor performance deficits. Both neuromorphological and neurobehavioral assessments are needed for a full description of alcohol-related neurodevelopmental disorders.

Altered development of responsiveness to clonidine in severely malnourished rats.

To examine the effects of malnutrition on the ontogeny of alpha 2 noradrenergic receptor function, we compared the effects of clonidine during early development in severely malnourished and well-nourished rat pups. Independent groups of pups from dams given either 6% or 25% casein diets received one of five doses of clonidine at 5, 10, 15, 20 or 25 days of age and dose-response relationships for motor activity were determined. In the 25% pups the clonidine-induced locomotor activity was greatest at 5 and 10 days, intermediate at 15 days and not elevated at 20 and 25 days. The malnourished pups exhibited a significant delay in the transition from hyperactivity to hypoactivity, being activated by clonidine until at least 25 days. Wall-climbing measures indicated similar developmental trends as overall activity. These results are discussed in terms of the proposed mechanisms mediating the developmental change in the effects of alpha 2 receptor stimulation.

Neonatal binge ethanol exposure using intubation: timing and dose effects on place learning.

Neonatal rats were given ethanol using an acute intubation procedure that resulted in daily binge-like exposure with minimal effects on somatic growth. Acquisition of place learning in the Morris water maze was evaluated on postnatal days (PD) 26-31. In Experiment 1, a total of 5.25 g/kg/day of ethanol was administered in two daily intubations on PD 4-6, PD 7-9, or PD 4-9, producing mean peak BACs of 265 mg/dL. Place learning acquisition deficits in a 114-cm-diameter tank were found for the PD 4-9 and PD 7-9 groups, but not the PD 4-6 group. In Experiment 2, either 4.5 or 5.25 g/kg/day of ethanol was administered on PD 7-9 and place learning was tested in a 171-cm-diameter tank. Significant acquisition deficits resulted from the higher dose, and probe trial search patterns for both ethanol groups were significantly less localized than controls. In Experiment 3, no significant effects of either PD 7-9 dose were found on a visible platform task. These findings reveal selective place learning deficits in this intubation model of neonatal binge exposure, and confirm a temporal window of vulnerability to spatial learning deficits during the second neonatal week.