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A new rat model for investigating the effects of alcohol on the adolescent brain: Acute and long-term effects
Get PDFAdolescence represents a time of unique vulnerability to improper brain maturation, due to continued development of some frontal regions of the brain and a propensity for risky behaviours including alcohol use. Recently, in New Zealand and overseas, increases in binge drinking during adolescence have necessitated further research into alcohol’s short- and long-term effects on the brain and behaviour. Important research using animal models has revealed gross changes in brain structure and function in response to large alcohol doses, however it is still not known what subtle changes, if any, may result from binge drinking at the levels that are seen in the majority of the population.
The current study aims to address this by using Long-Evans rats to model moderate- to high-levels of weekly binge drinking in human adolescents. Male and female rats were given 9.0 g/kg/day alcohol by intra-gastric gavage, every fourth day from PN28 to 48, resulting in a mean peak BEC on alcohol dosing days of 277 mg/dL. Control animals were not given alcohol.
The brains of the rats were investigated immediately following alcohol treatment (ages PN29, PN33, PN37, PN41, PN45 and PN49) for presence of apoptotic cell death and gliosis. A battery of behavioural tests was completed up to a year of post-natal age in order to investigate changes in brain function that persist in the long-term Histological survey throughout the cerebrum revealed no apoptosis, which could be consistent with an insult, after any of the 6 alcohol treatments. There were, however, subtle differences in astrocyte number in the rostral cingulate cortex, which require further study. Behavioural tests revealed subtle differences in performance between treatment/sex groups in key areas, despite a considerable period of abstinence. Of particular interest was an alcohol-induced reduction in working memory performance, during recognition of a novel object, that affected male but not female rats. Male, alcohol-treated rats also were less accurate at finding the platform in the initial stages of the MWM probe trial, as evidenced by fewer crossings of the previous platform location. This finding was not replicated in the female rats. Female rats that had been treated with alcohol during adolescence did, however, spent a significantly greater percentage of their path length in the outer zone of the MWM during the probe trial and this behaviour is normally indicative of a non-problem solving, panic response
Study of CA1 Pyramidal Cell Dendritic Spines of the Rat Hippocampus by Serial Electron Microscopy Following a Single Ethanol Exposure on Postnatal Day 6
No full textExposure of the developing fetus to ethanol has numerous and wide-ranging effects including hippocampal damage resulting in difficulties with spatial learning and memory. Children and animal models tested on these types of tasks are able to perform them but not to the same standard as controls. This suggests that although the brains of these subjects still contain the same basic circuitry, it appears to have been altered in some way that decreases performance. This has been previously investigated with cell count studies that found ethanol exposure during development significantly decreases the number of CA1 but not CA3 cells in the hippocampus. The axons from CA3 pyramidal neurons are an important input to the CA1 cells and it is likely that the synaptic complement of the CA1 apical dendritic tree will be altered to accommodate this imbalance. The current study investigated whether there were any changes to the spines, synapses and mitochondria of the CA1 dendritic tree in the upper stratum radiatum following ethanol exposure.
Rat pups were exposed to a single-binge ethanol dose (6g/kg body weight) on PN6 to investigate brain tissue at PN26-30. The animals underwent intra-cardiac perfusion with a paraformaldehyde/glutaraldehyde fixative solution to obtain hippocampal tissue for transmission electron microscopy. An initial series of fixation trials were performed to optimise the quality of tissue fixation then sets of serial sections were used with 3-dimensional reconstruction technology (Synapse Web) to analyse components of the CA1 dendritic tree. The total number of spines/μm of dendrite was not found to be significantly different between ethanol-exposed and control animals, (p=0.8894) and both groups had significantly more normal spines (F(1, 27)=0.2369, P=0.6304) than other types. The total number of synapses was not significantly different between treatment groups (p=0.5677) and each group had significantly more non-perforated than perforated synapses (F(1,20)=26.15, P<0.0001). There was also no significant difference in the volume fraction of mitochondria within the length of dendrite analysed, between groups (P=0.9593). However, the length of total apical dendritic tree of the CA1 pyramidal cells was significantly longer (p<0.001) in the alcohol treated group (294.1±4.9µm) compared to the control group (267.4±4.3µm).
This study has shown that the density of dendritic spines and synapses on the spiny branchlets of CA1 pyramidal cells in the upper stratum radiatum was preserved following a single ethanol exposure on postnatal day six, but that the overall length of the dendrite tree was increased. This is thought to be an attempt to compensate for CA1 pyramidal cell loss with reduced plasticity of the existing dendrite and suggests that instead the total number of spines and synapses per CA1 cell may have been altered
Temporal Changes in the Expression of BAX and BCL-2, and the Sub-regional Vulnerability to Cellular Apoptosis after a Binge-like Ethanol Exposure in the Developing Rattus norvegicus Hippocampus
Get PDFExposure to ethanol in utero can result in life-long disability. Ethanol is neurotoxic, and exposure of high doses during development results in widespread cell death. A particular brain region of interest that is affected is the hippocampus, which has implications in learning and memory. The present study uses a rat model of binge-like drinking, during the third trimester-equivalent stage of neural development of humans, to investigate cell death in the hippocampus. The optical fractionator method was used to estimate total acute apoptotic cell death in the CA1, CA3, and DG sub-regions of the hippocampus. Rat pups were given ethanol on PN6 and PN8, or on PN8 only, and tissue was collected 12 hours after the last ethanol exposure. Furthermore, relative expression of apoptotic proteins Bcl-2 and Bax was measured in the CA1/DG and CA3 sub-regions 12, 24, and 48 hours after ethanol exposure on PN6.
These experiments showed significant cell death in all sub-regions of the hippocampus after a single, exposure on PN8 only, with a lot less cell death following a PN8 exposure, which was preceded by a PN6 exposure. The Bcl-2:Bax ratio significantly increased at the 24 hour time point, and returned back to control levels by 48 hours. The results suggest that a prior ethanol exposure may affect subsequent acute apoptotic cell death induced by ethanol, perhaps by a protective mechanism as the Bcl-2:Bax ratio increases at 24 hours. This study increases our understanding of apoptotic cell death as a consequence of binge drinking during the third trimester equivalent of human foetal development
The Effects of Binge Alcohol Consumption on the Developing Brain
No full textExposure to ethanol during the third trimester brain growth spurt can have detrimental effects. Damage to the central nervous system being the most severe outcome resulting from prenatal ethanol exposure causing a range of deficits on a continuum of gross to subtle. Human data on the timing and dose of ethanol exposure that results in perturbation is limited, so the use of animal models in studying these precise effects is critical. This study aimed to investigate the effects of a double binge exposure to ethanol on post-natal day six (PN6) and post-natal day eight (PN8), in the third trimester equivalent of human brain development, using a rat model. The acute affects of ethanol exposure and withdrawal were assessed after the first binge on PN6. At weaning the animals were placed in a reversed light cycle housing environment and activity, anxiety, learning and memory experiments were conducted during the dark cycle, the period of normal activity for the rat. These experiments revealed a marked decrease in locomotion twenty-four and thirty-two hours following the first binge exposure on PN6, with severe seizure activity being greatest at twenty four hours post ethanol exposure. Habituation to an unfamiliar environment was not effected by ethanol exposure. Hyperactivity was observed in the open field and no difference in anxiety related behaviour was apparent following open-field testing. However, elevated T-maze testing revealed a difference in anxiety related behaviour. Ethanol exposed animals had a decreased learned fear response but no difference in innate fear. Learning and memory deficits were observed in novel object recognition testing and both the reference memory and working memory versions of the Morris water maze. Following behavioural testing, a pilot study was carried out to determine the number of synapses on the apical dendrites of the CA1 pyramidal cells was using the physical disector method on electron micrographs. There was a significant decrease in synapses per unit volume in the ethanol treated animals compared to controls. This study provides an important link between structural changes and functional deficits associated with a double binge-like ethanol exposure during the third trimester brain growth spurt
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Study of CA1 Pyramidal Cell Dendritic Spines of the Rat Hippocampus by Serial Electron Microscopy Following a Single Ethanol Exposure on Postnatal Day 6
Get PDFExposure of the developing fetus to ethanol has numerous and wide-ranging effects including hippocampal damage resulting in difficulties with spatial learning and memory. Children and animal models tested on these types of tasks are able to perform them but not to the same standard as controls. This suggests that although the brains of these subjects still contain the same basic circuitry, it appears to have been altered in some way that decreases performance. This has been previously investigated with cell count studies that found ethanol exposure during development significantly decreases the number of CA1 but not CA3 cells in the hippocampus. The axons from CA3 pyramidal neurons are an important input to the CA1 cells and it is likely that the synaptic complement of the CA1 apical dendritic tree will be altered to accommodate this imbalance. The current study investigated whether there were any changes to the spines, synapses and mitochondria of the CA1 dendritic tree in the upper stratum radiatum following ethanol exposure.
Rat pups were exposed to a single-binge ethanol dose (6g/kg body weight) on PN6 to investigate brain tissue at PN26-30. The animals underwent intra-cardiac perfusion with a paraformaldehyde/glutaraldehyde fixative solution to obtain hippocampal tissue for transmission electron microscopy. An initial series of fixation trials were performed to optimise the quality of tissue fixation then sets of serial sections were used with 3-dimensional reconstruction technology (Synapse Web) to analyse components of the CA1 dendritic tree. The total number of spines/μm of dendrite was not found to be significantly different between ethanol-exposed and control animals, (p=0.8894) and both groups had significantly more normal spines (F(1, 27)=0.2369, P=0.6304) than other types. The total number of synapses was not significantly different between treatment groups (p=0.5677) and each group had significantly more non-perforated than perforated synapses (F(1,20)=26.15, P<0.0001). There was also no significant difference in the volume fraction of mitochondria within the length of dendrite analysed, between groups (P=0.9593). However, the length of total apical dendritic tree of the CA1 pyramidal cells was significantly longer (p<0.001) in the alcohol treated group (294.1±4.9µm) compared to the control group (267.4±4.3µm).
This study has shown that the density of dendritic spines and synapses on the spiny branchlets of CA1 pyramidal cells in the upper stratum radiatum was preserved following a single ethanol exposure on postnatal day six, but that the overall length of the dendrite tree was increased. This is thought to be an attempt to compensate for CA1 pyramidal cell loss with reduced plasticity of the existing dendrite and suggests that instead the total number of spines and synapses per CA1 cell may have been altered
The Effects of Binge Alcohol Consumption on the Developing Brain
No full textExposure to ethanol during the third trimester brain growth spurt can have detrimental effects. Damage to the central nervous system being the most severe outcome resulting from prenatal ethanol exposure causing a range of deficits on a continuum of gross to subtle. Human data on the timing and dose of ethanol exposure that results in perturbation is limited, so the use of animal models in studying these precise effects is critical. This study aimed to investigate the effects of a double binge exposure to ethanol on post-natal day six (PN6) and post-natal day eight (PN8), in the third trimester equivalent of human brain development, using a rat model. The acute affects of ethanol exposure and withdrawal were assessed after the first binge on PN6. At weaning the animals were placed in a reversed light cycle housing environment and activity, anxiety, learning and memory experiments were conducted during the dark cycle, the period of normal activity for the rat. These experiments revealed a marked decrease in locomotion twenty-four and thirty-two hours following the first binge exposure on PN6, with severe seizure activity being greatest at twenty four hours post ethanol exposure. Habituation to an unfamiliar environment was not effected by ethanol exposure. Hyperactivity was observed in the open field and no difference in anxiety related behaviour was apparent following open-field testing. However, elevated T-maze testing revealed a difference in anxiety related behaviour. Ethanol exposed animals had a decreased learned fear response but no difference in innate fear. Learning and memory deficits were observed in novel object recognition testing and both the reference memory and working memory versions of the Morris water maze. Following behavioural testing, a pilot study was carried out to determine the number of synapses on the apical dendrites of the CA1 pyramidal cells was using the physical disector method on electron micrographs. There was a significant decrease in synapses per unit volume in the ethanol treated animals compared to controls. This study provides an important link between structural changes and functional deficits associated with a double binge-like ethanol exposure during the third trimester brain growth spurt
Drinking During the Third Trimester of Pregnancy – a Cause for Concern
Get PDFIntroduction: Foetal Alcohol Spectrum Disorder (FASD) is an array of disorders attributed to CNS damage, behavioural and social manifestations after varying degrees of prenatal exposure to alcohol. Due to the majority of effects being phenotypically subtle, many cases are not diagnosed, and subsequently do not receive support and treatment. Drinking during pregnancy is an on-going issue in New Zealand, in particular binge-like episodes resulting in high blood alcohol concentrations. These episodes have been shown to continue throughout pregnancy in some women, and a lack of evidence correlating specific structural and functional outcomes after a single alcohol binge during the third trimester leaves room for question about what timing and quantity of alcohol induces lasting abnormalities in early life and adulthood.
Methods: Two cohorts of animals were used – (1) for stereological studies and (2) for behavioural studies. On Post Natal day 6 (PN6), the human third-trimester equivalent, male and female Long Evans rats were randomly assigned into one of three treatment groups and administered an intragastric intubation; Ethanol 6.0g/kg (E6), 5.25g/kg (E5), 4.5g/kg (E4); and two control groups - Intubation control (IC) and Suckle Control (SC). Cohort 2 underwent social play behaviour analysis via video recording from P32-34, and again on PN80-82. Animals were later analysed on the Elevated T-maze (ETM) for anxiety-related responses to an aversive environment. Cohort 1 were deeply anaesthetised and perfused on PN365. Brains were removed, cryoprotected, frozen and sectioned in the coronal plane at 60μm. A random systematic set of sections were stained with thionin followed by unbiased stereological methods using the optical disector to determine the number of neurons in the Anterior Cingulate Cortex (Acc) and hippocampal CA1 area.
Results: Cohort 1 revealed a dose-dependent loss of hippocampal CA1 cells across all alcohol-exposed groups (P<0.01); along with E6 and E5 animals showing a 39.6% and 30.8% (P<0.05) mean decrease in Acc cells relative to IC animals. The E6 group also had significantly less hippocampal CA1 and Acc neurons than the E4 group (P<0.05). Play behaviour analysis in cohort 2 revealed increased rates of attack by the alcohol-exposed and IC groups compared with controls (P<0.05). SC animals did not display the normal reduction in attack frequency between adolescence and adulthood, while E6 animals preferred the evasive form of defence significantly more often than all other groups during adulthood (P<0.05). ETM testing did not reveal any definitive anxiogenic / anxiolytic effects of prenatal alcohol exposure (P = 0.058), however E6 animals showed a significant lack of response inhibition from trial 1 to trial 2 on the inhibitory avoidance task relative to IC and SC animals (P<0.05). The one-way escape task revealed E6 animals took significantly less time than E4 animals to escape into the closed arm, however as no difference was found with relation to control groups this result was equivocal.
Conclusion: It is clear that a single ethanol binge on PN6 induces significant neuronal death in both the hippocampal CA1 (E6 = 49%, E5 = 38%, E4 = 26%) and Acc (E6 = 28%, E5 = 24%, E4 = 5%) areas, in a dose dependent fashion. Both of these areas are involved with social functioning, and corresponding social play abnormalities were observed, however not in the fashion hypothesized. During adolescence, both ethanol-exposed and control groups were behaving abnormally and showing different rates of defensive patterns than previously reported in the literature. This may be due to a “rub off” effect of the alcohol animals being housed with the control animals. These social play behaviour anomalies seen in adolescence were not evident in adulthood, indicating some level of functional recovery to appropriate levels of attack and defence during a ten-minute play period. However E6 animals showed a significantly higher probability of evading an attack than SC animals, suggesting a preference for bout termination possibly due to intimidation by the more cognitively intact SC animals. Anxiety testing revealed anomalies in response inhibition in E6 animals, however further refined studies are needed to definitively elucidate these putatively subtle changes.
These data contribute to the growing pool of evidence that any amount of alcohol during any stage of pregnancy may have serious repercussions, and only through continuing research will we begin to build an evidence base for public health policy and guidelines
Temporal Changes in the Expression of BAX and BCL-2, and the Sub-regional Vulnerability to Cellular Apoptosis after a Binge-like Ethanol Exposure in the Developing Rattus norvegicus Hippocampus
No full textExposure to ethanol in utero can result in life-long disability. Ethanol is neurotoxic, and exposure of high doses during development results in widespread cell death. A particular brain region of interest that is affected is the hippocampus, which has implications in learning and memory. The present study uses a rat model of binge-like drinking, during the third trimester-equivalent stage of neural development of humans, to investigate cell death in the hippocampus. The optical fractionator method was used to estimate total acute apoptotic cell death in the CA1, CA3, and DG sub-regions of the hippocampus. Rat pups were given ethanol on PN6 and PN8, or on PN8 only, and tissue was collected 12 hours after the last ethanol exposure. Furthermore, relative expression of apoptotic proteins Bcl-2 and Bax was measured in the CA1/DG and CA3 sub-regions 12, 24, and 48 hours after ethanol exposure on PN6.
These experiments showed significant cell death in all sub-regions of the hippocampus after a single, exposure on PN8 only, with a lot less cell death following a PN8 exposure, which was preceded by a PN6 exposure. The Bcl-2:Bax ratio significantly increased at the 24 hour time point, and returned back to control levels by 48 hours. The results suggest that a prior ethanol exposure may affect subsequent acute apoptotic cell death induced by ethanol, perhaps by a protective mechanism as the Bcl-2:Bax ratio increases at 24 hours. This study increases our understanding of apoptotic cell death as a consequence of binge drinking during the third trimester equivalent of human foetal development
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