Don W. Walker

PRENATAL ETHANOL EXPOSURE PERMANENTLY REDUCES THE NUMBER OF PYRAMIDAL NEURONS IN RAT HIPPOCAMPUS

The effect of prenatal ethanol exposure in the rat on the development of dorsal hippocampal pyramidal and dentate gyrus granule cells was examined. Ethanol was administered in a nutritionally adequate liquid diet to pregnant rats during days 10--21 of gestation. Control groups were either pair-fed the same liquid diet, except for equicaloric substitution of sucrose for ethanol, or received free access to pelleted laboratory food. The brains of 60-day-old offspring exposed to ethanol during gestation were found to have 20% fewer dorsal hippocampal pyramidal cells than did those of controls. Prenatal ethanol exposure, however, did not affect the number of dentate gyrus granule cells. Prenatal exposure to ethanol permanently reduced the number of prenatally formed hippocampal neurons without altering physical growth, which suggested that the developing nervous system is particularly sensitive to the toxic effects of ethanol.

Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative

Biocytin, a naturally occurring low molecular weight analog of biotin, was evaluated as a neuroanatomical tract-tracing marker in the adult rat brain. Since it retains high-affinity binding to avidin, biocytin can be labelled with avidinylated visualization reagents. Iontophoretic or pressure injections resulted in filling of cell bodies and dendrites around the injection site and their efferent axonal processes and boutons. Retrogradely labelled neurons were occasionally observed at a distance but only with large injections. Anterograde tracing with biocytin is successful even in animals that are quite old, in contrast to lectins and HRP conjugates, and offers advantages in delivery, tissue processing, selection of light and/or electron microscopic labels, time to obtain results, and cost over many conventional tracers.

Impairment of shuttle box avoidance learning following prolonged alcohol consumption in rats

Abstract Prolonged consumption of alcohol (6 months in Experiment 1; 9 months in Experiment 2) concomitant with adequate nutrition was found to impair the acquisition of shuttle box avoidance in rats. Alcohol was administered in the form of a liquid diet as the only source of fluid and calories. Ethanol constituted 35 per cent of the total caloric value of the liquid diet. One control group received the identical liquid diet with isocaloric substitution of sucrose for ethanol, and another control group received lab Chow and water. Two weeks after alcohol was omitted from the diet, the alcohol-consuming rats were severely impaired in learning the avoidance task as compared to the sucrose-consuming and lab Chow control groups. The latter two control groups did not differ from one another.

Alcohol withdrawal in mice: Electroencephalographic and behavioral correlates ☆

Abstract Chronic bipolar electrodes were implanted in forebrain structures of mice including dorsal hippocampus, dorsal-frontal cortex, dorsal-lateral thalamus and septum. Two weeks after surgery, the mice were reduced to 85–90% of free-feeding weight, and physical dependence on alcohol was established by maintaining the mice on an alcohol-containing liquid diet for 6 days. A control group was pair-fed an identical liquid diet except sucrose was substituted isocalorically for alcohol. Following 6 days of consumption alcohol was withdrawn and the development of both EEG and behavioral sequelae of withdrawal was monitored for 6–8h. Only mice withdrawn from alcohol exhibited behavioral signs of withdrawal which included hyperactivity, severe tremor, ataxia, sudden sprawling movements, and clonic-tonic convulsions. No abnormal behavior and no abnormal EEG activity resulted from the withdrawal of the sucrose control diet. EEG recordings during withdrawal from alcohol, however, indicated a progressive development of widespread neural epileptiform activity beginning with EEG slowing and increased amplitudes, followed by single spike events and often leading to sustained epileptic seizure discharge. Similar abnormal EEG patterns were observed throughout the forebrain including cortex, thalamus, hippocampus and septal area. The development of abnormal EEG activity was more severe during the second alcohol withdrawal period following 4 days of alcohol exposure than during the first alcohol withdrawal period following 6 days of alcohol exposure. It is hypothesized that the widespread forebrain epileptiform activity originates in, or is organized from, a central pacemaking region such as midline thalamus or reticular formation.

Ethanol dependence in the rat: a parametric analysis.

Rats were maintained on liquid diets as their sole source of calories and fluid for 10, 15, 20, and 30 days. The diets consisted 35-40% of total calories in the form of ethanol. This procedure resulted in substantial ethanol intake leading to behavioral intoxication. Blood ethanol concentrations were found to be elevated throughout the day with a peak during the dark phase of the light cycle. The removal of ethanol resulted in evidence of physiological dependence, including behavioral manifestation of autonomic and somatic dysfunction and an increased susceptibility to audiogenic convulsions. Ten days of ethanol exposure was found to be sufficient for the reliable induction of ethanol dependence. Further increases in ethanol exposure resulted in increased hyperexcitability as measured by susceptibility to audiogenic convulsions. The severity of withdrawal behavior was found to be correlated with the blood ethanol concentration measured upon ethanol removal. A behavioral rating scale for the evaluation of alcohol withdrawal intensity in rats is described.

Alterations and recovery of dendritic spine density in rat hippocampus following long-term ethanol ingestion

Neuronal loss and dendritic pathology are often observed in humans and animals after long-term ethanol ingestion. It is not known, however, if surviving but damaged neurons can recover normal structure during ethanol abstinence. We quantified dendritic spine density in two neuronal populations in rat hippocampus to investigate whether reversibility from the cellular neurotoxic sequelae of chronic ethanol exposure was possible. Male Long-Evans rats were maintained for 20 weeks on an ethanol-containing liquid diet. Controls were pair-fed a liquid diet with sucrose substituted isocalorically for ethanol. One-half of each group was sacrificed at the end of the 20-week treatment and one-half was given a 20-week ethanol-free recovery period period to sacrifice. Analysis of rapid Golgi material revealed a decreased spine density in CA1 pyramidal cells that increased to control level during abstinence, and an increased spine density in dentate gyrus granule cells that was reduced toward control level during abstinence. Thus, despite the fact that chronic ethanol exposure produced differential initial effects, the return toward normal spine density in each region is consistent with the concept of neuronal recovery and reorganization during abstinence from ethanol.

Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus.

We have previously demonstrated that chronic ethanol consumption decreases neurotrophic activity in hippocampal extracts, as assessed by a chick dorsal root ganglia bioassay, but has no effect on hippocampal NGF mRNA or NGF protein levels. We presently report that hippocampal mRNAs encoding neurotrophin-3 and basic fibroblast growth factor are also unaffected. However, in contrast, brain-derived neurotrophic factor mRNA is reliably decreased, thereby suggesting that ethanol-induced damage of the septohippocampal system may at least partially result from an ethanol-induced decrease in hippocampal brain-derived neurotrophic factor expression.

Short-term memory impairment following chronic alcohol consumption in rats

Abstract Prolonged alcohol consumption (20 weeks), concomitant with adequate nutrition, was found to result in a residual short-term memory deficit after a 2-month alcohol-free period. Alcohol was administered in a liquid diet containing 35–37% ethanol-derived calories. One control group was pair-fed the same diet, except that sucrose was isocalorically substituted for ethanol. A second control group received pelleted laboratory food. After a 60-day alcohol-free period, short-term memory was assessed by training the rats on a discrete-trial temporal-alternation task in which bar presses were reinforced on alternate trials. Performance of the alternation problem was evaluated under conditions of short (20 sec) and long (50 sec) between-trial retention intervals. Although alcohol-treated-rats were relatively unimpaired when the retention interval was short, they were severely impaired with the long retention interval. In addition, the performance of alcohol-treated rats was severely disrupted when a distractor task was introduced during the short retention interval. The results were discussed relative to the similar short-term memory impairments of chronic alcoholic and alcoholic Korsakoff patients.

Ethanol-induced alterations in the expression of neurotrophic factors in the developing rat central nervous system.

Neonatal rats were exposed to ethanol throughout gestation, or during the early postnatal period (postnatal days 4-10 (P4-10)), and enzyme-linked immunoabsorbent assays were subsequently conducted in order to assess nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) protein content in hippocampus, septum, cortex/striatum and cerebellum. These determinations revealed that following prenatal ethanol treatment, there were significant ethanol-induced increases in NGF in P1 cortex/striatum, but no changes in any of the three neurotrophic factors (NTFs) in the other brain regions. Cortex/striatal NGF protein returned to control levels by P10. Following early postnatal exposure, BDNF was elevated in hippocampus and cortex/striatum (assessed on P10), and NGF was also enhanced in cortex/striatum at this age. Hippocampal and cortex/striatal BDNF returned to control levels by P21, but cortex/striatal NGF levels remained enhanced at this age. This NTF did not differ in ethanol and control animals by P60, however. The possible significance of elevated levels of NTFs as a function of ethanol exposure is discussed, and it is speculated that while such alterations could play a protective role, increases in these substances during critical developmental periods could also prove to be deleterious, and could even contribute to certain of the neuropathologies which have been observed following developmental ethanol exposure.

Modulation of ethanol neurotoxicity by nerve growth factor.

Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.