Effects of pinealectomy and melatonin on the retrograde degeneration of retinal ganglion cells in a novel model of intraorbital optic nerve transection in mice

The effects of pinealectomy and of intraperitoneally administered melatonin on the retrograde degeneration of retinal ganglion cells (RGCs) were examined in a novel model of optic nerve (ON) transection in C57BL/16J mice. RGCs were prelabeled with the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (Di-I), and the ON was cut inside the orbital cavity 7 days later. The degree of RGC injury was assessed by counting viable Di-I labeled RGCs in various locations of the retina. In unlesioned control eyes, a mean ganglion cell density of 1,891 +/- 30/mm2 (mean +/- S.E.M.) was determined. The cell density markedly declined at 14 days after axotomy (295 +/- 9 cells/mm2; 15.6% of contralateral). Sham-pinealectomy did not influence the density of RGCs at 14 days after ON transection (382 +/- 37 cells/mm2). In pinealectomized animals, on the other hand, the RGC number was significantly reduced as compared with untreated and sham-pinealectomized animals (91 +/- 33 RGCs/mm2). The effect of pinealectomy was reversed after i.p. administration of melatonin (4 mg/kg bw bolus followed by continuous infusion of 8 mg/kg bw/day) (286 +/- 27 cells/mm2). In nonpinealectomized animals, on the contrary, i.p. melatonin did not influence the RGC density (344 +/- 20 cells/mm2). The present results suggest that endogenous melatonin prevents the delayed degeneration of adult central nervous system (CNS) neurons in vivo, and that exogenous substitution of melatonin may be useful to protect injured neurons against cell death under conditions of melatonin deficiency, e.g. in the aged brain, when melatonin synthesis and secretion have decreased.     

General design principle for scalable neural circuits in a vertebrate retina

Unlike mammals, fish continue to grow throughout their lives, to increase the size of their eyes and brain, and to add new neurons to both. As a result of visual system growth, the ability to detect small objects increases with the age and size of the fish. In addition to the birth of new retinal ganglion cells (RGCs), existing cells increase the size of their dendritic arbors with retinal growth. We have used this system to learn design principles a vertebrate retina uses to construct its neural circuits, and find that the size of RGC arbors changes with retina and eye size according to a power law with an exponent close to 1/2. This power law is expected if the retina uses a strategy that, independent of eye size, simultaneously optimizes both the accuracy with which each RGC represents light intensity and the image spatial resolution provided to the fish's brain.     

Expression of an intrinsic growth strategy by mammalian retinal neurons.

Postnatal cat retinal ganglion cells (RGCs) were retrogradely labeled with fluorescent microspheres, dissociated from the retina using a peeling procedure, and monitored in cell culture with a time-lapse video microscopy system. The spatial patterns formed by the growing neurites were analyzed using conventional and fractal measures (Hausdorff dimension, H) of their extent and complexity. The results presented were obtained from the arborizations formed by the neurites of 48 labeled and isolated ganglion cells growing separate from each other and separate from a feeder layer of astrocytes. Cells were obtained from animals when the RGCs were postmitotic and after dendritic differentiation in vivo at age 0-1 week (4/48), 2-5 weeks (35/48), or 6-8 weeks (9/48). By 48 hr after plating, the number of surviving labeled RGCs was reduced to 22-28% of its initial value. After removal of all processes and isolation in vitro, these RGCs expressed neurite patterns strikingly similar to those seen in the intact retina, although the RGCs had been deprived of potential cues from the intact retina and target tissue. Self crossings of the growing neurites were rare (less than 0.5%, 20 cells, n = 2500 neurites). Calculation of the Hausdorff dimension, a metric for the space-filling capacity of the neurite patterns, revealed that after 3-day culture 77% (n = 56) of the RGCs achieved relatively uniform coverage of territory (1.6 less than H less than 1.9). This coverage was independent of the number of interbranchpoint segments and/or the total neurite length of a particular neurite pattern. A sample of dendritic arbors from RGCs in intact retina yielded similar values for the Hausdorff dimension (H = 1.73, SD = 0.12, n = 18, range 1.54-1.94). These results reveal that a mammalian central nervous system neuron, for at least 8 postnatal weeks, has the intrinsic capacity for reexpression of in vivo structure characteristic of that cell type in the absence of interaction with neighboring neurons, afferent input, and target tissue. These neurons exhibit stereotyped growth resulting in uniform coverage of a restricted territory by the strategic selection of the length, location, and orientation of interbranchpoint segments.     

Blockade of electrical activity promotes the death of mammalian retinal ganglion cells in culture.

During the first 2 postnatal weeks, up to 50% of the ganglion cells in the mammalian retina normally die. Natural cell death may result from several factors, and electrical activity has been proposed as one critical element. Recent experiments in vivo using intraocular injection of tetrodotoxin (TTX) have suggested that competition for survival between ganglion cells from the two eyes is mediated by their degree of neuronal activity. In addition, the level of activity of afferents to the ganglion cells has been postulated to be an important variable in determining their survival. To investigate the mechanism of cell death engendered by altered activity, I studied the effect of electrical blockade with TTX (to block sodium channels and thus action potentials) or low Ca/high Mg (to block transmitter release and hence synaptic activity) on individual neurons in vitro. For this purpose, identified retinal ganglion cells (RGCs) from postnatal rats were maintained in culture. Unlike the previous in vivo experiments, this approach permitted the exact concentration of each agent to be controlled and the electrical activity of the RGCs to be recorded. In cultures from animals of postnatal day 2-10 (P2-10), 1 microM TTX or 0.2 mM Ca/20 mM Mg resulted in the death of about 50% of the RGCs, representing those cells that had displayed spontaneous electrical activity, but did not affect RGCs that lacked activity. However, the death of RGCs with spontaneous activity from P11-13 animals was not influenced by these drugs. These findings suggest that during a critical period of development neurons become dependent upon electrical activity, and the cessation of this activity can result in their death. In addition, conditioned medium, collected from cultures lacking TTX, rescued from death a large proportion of TTX-treated RGCs. Thus, the critical element for survival may represent modulation of a trophic factor related to the level of activity rather than electrical activity itself. Since, in vivo, natural cell death occurs in neurons of similar type and age, and in the same proportion as that induced by the artificial blockade of electrical activity in culture, these findings may be germane to the mechanism of natural cell death in the retina.     

Substance P-containing ganglion cells become progressively less detectable during retinotectal development in the frog Rana pipiens.

Substance P-like immunoreactivity (SPLI) was immunohistochemically analyzed in the retinae and optic tecta of Rana pipiens embryos and tadpoles between stages 25 of Shumway (S25) and XXV of Taylor and Kollros (TKXXV). A population of retinal ganglion cell (RGC) somata display SPLI. The number of labeled cell bodies increases in proportion and staining intensity between S25 and TKX and progressively decreases toward the end of metamorphosis. At TKXXV, only occasional cells in the periphery of the retina displaying SPLI can be observed in the RGC layer, heralding the adult condition, in which SPLI can only be seen rarely in occasional RGCs. An increasing proportion of optic nerve axons display SPLI from S25 through TKXVI, decreasing progressively thereafter toward the end of the larval period. Concurrently, SPLI appears for the first time in the superficial tectal neuropil between TKIII and TKV, with progressively increasing staining intensity and in a discrete lamina previously shown to contain retinofugal terminals in the adult. These observations corroborate inferences from previous studies indicating the existence of populations of peptidergic RGCs that terminate within precisely restricted synaptic loci in the tectum and presumably perform different functional operations in the adult. Previous observations, however, necessitated various experimental manipulations involving injuries to the visual system in order to demonstrate neuroactive peptide-like immunoreactivity in RGCs, thus allowing the possibility of posttraumatic expression of anomalous peptide phenotypes that may not reflect normal features of RGCs. The present study eliminates this variable and provides further evidence of the existence of peptidergic RGCs.     

Neuroprotective effect of melatonin against hypoxia‐induced retinal ganglion cell death in neonatal rats

The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) concentrations, expression of vascular endothelial growth factor receptors, Flt‐1 and Flk‐1, release of cytochrome c from mitochondria, and caspase‐3 expression were examined in the retinas of 1‐day‐old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt‐1 and Flk‐1 and the tissue concentration of LPO, TNF‐α, and IL‐1β were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF‐α and IL‐1β expression was specifically located on microglial cells, whereas Flt‐1 and Flk‐1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase‐3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti‐inflammatory, and anti‐apoptotic effects. Melatonin suppressed Flt‐1 and Flk‐1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity.     

Ethanol induces cell death and cell cycle delay in cultures of pheochromocytoma PC12 cells

Animal models have clearly established that ethanol exposure can deplete neurons in the developing nervous system. However, the mechanism by which ethanol reduces cell number is unclear. In our study, cultures of pheochromocytoma cells, a neuronal-like cell line, were maintained in media, which supported cell proliferation. Although cell numbers continued to increase in the presence of ethanol, this increase was partially inhibited by ethanol exposure. This inhibitory effect was concentration and duration dependent. Cell proliferation was still partially inhibited after removal of ethanol, but this inhibition was temporary and disappeared after a 24-hr recovery period in ethanol-free conditions. Further study indicated that ethanol partially inhibited the increase in cell numbers by two mechanisms: (1) studies with vital stains indicated that ethanol induced cell death; (2) experiments using synchronized pheochromocytoma cell cultures showed that ethanol can induce cell cycle delay, thereby lengthening the doubling time of the cells. Analysis by flow cytometry indicated that with ethanol exposure, the cells accumulated in the G1 phase of the cell cycle. Our results suggest that in the developing nervous system, ethanol may limit the numbers of proliferating, neuronal precursor cells by two simultaneous mechanisms, cell death and cell cycle delay.     

Palmitic acid triggers cell apoptosis in RGC-5 retinal ganglion cells through the Akt/FoxO1 signaling pathway

Hallmarks of the pathophysiology of glaucoma are oxidative stress and apoptotic death of retinal ganglion cells (RGCs). Lipotoxicity, involving a series of pathological cellular responses after exposure to elevated levels of fatty acids, leads to oxidative stress and cell death in various cell types. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O1 (PI3K/Akt/FoxO1) pathway is crucial for cell survival and apoptosis. More importantly, FoxO1 gene has been reported to confer relatively higher risks for eye diseases including glaucoma. However, little information is available regarding the interaction between FoxO1 and RGC apoptosis, much less a precise mechanism. In the present study, immortalized rat retinal ganglion cell line 5 (RGC-5) was used as a model to study the toxicity of palmitic acid (PA), as well as underlying mechanisms. We found that PA exposure significantly decreased cell viability by enhancing apoptosis in RGC-5 cells, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. PA also induced a remarkable increase in reactive oxygen species and malondialdehyde. Moreover, PA significantly decreased the level of phospho-Akt and phospho-FoxO1 in cells. Finally, shRNA knockdown and plasmid overexpression studies displayed that downregulation of Akt protein or upregulation of FoxO1 protein augmented cell death, while knockdown of FoxO1 or overexpression of Akt1 abolished PA-induced cell death. Collectively, our results indicated that PA-induced cell death is mediated through modulation of Akt/FoxO1 pathway activity.

     

Fetal alcohol exposure and temporal vulnerability: regional differences in cell loss as a function of the timing of binge-like alcohol exposure during brain development

This study was conducted to determine the temporal and regional vulnerability of the brain as a function of exposure to alcohol during brain development. Our goal was to manipulate the timing of alcohol exposure and assess the relative risk of cell loss in two different brain regions. Groups of timed pregnant Sprague-Dawley rats received binge-like alcohol exposure during either the first 10 days (first-trimester equivalent) or second 10 days of gestation (second-trimester equivalent), or the combination of first- and second-trimester equivalents for prenatal treatments. Offspring from some of the animals exposed to alcohol during the combined first- and second-trimester equivalent were reared artificially from postnatal days (P) 4 through 9 (part of the third-trimester equivalent) and also received binge-like alcohol during this period, producing animals that were exposed to alcohol during all three trimesters equivalent. Offspring from untreated dams were also reared artificially and received alcohol from only P4-9, thus creating animals that were exposed to alcohol only during part of the third-trimester equivalent. All pups were perfused on P10. Appropriate controls (nutritional and normally reared) were matched to every alcohol treatment combination. Peak blood alcohol concentrations were not different among the treatment groups for a given sampling time. Total cell numbers in the cerebellum (Purkinje and granule cells) and the olfactory bulb (mitral and granule cells) were estimated by the unbiased stereological technique, the optical disector. In terms of temporal vulnerability, alcohol exposure during the equivalent of all three trimesters resulted in a greater reduction in cerebellar Purkinje cell numbers compared with exposure to alcohol during the third-trimester equivalent, whereas both groups had a significant reduction in cell number compared with all other timing groups. Cerebellar granule cell number was reduced after alcohol exposure during all three trimesters equivalent, compared with all other timing groups. Alcohol exposure during the third-trimester equivalent resulted in a decrement in the number of olfactory bulb mitral cell numbers compared with all other groups, but there were no differences among the timing groups in numbers of olfactory bulb granule cells. When the cell loss in the two regions was compared within each alcohol treatment group to determine the relative regional vulnerability, the primary salient finding was that cerebellar Purkinje cells were more vulnerable to alcohol-induced loss subsequent to exposure during all three trimesters equivalent. No other regional differences were detected. These results extend earlier findings by showing that alcohol exposure during different periods of brain development results in regional differences in cell loss as a function of the timing of alcohol exposure during brain development and illustrate the variability of alcohol-induced neuronal loss.     

Increased preference for ethanol in the infant rat after prenatal ethanol exposure, expressed on intake and taste reactivity tests

BACKGROUND: Previous studies have shown that prenatal exposure during gestational days 17 to 20 to low or moderate doses of ethanol (1 or 2 g/kg) increases alcohol intake in infant rats. Taking into account that higher consumption does not necessarily suggest a preference for alcohol, in the present study, the hedonic nature of the prenatal experience was analyzed further with the use of a taste reactivity test. METHOD: General activity, wall climbing, passive drips, paw licking, and mouthing in response to intraoral infusions of alcohol, water, and a sucrose-quinine solution (which resembles alcohol taste in rats) were tested in 161 preweanling 14-day-old rat pups that were prenatally exposed to 0, 1, or 2 g/kg of alcohol during gestational days 17 to 20. Consumption of those substances was measured during the taste reactivity test and on postnatal day 15. RESULTS: Pups that were prenatally exposed to both doses of ethanol displayed lower levels of general activity and wall climbing than controls in response to ethanol. Infant rats that were treated prenatally with both doses of ethanol showed higher intake of the drug and also more mouthing and paw licking in response to ethanol taste. Only pups that were exposed to the higher ethanol dose in utero generalized those responses to the sucrose-quinine compound. CONCLUSIONS: These results seem to indicate that for the infant rat, the palatability of ethanol is enhanced after exposure to the drug during the last days of gestation.     

Chronic ethanol treatment and withdrawal alter ACPD-evoked calcium signals in developing Purkinje neurons

BACKGROUND: Alcohol exposure during human fetal development can result in fetal alcohol syndrome, a condition characterized by central nervous system dysfunction. Detailed studies in animal models of fetal alcohol syndrome show that the cerebellar region is particularly sensitive to alcohol exposure during early development; however, the cellular mechanisms underlying the alcohol sensitivity of the immature cerebellum are poorly understood. METHODS: Primary neuronal cultures of cerebellar cells were prepared from embryonic day 20 rat pups. Cultures were exposed to ethanol (33 mM; 150 mg/100 ml) during the main period of morphological development of the Purkinje neurons, from 6 to 17 days in vitro. After the ethanol treatment, the response of Purkinje neurons to the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 microM) was examined in parallel fura-2 Ca2+ imaging and current-clamp experiments. In an additional set of experiments, ethanol-treated cultures were allowed to withdraw from ethanol overnight before recordings were performed. RESULTS: In Ca2+ imaging studies, the mean peak amplitude of ACPD-evoked Ca2+ signals was depressed in the dendritic region of chronic ethanol-treated Purkinje neurons compared with control neurons (p < 0.05, unpaired t test), whereas there was no apparent difference in the somatic region. In contrast, peak ACPD-evoked Ca2+ signals were enhanced in both the somatic and dendritic regions of withdrawn Purkinje neurons compared with control neurons. Parallel current-clamp studies showed no consistent effect of chronic ethanol treatment or ethanol withdrawal on the membrane response to ACPD. CONCLUSIONS: These results show that prolonged ethanol exposure and early withdrawal lead to alterations in mGluR-evoked Ca2+ signaling in cerebellar Purkinje neurons. Metabotropic GluRs in the Purkinje neuron play important roles in cerebellar development and function, suggesting that alterations of mGluR signaling pathways by ethanol may play a key role in the actions of ethanol on the developing cerebellum.     

Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells.

Optic nerve transection in adult rats results inthe death of approximately 50% of the axotomized retinal ganglion cells (RGCs)by 1 week and nearly 90% by 2 weeks after injury. The capacity of brain-derivedneurotrophic factor (BDNF) to prevent this early, severe loss of RGCs wasinvestigated in vivo by intravitreal injections of BDNF [5 micrograms in 5microliters of bovine serum albumin/phosphate-buffered saline (BSA/PBS)] orvehicle (5 microliters of BSA/PBS). Using quantitative anatomical techniques, weshow that (i) all RGCs survived 1 week after a single injection of BDNF at thetime of axotomy. (ii) RGC densities decreased in the BDNF-treated retinas by 2weeks but remained significantly greater than in the untreated controls. (iii)An enhanced RGC survival was obtained with single injections of BDNF from 6 daysbefore to 5 days after axotomy. (iv) Repeated injections resulted in greaternumbers of surviving RGCs, an effect that declined to undetectable levels by 6weeks. (v) There were indications for an endogenous local source of trophicsupport whose expression was triggered by ocular injury, particularly to theanterior part of the eye. (vi) With multiple BDNF injections, there was profuseaxonal sprouting around the optic disc. This remarkable intraretinal growth wasnot, however, reflected in increased RGC innervation of the peripheral nervegrafts, which are known to facilitate regeneration when used as optic nervesubstitutes.     

Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma

Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal ganglion cells (RGCs) and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. We evaluated the effects of NGF eye drops in a rat model of glaucoma. We also treated 3 patients with progressive visual field defects despite IOP control. Glaucoma was induced in rats through injection of hypertonic saline into the episcleral vein. Initially, 2 doses of NGF (100 and 200 μg/mL) were tested on 24 rats, and the higher dose was found to be more effective. Glaucoma was then induced in an additional 36 rats: half untreated and half treated with 200 μg/mL NGF QID for 7 weeks. Apoptosis/survival of RGCs was evaluated by histological, biochemical, and molecular analysis. Three patients with advanced glaucoma underwent psychofunctional and electrofunctional tests at baseline, after 3 months of NGF eye drops, and after 3 months of follow-up. Seven weeks of elevated IOP caused RGC degeneration resulting in 40% cell death. Significantly less RGC loss was observed with NGF treatment (2,530 ± 121 vs. 1,850 ± 156 RGCs/mm²) associated with inhibition of cell death by apoptosis. Patients treated with NGF demonstrated long lasting improvements in visual field, optic nerve function, contrast sensitivity, and visual acuity. NGF exerted neuroprotective effects, inhibiting apoptosis of RGCs in animals with glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF improved all parameters of visual function. These results may open therapeutic perspectives for glaucoma and other neurodegenerative diseases.     

Effects of Ethanol Exposure during the Third Trimester Equivalent on Neuron Number in Rat Hippocampus and Dentate Gyrus

An artificial rearing procedure was used to expose neonatal rats to a formula containing 3.74% ethanol during postnatal days 4 through 10. This treatment produced a mean blood ethanol concentration of 379.8 +/- 17.3 mg/dl. When the pups were killed on the afternoon of postnatal day 10, brain weight to body weight ratio in the ethanol-exposed rats was reduced 22.4% and 21.5% compared to suckle and pair-fed controls, respectively. Ethanol exposure also resulted in a 16% reduction of neurons in hippocampal field CA4, compared to controls, but did not produce deficits in fields CA1 or CA3. There was also a 10% increase in the number of neurons (a population of cells in the midst of a proliferative phase at the time of the exposure) in the granule cell layer of the dentate gyrus. The ethanol exposure did not affect cell size in any of the four neuron populations measured. These results suggest, that within the dose and timing parameters examined, ethanol exposure during the third trimester equivalent appears to be preferentially harmful to specific populations of developing neurons.     

Growth hormone expression and neuroprotective activity in a quail neural retina cell line

We have previously shown that growth hormone (GH) is produced within cells of the chick embryo retina where it appears to act as an autocrine/paracrine anti-apoptotic factor in the regulation of programmed cell death during retinal development. These investigations were carried out on cultured chick embryo retinal ganglion cells (RGCs) as well as on the chick embryo retina in ovo, using GH protein knock-down by immunoneutralization. We have now investigated the putative neuroprotective actions of GH using a quail embryo neural retina cell line (QNR/D) treated with GH siRNA to silence the local synthesis of GH. We now show that knock-down of GH by gene silencing in cells of this cultured embryonic neural retina cell line, using NR-cGH-1 siRNA, correlates with the increased appearance in the cultures of cells with apoptotic nuclear morphology. This result is consistent with our previous results using protein knock-down by immunoneutralization. We thus validate, using different technology and a different culture system, our contention that GH, produced locally by cells of the neural retina acts in an autocrine or paracrine manner to regulate cell survival in the retina.     

Ethanol withdrawal influences survival and morphology of developing rat hippocampal neurons in vitro

BACKGROUND: Previous studies in this laboratory have shown that, like their counterparts in vivo, fetal rat hippocampal pyramidal neurons in culture develop abnormally small dendritic arbors when exposed to ethanol. This study asked whether ethanol's inhibitory effects on dendritic development differ when the duration of ethanol exposure and timing of withdrawal are varied to correspond with early versus later stages of development and whether ethanol withdrawal influences survival of these neurons. METHODS: We compared neurons exposed continuously for 6 or 14 days to ethanol (70 mM) with neurons transferred from ethanol-containing medium to control medium either 1 day after adding ethanol (before dendrites elongated) or 6 days after adding ethanol (after dendrites began elongating). We then performed morphometric and cell density analyses at 6 and 14 days using digital images of neurons immunostained with microtubule-associated protein 2 (MAP2) to visualize dendrites. RESULTS: Continuous exposure to ethanol decreased the length and number of dendrites formed but had no effect on neuron survival compared with controls without ethanol. Dendritic length was less inhibited when ethanol was withdrawn after 1 day, but the number of dendrites per cell was unchanged compared with neurons continuously exposed to ethanol. Withdrawal from ethanol at 1 day slightly enhanced the survival of neurons assessed at 14 days compared with neurons in control medium and with neurons exposed continuously to ethanol. In contrast, withdrawal from ethanol at 6 days severely decreased the number of neurons at 14 days. CONCLUSIONS: These results suggest that dendrites can achieve normal length when ethanol exposure is limited to only 1 day and withdrawal occurs before dendrites begin elongating. However, a persistent reduction in dendrite number results in smaller overall dendritic arbor size. Although continuous exposure to ethanol has little effect on neuron survival in these cultures, and exposure limited to 1 day followed by withdrawal can be neuroprotective against cell death associated with increased time in culture, longer exposure before withdrawal can trigger cell death.     

Effects of Prenatal Ethanol Exposure on Parvalbumin-Expressing GABAergic Neurons in the Adult Rat Medial Septum

Exposure of human fetuses to ethanol often results in the fetal alcohol syndrome. Animal models of fetal alcohol syndrome have been developed and used to examine the consequences of prenatal ethanol exposure on the central nervous system. The objective of this study was to determine the long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA+) GABAergic neurons of the rat medial septum. Pregnant Long-Evans rats were maintained on 1 of 3 diets from gestational day 0 to 21: an ethanol-containing liquid diet in which ethanol accounted for 35% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, or a lab chow control diet. Offspring were killed on postnatal day 60, and their brains were prepared for parvalbumin immunocy to chemistry. Female rats exposed to the ethanol-containing diet during gestation had 42% fewer PA+ neurons in the medial septum and reduced PA+ cell density when compared with female rats exposed to the sucrose diet. Ethanol females also had fewer PA+ neurons per unit volume than sucrose females. Male rats exposed to ethanol did not display a similar reduction in PA+ neurons or density. No effect of prenatal diet was found on the area or volume of the medial septum, nor were cell diameters affected. As such, prenatal exposure to ethanol seems to reduce permanently the number of PA+ neurons in the female rat medial septum without affecting area, volume, or neuronal size. Functional implications and possible relations to the fetal alcohol syndrome are discussed.