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Biomedical subjects

S S Suarez

Publications and source records attributed to S S Suarez.

At least 19 recordsLinked to original sources

Hyperactivated motility in sperm.

Hyperactivation is a movement pattern seen in sperm at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated sperm appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, the two events are regulated by somewhat different pathways.

Animals↗

Hyperactivation of mammalian sperm.

Mammalian sperm commonly show hyperactivated motility just before fertilization. The movement of hyperactivated sperm appears different in fluids of different viscosity and elasticity and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Hyperactivation may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none have yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, divergent pathways regulate the two events.

Animals↗

Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir.

Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.

Acrosome↗

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.

1-Methyl-3-isobutylxanthine↗

Carbohydrate-mediated formation of the oviductal sperm reservoir in mammals.

Mammalian sperm are trapped in a reservoir in the oviduct until ovulation is imminent. Then, they are gradually released, such that a few meet the oocytes as they enter the ampulla of the oviduct. In the three eutherian species studied to date, sperm are trapped in the reservoir by carbohydrate-mediated binding to the oviductal mucosa. Evidence indicates that a molecule on the surface of the plasma membrane overlying the acrosome binds to a carbohydrate moiety on the surface of the oviduct. While sperm remain bound, they appear to be protected from degradation. When sperm become capacitated, they lose binding affinity for the oviductal mucosa. The mechanism initiating capacitation in the reservoir is unknown; however, it must be tied to the hormonal signalling of ovulation. Hyperactivated motility may assist sperm in pulling off from the mucosal surface as binding affinity declines. The function of the reservoir appears to be to prevent polyspermy and ensure fertilization by providing a small number of sperm in the proper physiological condition for fertilization at the time the oocytes enter the oviduct.

Animals↗

Hyperactivation of mammalian spermatozoa: function and regulation.

Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry. Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified. There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct. Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. The process may also involve increases in intracellular cAMP, which at least is required to support motility in general. Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways.

Animals↗

Three-dimensional structure of the Golgi apparatus in mouse spermatids: a scanning electron microscopic study.

In this study, the three-dimensional organization of the Golgi apparatus in mouse spermatids was elucidated by preparing testicular tissue with the osmium-DMSO-osmium method and examining it by stereo-scanning electron microscopy. The cis-most saccule was found to be a regular network of anastomotic membranous tubules covered by a single cisterna of ER. The trans-Golgi network was seen to be composed of irregular saccules perforated by pores at the edge. It appears that the anastomosing trans-Golgi network breaks down into strings of connected vesicles which arise from the edge of the saccules during the cap phase of spermiogenesis. Many apparently individual vesicles seen in thin sections through the trans-Golgi network are actually joined in continuous strings. This was the first time that these structures could be visualized directly without three-dimensional image reconstruction. By correlating the morphology of the Golgi apparatus with the stage of acrosome formation, the Golgi cisternae were found to change dynamically in a cis-trans direction from fenestrated saccules to continuous strings of vesicles, which finally dissipated as transport vesicles at the trans aspect. This suggests that the hypothetical model of cisternal maturation, which dictates that cargo moves through the Golgi apparatus without leaving the cisternal lumen and the secretion occurs by progressive maturation of the Golgi cisternae as they move in the cis-trans direction, may be applicable to acrosome formation.

Acrosome↗

Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes Lewis-a trisaccharide.

Sperm binding to oviductal epithelium produces a reservoir in vivo that may serve to maintain sperm fertility and provide sperm for fertilization when ovulation occurs. Previously, it was determined that bull sperm binding could be blocked by fucoidan and its component fucose; furthermore, treatment of epithelium with fucosidase prevented binding. The present study was conducted to further characterize binding. Because fucose would probably be present on the epithelium as part of oligosaccharide moieties of glycoproteins and/or glycolipids, competitive binding inhibition activity was tested for fucose in five linkages commonly found in oligosaccharides. Binding inhibition was assayed by determining the concentration of motile, frozen/thawed sperm bound to fresh epithelial explants in the presence of test inhibitors. Initially, 5 monosaccharides were tested at 30 mM (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose), and only fucose significantly reduced sperm binding compared to vehicle control (p = 0.03). Of the oligosaccharides tested (lacto-N-fucopentaose I, 3-fucosyllactose, Lewis-X, Lewis-a, and GlcNAcbeta1-4[Fucalpha1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.

Animals↗

Cultured postnatal rat septohippocampal neurons change intracellular calcium in response to ethanol and nerve growth factor.

Ethanol exposure affects cellular mechanisms involved in the regulation of calcium (Ca2+) homeostasis. Neurotrophins, such as nerve growth factor (NGF), stabilize intracellular Ca2+([Ca2+]i) during a variety of neurotoxic insults. In this study, changes in [Ca2+]i during treatment with ethanol and NGF were measured at the cell body of neurons using the Ca2+ indicator indo-1. Cultured postnatal day-of-birth (P0) septohippocampal (SH) neurons that were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), increased [Ca2+]i in response to ethanol. This response was dose-related. P0 SH neurons treated with NGF had lower [Ca2+]i than neurons withdrawn from NGF, implying that NGF may modulate Ca2+ homeostasis in these neurons. NGF also prevented the dose-related increase in [Ca2+]i in ethanol-treated SH neurons. The SH neurons increased [Ca2+]i when they were stimulated with 30 mM potassium chloride (KCl). Ethanol inhibited the potassium-stimulated change in [Ca2+]i but the combination of ethanol and NGF caused [Ca2+]i to increase with 100 mg% and 400 mg% ethanol and to decrease to a lower level with 200 mg% ethanol. These data were compared to data from previously published similar aged medial septal (MS) neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Clin. Exp. Res. 20 (1996) 1385-1394) and with embryonic gestational day 21 (E21) SH neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Brain Res. 729 (1996) 176-189). Differences in [Ca2+]i responses were observed in ethanol and NGF-treated postnatal SH neurons compared with P0 MS neurons and E21 SH neurons. Of these differences, most occurred during the combined treatment with ethanol and NGF compared with either treatment alone.

Animals↗

Distribution of mucus and sperm in bovine oviducts after artificial insemination: the physical environment of the oviductal sperm reservoir.

There is a sperm reservoir in the caudal oviduct of cattle and other mammals. We had observed trapping of sperm by mucus produced by explants of bovine oviductal epithelium in vitro; therefore, we used techniques designed for preserving mucus and luminal dimensions to determine whether mucus is associated with the reservoir in vivo. Heifers were synchronized by prostaglandin injections and inseminated during estrus. Oviducts on the side of ovulation were surgically removed either 8-10 h after insemination (preovulatory) or 50-55 h after insemination (postovulatory). Segments (1 cm) taken from the uterotubal junction (UTJ), caudal 3 cm of isthmus, and mid ampulla were snap frozen. Frozen sections were coated with collodion and postfixed in phosphate-buffered formaldehyde containing cetyl-pyridinium chloride. Sections were alternately stained with periodic acid-Schiff stain (PAS) or alcian blue/PAS. Most of the oviductal lumen was highly branched with passages that measured only a few microns across and were filled with mucus. In limited areas, the lumen opened to 100 microm across and was only lightly stained for mucus. Overall, the lumen was much narrower than in sections prepared by standard fixation and paraffin embedding. Sperm were found scattered throughout the lumen of the UTJ and isthmus, in both the narrow, deeply stained luminal areas and the wider, lightly stained areas. The numbers tapered off cranially, especially prior to ovulation. In conclusion, the combination of narrow passages and mucus would appear to impede sperm progress, contributing to the creation of a reservoir.

Alcian Blue↗

Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro.

Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca2+]i in spermatozoa still attached to OEC. In addition, [Ca2+]i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca2+]i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca2+]i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca2+]i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca2+]i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca2+]i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization.

Acrosome↗

Bovine sperm binding to oviductal epithelium involves fucose recognition.

Sperm binding to oviductal epithelium probably serves to form the isthmic sperm reservoir. This interaction of sperm and oviductal epithelium may involve species-specific carbohydrate recognition. We tested a series of carbohydrates and glycoproteins for inhibition of bovine sperm binding to oviductal epithelium in vitro. Explants of isthmic and ampullar epithelium were obtained from oviducts that had been surgically removed from preovulatory heifers. The explants were incubated (39 degrees C, 5% CO2) with fetuin, asialofetuin, ovalbumin, fucoidan, fucose, N-acetyl glucosamine, or N-acetyl glucosamine sulfate dissolved in a modified Tyrode's balanced salt solution, termed sperm-TALP (pH 7.4, 295 mOsm) for 10 min before frozen-thawed motile sperm obtained by swim-up were added. After 15 min, the explants were rinsed, and sperm binding density was evaluated. Oviductal explants treated with fucoidan (3 mg/ml; p < 0.001, n = 5) or fucose (31 mM, p < 0.01, n = 6) had reduced densities of bound sperm compared to the controls. Incubation of explants in increasing concentrations of fucose (4-62 mM) resulted in increased inhibition of sperm binding. Pretreating explants with fucosidase also reduced sperm binding (p < 0.001, n = 3) compared to that in controls containing the fucosidase inhibitor deoxyfuconojirimycin. The presence of fucosylated molecules on the surface of the oviductal epithelium was confirmed by labeling with fucose lectins from Ulex europeus and Lotus tetragonolobus. We conclude that fucose is involved in a specific interaction between bovine sperm and oviductal epithelium.

Animals↗

Calcium homeostasis in cultured embryonic rat septohippocampal neurons is altered by ethanol and nerve growth factor before and during depolarization.

Ethanol and nerve growth factor (NGF) affect the survival of septohippocampal (SH) neurons. The effect of ethanol and NGF on calcium (Ca2+) homeostasis in these neurons was investigated in this study. Changes in intracellular-free Ca2+ concentration ([Ca2+]i) were measured using indo-1 in cultured embryonic (E21) SH neurons before stimulation (basal) and during stimulation with 30 mM potassium cloride (KCl+). SH neurons were treated with 0, 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). NGF treatment decreased, while ethanol did not affect basal [Ca2+]i. The combination of ethanol and NGF treatment led to increases in basal [Ca2+]i. While [Ca2+]i was lower during stimulation with KCl+ following ethanol or NGF treatment, ethanol and NGF treatment together led to significantly greater increases or decreases in [Ca2+]i compared to similarly treated NGF neurons. Responses of SH neurons were compared to those of medial septal (MS) neurons. Changes in [Ca2+]i during treatment with ethanol and/or NGF were reduced in SH neurons compared with MS neurons. We conclude that changes in Ca2+ homeostasis can occur in SH neurons in the presence of ethanol and/or NGF. The changes following ethanol treatment are enhanced by NGF. By altering Ca2+ homeostasis, NGF may enhance the survival of SH neurons during ethanol-induced neurotoxicity.

Animals↗

Effect of capacitation on bull sperm binding to homologous oviductal epithelium.

Sperm binding to oviductal epithelium is thought to be an important mechanism regulating sperm reservoir formation in the oviduct. On the basis of evidence in the hamster, we hypothesized that capacitation affects release of bovine sperm, allowing them to fertilize. Oviducts were obtained from the ovulatory side of estrous Holstein heifers. The isthmic and ampullar epithelia were milked out and reduced to fragments, which formed everted vesicles (explants). Explants were placed in tissue culture wells in TALP medium and incubated at 39 degrees C in 5% CO2. Frozen-thawed sperm were prepared by swim-up in TALP and capacitated by incubation for 4 h in TALP with 20 micrograms/ml heparin (without glucose). Uncapacitated sperm were used immediately after dilution into capacitation medium. Within 2 h of surgery, sperm were added to the explants and incubated with them for 15 min. Sperm and explants were videotaped, and the tapes were analyzed to determine the numbers of sperm bound per surface area. ANOVA did not show a difference between the number of sperm bound/0.1 mm2 in the isthmus and ampulla (p > 0.05); however, an effect of capacitation was detected (p = 0.0015). Also, the percentage of capacitated sperm, determined by chlortetracycline labeling, was greater in sperm that remained free-swimming in the presence of explants than in the absence of explants (p = 0.001). In conclusion, capacitation appears to be involved in the release of bovine sperm from oviductal epithelium and therefore could enable sperm to leave the reservoir and fertilize oocytes.

Animals↗

Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro.

Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and at 0.5, 2, 4, and 6 h of incubation by ratio image analysis of fluorescent images captured at 405 nm and 490 nm. At each time point, [Ca2+]i was lower in motile spermatozoa attached to OEC than in free-swimming spermatozoa. [Ca2+]i in spermatozoa attached to Matrigel was lower than in free-swimming spermatozoa and was comparable to [Ca2+]i in spermatozoa attached to OEC only at 0.5 h incubation. Beyond 0.5 h of incubation, [Ca2+]i was higher in spermatozoa attached to Matrigel than in spermatozoa attached to OEC. These results indicate that spermatozoa with low [Ca2+]i might preferentially attach to OEC and to Matrigel, but that [Ca2+]i is maintained at this basal level only in spermatozoa attached to OEC. The reduced [Ca2+]i in spermatozoa associated with OEC may prevent premature capacitation and acrosomal exocytosis of spermatozoa stored in the isthmic sperm reservoir.

Acrosome↗

Cultured postnatal rat medial septal neurons respond to acute ethanol treatment and nerve growth factor by changing intracellular calcium levels.

Ethanol neurotoxicity results in the loss of neurons during the development of the nervous system. Nerve growth factor (NGF) can ameliorate the neurotoxic effects of ethanol (EtOH) in rat medial septal (MS) neurons. These experiments study the effects of EtOH and NGF on neuronal calcium (Ca2+) homeostasis in cultured postnatal day of birth (PO) rat MS neurons. Previously, we observed that EtOH and NGF modulate intracellular Ca2+ levels [Ca2+]i) in unstimulated and high potassium stimulated (30 mM KCl) cultured rat embryonic day 21 (E21) MS neurons (Webb et al., Brain Res 701:61-74, 1995). The purpose of the present study was to explore whether the effects of EtOH and NGF on Ca2+ homeostasis were altered by developmental stage. The hypotheses tested were the following: treatment with EtOH affects Ca2+ homeostasis in postnatal day of birth (PO) rat MS neurons by causing transient and persistent changes in [Ca2+]i; NGF modulates Ca2+ homeostasis in MS neurons by regulating [Ca2+]i; the action of NGF changes the response of MS neurons to EtOH, thus altering Ca2+ homeostasis; and that EtOH and/or NGF effects on Ca2+ homeostasis are developmentally regulated. Our results indicated that behaviorally relevant levels of EtOH caused a rapid transient increase in basal [Ca2+]i, whereas there was no effect of NGF on basal [Ca2+]i. Ethanol and NGF interacted, resulting in the lowering of [Ca2+]i. During stimulation with high K+, EtOH inhibited the change in [Ca2+]i. NGF partially ameliorated this effect of higher levels of EtOH, allowing [Ca2+]i to increase. NGF and the lowest level of EtOH potentiated the high K+ stimulated increase in [Ca2+]i. Ethanol and NGF effects on [Ca2+]i were different in the PO neurons compared with our previously published observations in E21 neurons. Therefore, these data suggest that EtOH neurotoxicity and NGF protection involve mechanisms that regulate neuronal Ca2+ homeostasis, and the magnitude of these effects depend on developmental stage.

Animals↗

Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization.

Ethanol and nerve growth factor (NGF) affect the survival of cholinergic neurons in the rat medial septum. To investigate whether calcium (Ca2+) homeostasis in these neurons is affected by ethanol or NGF treatment, changes in intracellular free Ca2+ concentration ([Ca2+]i) were studied in embryonic (E21) cultured medial septal neurons before stimulation (basal) and during stimulation with high potassium (K+). Changes in [Ca2+]i across time were measured in cultures of neurons treated without ethanol or with 100, 200, 400, or 800 mg% ethanol with NGF (+NGF) or without NGF (-NGF). Changes in [Ca2+]i were analyzed from fluorescence images, using indo-1. The effect of ethanol or NGF treatment was to reduce the rise in basal [Ca2+]i. The combination of ethanol and NGF treatment in +NGF neurons led to increases in basal [Ca2+]i with the greatest increase in basal [Ca2+]i occurring with 200 mg% ethanol. The effect of ethanol or NGF was to increase [Ca2+]i during stimulation with high K+. The greatest increases in [Ca2+]i occurred with 100 and 800 mg% ethanol. Together, ethanol and NGF treatment in +NGF-treated neurons led to significantly greater increases or decreases in K+ stimulated changes in [Ca2+]i compared to similarly treated -NGF neurons. We conclude that in medial septal neurons (before and during depolarization) changes in Ca2+ homeostasis occur in the presence of ethanol or NGF. The changes in [Ca2+]i following ethanol treatment are greater when NGF is present.

Animals↗