Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices.

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.

Prenatal ethanol exposure diminishes activity-dependent potentiation of amino acid neurotransmitter release in adult rat offspring.

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies suggest that these deficits are, in part, linked to neurochemical abnormalities that reduce the ability to sustain long-term potentiation (LTP) in hippocampal formation of adult offspring. One presynaptic component of LTP that manifests during the first half-hour after tetanic stimulation is an enhancement of amino acid neurotransmitter release. Given that the onset of enhanced neurotransmitter release correlates temporally with the decay of hippocampal LTP in prenatal ethanol-exposed offspring, we tested the hypothesis that prenatal ethanol exposure reduces tetanus stimulus-induced potentiation of electrically evoked amino acid release in hippocampal slices. Rat dams consumed 1 of 3 diets throughout gestation: (1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories) that produces a maternal peak blood ethanol concentration of 83 mg/dl; (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) Purina rat chow ad libitum. Hippocampal slices were prepared from adult offspring from each experimental diet group. Neither the amount of hippocampal slice tissue protein nor the incorporation of [3H]-D-aspartate (D-ASP) was affected by prenatal ethanol exposure. Furthermore, spontaneous efflux and electrically evoked D-ASP release were similar among the three diet groups. However, tetanus stimulus-induced potentiation of evoked D-ASP release in prenatal ethanol-exposed offspring was reduced to about one-third of the potentiation of D-ASP release observed in the control diet groups. These results suggest that prenatal ethanol exposure produces long-lasting deficits in the neurochemical mechanisms responsible for activity-dependent potentiation of amino acid transmitter release without affecting the synaptic machinery responsible for amino acid uptake, storage, and release.

Prenatal ethanol exposure alters the modulation of the gamma-aminobutyric acidA1 receptor-gated chloride ion channel in adult rat offspring.

We examined the effect of prenatal ethanol exposure on gamma-aminobutyric acid (GABA)-stimulated 36Cl- flux. Sprague-Dawley rat dams were fed either a liquid diet containing 5% ethanol, pair-fed an isocalorically equivalent 0% ethanol diet or rat chow ad libitum throughout gestation. Membrane vesicles were prepared from medial frontal cortex, cerebellum and hippocampal formation of adult offspring in each diet group. GABA-stimulated 36Cl- flux was not significantly affected by prenatal ethanol exposure in any of the three brain regions examined. Positive allosteric modulation of GABA-stimulated 36Cl- flux by flunitrazepam or alphaxalone, as well as negative modulation by FG-7142 or pregnenolone, were all diminished in medial frontal cortex of 5% ethanol diet offspring compared with both ad libitum and pair-fed control groups. In cerebellum, prenatal ethanol exposure attenuated the modulatory effects of both benzodiazepines, but did not affect neurosteroid modulation. In hippocampus, prenatal ethanol exposure enhanced the effects of flunitrazepam and alphaxalone, whereas negative modulatory effects were either decreased (FG-7142) or unchanged (pregnenolone). These results indicate that moderate ethanol consumption during gestation can produce long-lasting alterations in neuromodulatory influences on GABAA receptor-mediated inhibitory neurotransmission in adult offspring. In hippocampal formation, the heightened sensitivity to positive modulatory influences may contribute to synaptic plasticity deficits in fetal ethanol-exposed rat offspring. We speculate that these prenatal ethanol-induced changes may be either a consequence of differential GABAA receptor subunit expression or receptor uncoupling in different brain regions. Furthermore, offspring exposed to ethanol in utero may display differential sensitivities to benzodiazepines and possibly other centrally active therapeutic agents.

Flanking sequences for the human intercellular adhesion molecule-1 NF-kappaB response element are necessary for tumor necrosis factor alpha-induced gene expression.

The regulated expression of intercellular adhesion molecule-1 (ICAM-1) by cytokines such as tumor necrosis factor alpha (TNF-alpha) plays an important role in inflammation and immune responses. Induction of ICAM-1 gene transcription by TNF-alpha has previously been shown to be dependent upon a region of the ICAM-1 5'-flanking sequences that contains a modified kappaB site. We demonstrate here that this modified kappaB site alone is insufficient for induction of transcription by TNF-alpha. Site-directed mutagenesis of both the kappaB site and specific flanking nucleotides demonstrates that both the specific 5'- and 3'-flanking sequences and the modified kappaB site are necessary for TNF-alpha induction. Further, site-directed mutagenesis of this modified kappaB site to a consensus kappaB site allows it to mediate transcriptional activation in response to TNF-alpha, even in the absence of specific flanking sequences. Transcription through this minimal ICAM-1 TNF-alpha-responsive region can be driven by co-expression of p65, and the minimal response element interacts with p65 and p50 in supershift mobility shift assays. However, when in vitro transcription/translation products for the Rel proteins are used in an electrophoretic mobility shift assay, only p65 is capable of binding the minimal response element while both p50 and p65 bind a consensus kappaB oligonucleotide. Additionally, in the absence of the specific flanking nucleotides, the ICAM-1 kappaB site is incapable of DNA-protein complex formation in both electrophoretic mobility shift assay and UV cross-linking/SDS-polyacrylamide gel electrophoresis analysis. These results demonstrate the requirement for specific flanking sequences surrounding a kappaB binding site for functional transcription factor binding and transactivation and TNF-alpha-mediated induction of ICAM-1.

Pervanadate mimics IFNgamma-mediated induction of ICAM-1 expression via activation of STAT proteins.

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the epidermis plays a critical role in the regulation of cutaneous inflammation, immunologic reactions, and tissue repair. Transcriptional upregulation of ICAM-1 in response to interferon-gamma (IFNgamma) occurs through a palindromic response element pIgammaRE. pIgammaRE is homologous to IFNgamma-activated sequences, which bind to tyrosine phosphorylated members of the transcription factor family known as signal transducers and activators of transcription (STAT). The importance of tyrosine phosphorylation events in the STAT pathway led us to investigate the effect of the protein tyrosine phosphatase inhibitor, pervanadate, on ICAM-1 expression. We show that treatment of A431 cells and human keratinocytes with pervanadate stimulates protein complex formation on pIgammaRE in a time- and concentration-dependent manner. As demonstrated by mobility supershift assays, the pervanadate-stimulated complex is similar to the IFNgamma-stimulated complex and contains Stat1. Pervanadate treatment also led to an increase in overall protein tyrosine phosphorylation and phosphorylation of Stat1, as well as the subsequent increase in ICAM-1 mRNA and cell surface protein levels. These data show that pervanadate can mimic each step in the IFNgamma-mediated pathway leading to ICAM-1 expression, demonstrate the ability of a pharmacologic agent to bypass the standard cytokine-receptor interaction required for increased ICAM-1 expression, and emphasize the importance of protein tyrosine phosphatases and protein tyrosine kinases in mediating inflammatory responses in the skin.

Retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells.

The regulated expression of cell adhesion molecules (CAM) on endothelial cells is central to the pathogenesis of various inflammatory processes. Retinoic acid and synthetic derivatives have been demonstrated to exert antiinflammatory effects in cutaneous diseases. To determine modes of retinoid action in the modulation of inflammatory responses, we explored effects of all-trans-retinoic acid (t-RA) on the TNFalpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in cultured human dermal microvascular endothelial cells. Pretreatment with t-RA specifically prevented TNFalpha-induced VCAM-1 expression, but not ICAM-1 and E-selectin induction. t-RA significantly reduced VCAM-1-dependent T cell binding to TNFalpha-treated human dermal microvascular endothelial cells as well. This differential modulation of TNFalpha-induced CAM expression by t-RA was reflected at steady state mRNA levels and in nuclear run-on studies. In transcriptional activation studies, the TNFalpha-mediated activation of the human VCAM-1 promoter was inhibited after t-RA treatment, while the ICAM-1 promoter activation was unaffected, indicating that the selective inhibition of CAM expression is regulated in part at the level of gene transcription. Furthermore, the transcriptional inhibition by t-RA appears to be mediated by its effects upon the activation of NF-kappaB-dependent complex formation. Analysis of protein-DNA binding assays revealed marked inhibition of specific NF-kappaB-dependent binding to the tandem NF-KB sites of the VCAM-1 promoter, but not to the functional NF-kappaB motif of the ICAM-1 promoter. The specific inhibition of cytokine-mediated VCAM-1 gene expression in vitro may provide a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo.

Interferon gamma-dependent induction of human intercellular adhesion molecule-1 gene expression involves activation of a distinct STAT protein complex.

In response to interferon gamma (IFNgamma), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5' regulatory region palindromic response element, pIgammaRE, has been shown to confer IFNgamma-dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pIgammaRE forms a distinct complex with proteins from IFNgamma-treated human keratinocytes, termed gamma response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pIgammaRE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1alpha-like protein; however, non-ICAM-1 IFNgamma-responsive elements (REs) known to bind Stat1alpha homodimers fail to compete for GRF binding in EMSA, and pIgammaRE does not cross-compete with these REs that complex with homodimeric stat1alpha. The pIgammaRE x GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFNgammaREs complexed to homodimeric Stat1alpha. These findings indicate that a distinct complex containing a Stat1alpha-like protein mediates IFNgamma-induced ICAM-1 gene transcription and identifies a subset of IFNgamma-responsive genes that appear to be regulated by this complex.

Dose- and age-dependent effects of prenatal ethanol exposure on hippocampal metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis.

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal formation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP1) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP1 accumulation in 10- to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP1 accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP1 accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in spleen norepinephrine and lymphocyte [3H]dihydroalprenolol binding site number in genetically epilepsy-prone rats.

Noradrenergic neurotransmission plays an important role in normal immune reactivity. Genetically epilepsy-prone (GEPR-9) rats exhibit deficits in central noradrenergic systems and diminished plaque-forming cell responses following immunization in vivo. In the present study we examined the hypothesis that immunosuppression in GEPR-9 rats is associated with alterations in the splenic noradrenergic system. The content of norepinephrine (NE) in spleens of GEPR-9 age-matched nonepileptic Sprague-Dawley control rats was determined by high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC). In addition, we measured the number of beta-adrenergic receptors on splenic lymphocyte membranes of GEPR-9 and control rats using the beta-adrenergic receptor antagonist dihydroalprenolol ([3H]DHA). HPLC-EC analysis revealed that splenic norepinephrine content was significantly greater in GEPR-9 rats than in controls. Results from receptor binding studies indicated a 33% reduction in specific binding of [3H]DHA to splenic lymphocyte membranes of GEPR-9 rats. Saturation of binding studies revealed a significant decrease in the maximum number of [3H]DHA binding sites on splenic lymphocyte membranes from GEPR-9 rats. These results indicate that the noradrenergic system in GEPR-9 rat spleen is altered. Whether either or both of these changes contribute to reduced immune reactivity in GEPR-9 rats remains to be determined.

Perinatal hypothyroidism decreases hippocampal mossy fiber zinc density in rats.

The effect of perinatal hypothyroidism on hippocampal mossy fiber zinc density was examined in rats. Timed pregnant Sprague-Dawley rat dams were given water containing either 0.02% propylthiouracil (PTU) or vehicle from gestational day 18 until their litters were weaned on postnatal day 31. Hippocampal mossy fiber zinc density was reduced by 75% in both the dorsal and ventral hippocampal formation CA3 stratum lucidum region of 31-day-old PTU-treated rats compared to untreated controls. Perinatal hypothyroidism did not alter hippocampal tissue zinc concentration, indicating that the PTU-induced reduction in mossy fiber zinc was not a consequence of reduced hippocampal zinc concentration. At 120 days of age, 3 months after discontinuation of PTU treatment, hippocampal mossy fiber zinc density remained significantly reduced by 33-45% in PTU-treated rats compared to control. These data indicate that perinatal hypothyroidism causes a long-lasting reduction in hippocampal mossy fiber zinc density.

Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring.

The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal ethanol exposure decreases hippocampal NMDA-sensitive [3H]-glutamate binding site density in 45-day-old rats.

The effect of prenatal ethanol exposure on N-methyl-D-aspartate (NMDA)-sensitive [3H]-glutamate receptor binding site density was studied in rat brain. Pregnant Sprague-Dawley rats were fed a liquid diet containing 3.35% ethanol throughout gestation. This diet produced maternal peak blood ethanol levels of about 39 mg/dl eight hours after the administration of the liquid diet. Pair-fed dams received an isocalorically matched liquid diet and an ad lib lab chow group served as control for the paired feeding technique. At 45 days of age, offspring from each of the three diet groups were sacrificed and brain NMDA-sensitive [3H]-glutamate binding site density measured using in vitro radiohistochemical techniques. NMDA-sensitive [3H]-glutamate binding site density was reduced significantly by 19 to 29% in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation of fetal alcohol rats compared to pair-fed and ad lib controls. NMDA-sensitive [3H]-glutamate binding site density was not significantly different among the three groups in the ventral hippocampal formation, posterior neocortex, lateral entorhinal cortex or cerebellum. These results are consistent with our previous observations of a reduction in total [3H]-glutamate receptor binding site density in the dorsal hippocampal formation of fetal alcohol rats, as well as more recent electrophysiological observations of a decrease in the sensitivity of fetal alcohol hippocampal slices to NMDA.

Perinatal hypothyroidism decreases hippocampal 3H-vinylidene kainic acid binding in rats.

The effect of perinatal hypothyroidism on the number and distribution of hippocampal kainic acid binding sites was examined in rats. Timed pregnant Sprague-Dawley rat dams were given water containing either 0.02% propylthiouracil (PTU) or untreated water from gestational day 18 until their litters were weaned at postnatal day 31. The offspring were sacrificed at 31 or 120 days of age, blood samples collected and their brains frozen. In the 31-day-old rats, serum thyroxine, serum triiodothyronine, total body weight and whole brain weight all indicated that the PTU-treated rats were hypothyroid. Hippocampal kainic acid binding was analyzed in sections of dorsal and ventral hippocampal formation by in vitro 3H-vinylidene kainic acid (VKA) autoradiography. Compared to the untreated controls, specific 3H-VKA binding was reduced by 43% in the ventral hippocampal formation stratum lucidum of 31-day-old PTU-treated rats. Specific 3H-VKA binding was not different in the dorsal hippocampal formation. Saturation of 3H-VKA binding studies indicated that the decrease-induced by PTU treatment--in ventral hippocampal 3H-VKA binding was due to a reduction in the total number of 3H-VKA binding sites. At 120 days of age, 3 months after the cessation of the PTU treatment, serum thyroid hormone levels were not different than those of controls, indicating a recovery of thyroid hormone function after the perinatal PTU treatment. However, specific 3H-VKA binding remained significantly reduced in the ventral hippocampal formation of 120-day-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal ethanol exposure decreases hippocampal mossy fiber zinc in 45-day-old rats.

The long-term consequences of prenatal ethanol exposure on histochemically detectable hippocampal mossy fiber zinc was examined using a recently developed quantitative histofluorescence procedure. Pregnant Sprague-Dawley rats were maintained throughout gestation on one of three dietary regimens: (a) a liquid diet containing either 3.35% ethanol, (b) an isocalorically matched liquid diet pair-fed to the 3.35% ethanol group, or (c) lab chow ad libitum. At 45 days of age, offspring from each of the three diet groups were sacrificed for determination of hippocampal mossy fiber zinc and zinc analysis of selected tissues by atomic absorption spectroscopy. Hippocampal mossy fiber zinc was reduced by 36% in dorsal and 20% in ventral hippocampal formation stratum lucidum of rats exposed to the 3.35% ethanol diet compared to the offspring of the pair-fed control and ad libitum control dams. No significant differences in zinc:TS-Q histofluorescence were observed between the ad libitum and pair-fed control groups. No significant differences were observed among groups in tissue wet weight or tissue zinc concentration in any of the brain or other body regions analyzed. These results indicate a long lasting prenatal ethanol exposure-induced reduction in hippocampal mossy fiber zinc in the absence of changes in any indices of total body zinc nutriture. These results suggest that prenatal exposure to relatively low blood ethanol levels (30-40 mg/dl) has subtle, yet long-lasting effects in the hippocampal formation, a brain region important in the process of memory consolidation.

Prenatal ethanol exposure decreases hippocampal 3H-vinylidene kainic acid binding in 45-day-old rats.

The effect of prenatal ethanol exposure on the kainate-sensitive subtype of glutamate receptor binding sites was studied using in vitro 3H-vinylidene kainic acid (VKA) autoradiography. Pregnant Sprague-Dawley rats were fed a liquid diet containing either 3.35% or 6.7% ethanol throughout gestation. Pair-fed dams received isocalorically matched liquid diets and a lab chow ad lib group served as control for paired feeding. At 45 days of age, the offspring were sacrificed and their brains analyzed for specific 3H-VKA binding. Compared to pair-fed controls, specific 3H-VKA binding was reduced by 13% to 32% in dorsal and ventral hippocampal CA3 stratum lucidum, entorhinal cortex and cerebellum of 45-day-old rats whose mothers consumed either 3.35% or 6.7% ethanol diets. The binding site reductions were statistically significant only in the ventral hippocampal formation and entorhinal cortex of the 3.35% ethanol diet group rats. Saturation of binding studies in the ventral hippocampal formation of 3.35% ethanol rats indicated that the decrease in specific 3H-VKA binding was due to a decrease in the total number of binding sites. Given the excitatory effect of kainic acid on the spontaneous firing rate of hippocampal CA3 pyramidal neurons, the reduction of kainate-sensitive glutamate binding in this region is consistent with the electrophysiological observation of decreased spontaneous activity of CA3 pyramidal neurons in fetal alcohol rats.

Prenatal ethanol exposure decreases hippocampal 3H-glutamate binding in 45-day-old rats.

The effect of prenatal ethanol exposure on putative glutamate receptor binding sites in rat brain was studied using radiohistochemical techniques. Pregnant Sprague-Dawley rats were fed a liquid diet containing either 3% or 6% (vol./vol.) ethanol throughout gestation. Pair-fed dams received isocalorically matched liquid diets and a lab chow ad lib group served as control for paired feeding. At 45 days of age, the offspring were sacrificed and their brains analyzed by in vitro 3H-glutamate autoradiography. Compared to pair-fed controls, specific 3H-glutamate binding was reduced by 49-53% in regions of the dorsal hippocampal formation of 45-day-old rats whose mothers consumed either 3% or 6% ethanol diets. Specific 3H-glutamate binding was decreased also in the ventral hippocampal formation, entorhinal and posterior neocortex, but to a less consistent degree and magnitude than in dorsal hippocampal formation of fetal alcohol rats. The reduction in hippocampal 3H-glutamate binding 45 days after prenatal ethanol exposure suggests a long-lasting net decrease in glutamate-mediated excitatory neurotransmission within the hippocampal formation of fetal alcohol rats. This glutamate receptor binding site alteration may be one factor contributing to a decrease in long-term potentiation of hippocampal CA1 pyramidal neurons in fetal alcohol rats. In addition, this alteration may underlie learning and other behavioral deficits associated with functional defects of the hippocampal formation.

Chemotaxis as a preparative technique for human polymorphonuclear leukocytes.

Human peripheral blood phagocytic cells were prepared in large quantities utilizing their chemotactic characteristics. Cells were isolated using an enlarged version of the Boyden chemotactic chamber and were collected after migrating through a Millipore membrane into a solution containing chemotactic factor. Chemotactic factors consisted of normal human serum, casein-saturated medium, and a combination of human serum and casein-saturated medium. The most effective attractant was the mixture of casein and fresh human serum. Yields of up to 24.8 times 10(6) cells could be attained from a starting volume of approximately 12 c.c. of peripheral blood. These cells were greater than 95 per cent polymorphonuclear leukocytes with the remaining cells being monocytes. No erythrocytes or lymphocytes contaminated these preparations. The advantages of this technique are as follows: (1) large quantities of human peripheral blood phagocytes consisting of greater than 95 per cent polymorphonuclear cells can be obtained: (2) these cells are known to be biologically active sice chemotaxis is a requirement for their isolation; (3) the separative method is dependent on chemotactic properties rather than sedimentation characteristics; and finally (4) the resulting cell preparations are virtually devoid of lymphocytes and erythrocytes.