Improving the solubility of pseudo-hydrophobic Alzheimer's Disease medicinal chemicals through co-crystal formulation.

Natural products are ligands and potential inhibitors of Alzheimer's disease (AD) tau. Dihydromyricetin (DHM) is a CNS active natural product. Despite having signature polyphenolic character, DHM is ostensibly hydrophobic owing to intermolecular hydrogen bonds that shield hydrophilic phenols. Our research shows DHM becomes ionized at near-neutral pH allowing formulation of salts with transformed solubility. The MicroED co-crystal structure with trolamine reveals DHM salts as metastable solids with unlocked hydrogen bonding and a thermodynamic bent to solubilize in water. All salt formulations show better inhibitory activity against AD tau than the non-salt form, with efficacies correlating to enhanced solubilities. These results underscore the role of structural chemistry in guiding selection of solubilizing agents for chemical formulation. We propose DHM salts are appropriate formulations for research as dietary supplements to promote healthy aging by combating protein misfolding. Additionally, DHM is a suitable lead for medicinal chemistry and possible development of CNS pharmaceuticals.

Supplementation with sodium butyrate protects against antibiotic-induced increases in ethanol consumption behavior in mice.

BACKGROUND: We have recently reported that oral treatment of adult male C57BL/6J mice with a non-absorbable antibiotic cocktail resulted in an increase in ethanol intake and in significant reductions in butyrate-producing gut microbiota populations. This work led us to hypothesize that reduction in butyrate levels within the gut is linked to antibiotic-induced increases in voluntary ethanol consumption. OBJECTIVE: This study tested whether ad libitum sodium butyrate supplementation can prevent antibiotic-induced ethanol consumption in mice. METHODS: Sodium butyrate was provided to adult male C57BL/6J mice in drinking water alone or in combination with antibiotic cocktail. Effects on ethanol (20%) intake were measured using drinking in the dark and modified 2-bottle choice paradigms. Body parameters, food and liquid intake, cecum, and adipose tissues were measured during and/or at the conclusion of the drinking in the dark study. Cecal 16s rRNA was analyzed for microbiota diversity and changes in specific bacterial phyla/species. RESULTS: In drinking in the dark, sodium butyrate supplementation prevented antibiotic-induced increases in ethanol intake without altering basal ethanol consumption. Furthermore, sodium butyrate supplementation lowered ethanol preference in the 2-bottle choice study. Ethanol intake was correlated to specific bacterial phyla/species. Sodium butyrate did not affect the changes in microbiota diversity and composition induced by antibiotic cocktail. CONCLUSIONS: The findings support a role of gut microbiota-derived butyrate in regulating alcohol-induced behaviors. Additionally, the work contributes to efforts in development of novel microbiome-based strategies as novel preventative and intervention-based therapeutics to address alcohol use disorder.

Manipulations of extracellular Loop 2 in α1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol.

We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region.

Differential effects of propofol and ethanol on P2X4 receptors expressed in Xenopus oocytes.

The current study investigated the effects of propofol on P2X4 receptors expressed in Xenopus oocytes using two-electrode voltage clamp. We also tested the effects of 100 mM ethanol on the same oocytes used to test propofol. Propofol potentiated ATP-gated currents in a concentration dependent manner in P2X4 receptors. In agreement with our previous findings, ethanol inhibited P2X4 receptors. The opposite effects of propofol and ethanol on P2X4 receptor function suggest that these anesthetics act via different sites/mechanisms in P2X receptors as has been suggested for GABA(A) and glycine receptors.

Pressure-sensitive and -insensitive coupling in gamma-aminobutyric acid(A) receptors.

RATIONALE: Previous behavioral and biochemical studies suggest that allosteric coupling processes initiated by benzodiazepines, barbiturates and neuroactive steroids can be sub-categorized on the basis of their sensitivities to antagonism by increased atmospheric pressure. However, biochemical evidence supporting this hypothesis was limited to single concentration studies in long sleep (LS) mice. OBJECTIVE: The present paper addresses these issues by extending biochemical investigation of pressure effects on allosteric modulators across a range of concentrations that allosterically enhance gamma-aminobutyric acid (GABA)A receptor function and alter behavior using two mouse genotypes. In addition, the effects of pressure on ligand binding were explored to further investigate the mechanism of pressure antagonism of allosteric modulation. METHODS: The effects of 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) on allosteric modulation of GABA(A) receptor function and [3H]flunitrazepam binding was tested in LS and C57BL mouse brain membranes (microsacs) using chloride flux and high-affinity binding assays. RESULTS: In both genotypes, exposure to 12 ATA heliox antagonized the allosteric enhancement of GABA(A) receptor function by flunitrazepam (0.1-10 microM) and pentobarbital (0.1-50 microM) but did not affect allosteric modulation by 3alpha-hydroxy-5beta-pregnan-20-one (0.1-1 microM). Pressure did not affect benzodiazepine receptor affinity (Kd) or the number of benzodiazepine receptors (Bmax). THE RESULTS: (1) confirm that there are differences in sensitivity to pressure antagonism of allosteric coupling among GABA(A) allosteric modulators; (2) demonstrate that these differences are not concentration or genotype dependent; (3) add evidence that pressure antagonizes allosteric modulation by uncoupling the receptor and (4) support the hypothesis that allosteric modulation of receptor function can be sub-categorized on the basis of sensitivity to pressure antagonism.

Direct evidence for a cause-effect link between ethanol potentiation of GABA(A) receptor function and intoxication from hyperbaric studies in C57, LS, and SS mice.

BACKGROUND: This article uses a direct ethanol antagonist, increased atmospheric pressure, to further test the causative link between ethanol potentiation of gamma-aminobutyric acid (GABA) type A receptor function and ethanol's behavioral effects. This was done by determining whether initial biochemical findings in long-sleep (LS) mice extended to other genotypes and whether the previously reported insensitivity of short-sleep (SS) mice to pressure antagonism of ethanol-induced loss of righting reflex extended to a nonselected ethanol-induced behavior. METHODS: The effects of 12 times normal atmospheric pressure of helium-oxygen gas (heliox) versus ethanol (25-200 mM) potentiation of GABA-activated Cl- uptake in brain membranes (microsacs) from C57, LS, and SS mice were tested by using a 36Cl- flux assay. The effects of pressure versus ethanol's (2 g/kg) anticonvulsant effect in SS mice were tested by using time to onset of isoniazid-induced myoclonic seizures. RESULTS: Exposure to 12 times normal atmospheric pressure heliox antagonized ethanol potentiation of GABA-activated Cl- uptake in all three genotypes across a range of ethanol concentrations that cause ethanol's behavioral and anesthetic effects. Pressure did not affect baseline receptor function. The threshold for initiating ethanol potentiation differed between genotypes in accordance with their behavioral sensitivities to ethanol (C57 and LS, < or =25 mM; SS, >50 mM). Pressure antagonized ethanol's anticonvulsant effect in SS mice. CONCLUSIONS: The results add important direct evidence supporting the hypothesis that ethanol potentiation of GABA(A) receptor function is an initial action of ethanol causing its behavioral effects. These findings also provide insight into possible effects of selective breeding on GABA(A) receptor function.

Ethanol enhances GABAA receptor function in short sleep and long sleep mouse brain membranes.

BACKGROUND: SS and LS mice have been used to explore the genetic and neurochemical bases for differences in sensitivity to ethanol. The present study investigated the effects of ethanol on GABAA receptor function in microsacs from these genotypes. The purpose was to test a key element of the hypothesis that differences between these lines in sensitivity to ethanol-induced enhancement of GABAA receptor function underlie their selected differences in sensitivity to ethanol-induced loss of righting reflex (LORR). METHODS: The effects of ethanol on GABA-activated 36Cl- uptake in brain membranes (microsacs) isolated from male SS and LS mice were tested using a chloride flux filtration assay. RESULTS: Ethanol significantly enhanced GABA-activated 36Cl- uptake in SS microsacs at concentrations of 100-300 mM. Ethanol did not significantly affect GABA-activated chloride uptake in this preparation at concentrations of 25 and 50 mM. Ethanol significantly enhanced GABA-activated 36Cl- uptake in LS microsacs at concentrations of 25-100 mM, but not at 200 mM. CONCLUSION: The present studies are the first to show a statistically significant effect of ethanol on GABA-activated chloride uptake in both SS and LS mice with a clear difference between the genotypes in threshold. The relative threshold differences between SS and LS microsacs in sensitivity to ethanol indicate that selection for resistance to ethanol-induced LORR in SS mice has shifted the ethanol-GABAA receptor concentration-response curve to the right. The findings add key evidence that supports a cause-effect relationship between sensitivity to ethanol-induced potentiation of GABAA receptor function and genetically determined sensitivity to ethanol's behavioral effects.

Preparation of novel, moisture-stable, Lewis-acidic ionic liquids containing quaternary ammonium salts with functional side chains.

A range of novel, moisture-stable, Lewis-acidic ionic liquids has been prepared by mixing appropriate molar ratios of MCl2 (M = Zn and/or Sn) and quaternary ammonium salts of formula [Me3NC2H4Y]Cl (Y = OH, Cl, OC(O)Me, OC(O)Ph); the influence of substituent Y and metal M on the physical properties of the melts has been investigated.

Effects of posttraining ethanol on an appetitive task.

The present study examined the effects of posttraining ethanol administration upon retention of an appetitive task using a variety of retention behaviors associated with the task. Male C57BL/6J mice were individually trained to find a cheese pellet placed in the corner of an open field. Five behavioral measures were used including locomotor activity counts, rearings, grooming episodes, approaches to the cheese pellet, and latency to consume the cheese pellet. Immediately after training, mice were injected intraperitoneally with saline or 2.0 g/kg of ethanol and then returned to their home cage in which four "intruder" mice were added for 2 h after training. On subsequent testing days (1, 6, 14, and 51 days posttraining), mice were returned to the original training environment and the five behaviors were measured. Both saline- and ethanol-treated mice habituated to the initially novel test environment at similar rates as indicated by decreased exploratory behavior (locomotor activity and rearings). In contrast, a divergence in the latency to consume the cheese pellet was observed: Saline-treated mice behaved as though the cheese was rewarding (decreased latency to eat the pellet), while the ethanol group behaved as though the cheese was aversive (increased latency to eat the pellet). Taken with previous studies, these results demonstrate that posttraining ethanol can have strikingly different effects on retention depending on the task, the measure of retention used, and the underlying neural structures involved.

Biochemical and morphological effects of fumonisin B(1) on primary cultures of rat cerebrum.

Chronic dietary consumption of the mycotoxin fumonisin B(1) (FB(1)) is associated with leukoencephalomalacia and neuronal degeneration, but identification of the cellular mechanisms underlying this neurotoxicity is difficult due to concurrent adverse systemic changes. For this reason, the present investigation used an in vitro approach to assess the short-term consequences of direct FB(1) (0. 5-75 microM) exposure on astrocytes and oligodendrocytes in primary cultures of rat cerebrum. Beginning at 5 days in vitro, the cultures were exposed to FB(1) at five concentrations (0.5-75 microM), and the cultures were evaluated at 10 and 15 days in vitro. The levels of the sphingolipid-associated constituents sphingosine and sphinganine were determined with a high-performance liquid chromatography. Relative to untreated cultures, exposure to FB(1) diminished the levels of sphingosine at 15 days in vitro, whereas FB(1)-exposed cultures showed significantly increased sphinganine levels and sphinganine/sphingosine ratios. In addition to these changes in sphingolipid constituents, FB(1)-exposed (0.5-75 microM) cultures exhibited a two-fold increase in the number of process-bearing cells by 15 days in vitro. Also, the activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase, an enzyme associated with myelin and oligodendrocytes, was increased in FB(1)-treated cultures. This study suggests that short-term exposure to FB(1) may modify the proliferation or differentiation of glial cells.

Glomerular hyperfiltration, high renin, and low- extracellular volume in high blood pressure.

Abnormal renovascular resistance and glomerular filtration rate are characteristic of established hypertension and may also be involved in its pathogenesis. To determine renal and body fluid correlates of the predisposition to high blood pressure, we examined 100 healthy young adults with high or low blood pressure. Within each group, half had parents with high blood pressures, and half had parents with low blood pressures. Renal function and hemodynamics, body fluid volumes, and relevant hormones and genotypes were measured. Subjects with high personal and parental blood pressures had the highest levels of glomerular filtration rate (P<0.02) and plasma active renin concentration and low levels of exchangeable sodium and plasma volume (P<0.02). High glomerular filtration rate was not associated with differences in urinary kallikrein or prostaglandins. Polymorphisms of the renin, angiotensin-converting enzyme, and angiotensinogen genes were not associated with differences in glomerular filtration rate or renin. Subjects with high personal, but low parental, blood pressures had low exchangeable sodium and plasma volumes (P<0.02) but normal glomerular filtration rates. In this population, extracellular volume depletion and high renin are correlates of high blood pressure in early adulthood, and glomerular hyperfiltration is a feature of those who also have familial predisposition to high blood pressure.

Heterogeneity of astroglia cultured from adult human temporal lobe.

This study characterized the morphological and electrophysiological diversity of astroglia cultured from adult human cerebral temporal lobe, and explored the influence of the cytokine interleukin-1beta on these cells. The cultures contained astroglia positive for glial fibrillary acidic protein which were flat, bipolar or multipolar in shape and variable in size. A subpopulation of the bipolar and multipolar cells was positive for S100 protein. The most striking feature of these cultures was the presence of glia with long (600 micrometer) processes with few branches or only terminal branches. Patch clamp recordings of the non-stellate process bearing cells revealed prominent inward Na(+) and transient and sustained outward K(+) conductances. Distinct differences in the relative proportion of these conductances were evident among cells but did not appear to be correlated with cell morphology. Treatment of cultures with interleukin-1beta for 96 h did not change total protein content, but increased the content of S100beta protein and decreased the content of glial fibrillary acidic protein. The findings indicate that cultures of adult human cerebrum contain subpopulations of morphologically and electrophysiologically pleomorphic glial fibrillary acidic protein positive astroglia, exhibit increased levels of the neurotrophic factor S100beta when exposed to interleukin-1beta, and may serve as a useful model for investigation of glial involvement in neuropathology.

In vivo and in vitro hyperbaric studies in mice suggest novel sites of action for ethanol.

The present study uses increased atmospheric pressure as an ethanol antagonist to test the hypothesis that allosteric coupling pathways in the GABA(A) receptor complex represent initial sites of action for ethanol. This was accomplished using behavioral and in vitro measures to determine the effects of pressure on ethanol and other GABAergic drugs in C57BL/6 and LS mice. Behaviorally, exposure to 12 times normal atmospheric pressure (ATA) of a helium-oxygen gas mixture (heliox) antagonized loss of righting reflex (LORR) induced by the allosteric modulators ethanol and pentobarbital, but did not antagonize LORR induced by the direct GABA agonist 4,5,6,7-tetrahydroisoxazolo-pyridin-3-ol (THIP). Similarly, exposure to 12 ATA heliox antagonized the anticonvulsant effects verses isoniazid of ethanol, diazepam and pentobarbital. Biochemically, exposure to 12 ATA heliox antagonized potentiation of GABA-activated 36Cl-uptake by ethanol, flunitrazepam and pentobarbital in LS mouse brain preparations, but did not alter GABA-activated 36Cl- uptake per se. In contrast to its antagonist effect versus other allosteric modulators, pressure did not antagonize these behavioral or in vitro effects induced by the neuroactive steroid, 3alpha-hydroxy-5beta-pregnan-20-one (3alpha,5beta-P). These findings add to evidence that pressure directly and selectively antagonizes drug effects mediated through allosteric coupling pathways. The results fit predictions, and thus support the hypothesis that allosteric coupling pathways in GABA(A) receptors represent initial sites of action for ethanol. Collectively, the results suggest that there may be common physicochemical and underlying structural characteristics that define ethanol sensitive regions of receptor proteins and/or their associated membranes that can be identified by pressure within (e.g., GABA(A)) and possibly across (e.g., GABA(A), NMDA, 5HT3) receptors.

Changes in mRNA levels for heat-shock/stress proteins (Hsp) and a secretory vesicle associated cysteine-string protein (Csp1) after amphetamine (AMPH) exposure.

Damage to nerve terminals, reactive gliosis and somatic degeneration can result when pronounced hyperthermia occurs during amphetamine (AMPH) exposure. The effects of AMPH-induced hyperthermia and damage on the relative mRNA levels for several heat shock/stress proteins (Hsp27, Hsp60, Hsp70 and Hsc70), as well as secretory vesicle associated cysteinestring protein (Csp1) were determined in both the striatum and substantia nigra using reverse transcriptase polymerase chain reaction (RT-PCR). These changes were compared to changes in Hsp mRNA levels seen in primary rat cerebral astrocyte cultures after heat shock/stress. Striatal Hsp70 mRNA increased about 2-fold over control levels at 16 hr after AMPH-induced hyperthermia, and was the only Hsp species to significantly increase in response to AMPH. Hsp70 mRNA levels returned to control within 14 days after AMPH. Two-fold increases in Hsp70 mRNA were also seen in primary cultures of rat cerebrum 24 hr after heat shock. In primary cultures and brain tissue, the increased Hsp70 mRNA levels were still more than 500-fold less than constitutive Hsc70 mRNA and 50-fold less than Hsp60 levels. Hsp27 mRNA was not present in the striatum, nigra and primary cell cultures. Thus, the expression of Hsp species mRNA measured was very similar in brain tissue and primary cell cultures. Because only a modest induction of Hsp 70 mRNA occurred, the Hsp species evaluated may only play a minor role in AMPH neurotoxicity. However, further studies are necessary to determine whether large increases in Hsp70 are occurring in selected neurons or glia in the striatum. RT-PCR products for Csp1 were produced in total RNA obtained from brain but not from cultured astrocytes, suggesting that the Csp1 mRNA measured by RT-PCR is of neuronal origin. Csp1 mRNA levels were acutely downregulated in neurons in the substantia nigra, possibly in response to damage, but not the striatum after AMPH exposure. A slight long-term upregulation at 4 months of Csp1 mRNA may occur in the striatum but not in nigra.

Direct antagonism of ethanol's effects on GABA(A) receptors by increased atmospheric pressure.

Previous studies have shown that exposure to 12 times normal atmospheric pressure of helium-oxygen gas (heliox) directly antagonizes a range of ethanol's acute and chronic behavioral effects. The present study extends the investigation to the biochemical level by investigating the effects of pressure on ethanol-induced potentiation of GABA(A) receptor function in mouse membrane vesicles (microsacs). Exposure to 12 atmospheric pressure heliox significantly antagonized ethanol (25 to 100 mM) potentiation of GABA-activated 36Cl- uptake, but did not significantly alter baseline GABA(A) receptor function measured by the response of the system to GABA (10 to 100 microM), bicuculline (3 and 100 microM), or picrotoxin (100 microM). These findings add essential support for the hypothesis that hyperbaric exposure is a direct ethanol antagonist that can be used as a tool to help identify ethanol's initial cellular and molecular sites of action that cause its behavioral effects. Taken in context with previous behavioral studies, the present results also provide important new evidence for a cause-effect relationship between ethanol potentiation of GABA(A) receptor function and ethanol's anesthetic and behavioral effects.

Human alpha-adducin gene, blood pressure, and sodium metabolism.

The adducin genes contribute significantly to population variation in rat blood pressure and cell membrane sodium transport. The 460Trp mutation of the human alpha-adducin gene has been associated with hypertension, in particular hypertension sensitive to sodium restriction. We studied the relationship between the 460Trp mutation and population variation in blood pressure and sodium metabolism. From 603 Scottish families, we selected 151 offspring and 224 parents with blood pressures in either the upper (high) or bottom (low) 30% of the population distribution and measured the 460Trp mutation using allele-specific hybridization. In offspring, we also measured exchangeable sodium, plasma volume, and total body water. Plasma levels of components of the renin-angiotensin system, atrial natriuretic peptide, and cellular sodium and transmembrane sodium efflux were also estimated. The overall frequency of the 460Trp mutation was 27.1%. In offspring and parent groups, we found no difference in the genotype or allele frequencies of the 460Trp mutation between subjects with high or low blood pressure. There was no overall association between the alpha-adducin genotypes and blood pressure variation. In offspring, the 460Trp mutation was not associated with any significant differences in body fluid volumes or exchangeable sodium; levels of plasma renin, angiotensin II, aldosterone, or atrial natriuretic peptide; intracellular sodium; or ouabain-sensitive transmembrane sodium efflux. These findings suggest that in our Scottish population, the alpha-adducin 460Trp polymorphism is not related to blood pressure and does not affect whole body or cellular sodium metabolism.

Platelet sodium/hydrogen ion exchange in normal pregnancy and non-proteinuric pre-eclampsia.

In non-pregnant individuals, abnormalities in cation transport in vascular tissues have been linked to essential hypertension. In the present study, we consider whether Na+/H+ exchange (NHE) is affected in non-proteinuric pre-eclampsia (NPP). Platelet NHE characteristics and plasma cholesterol were measured in a cross-sectional study of normal primigravidae at 14 +/- 0.5 (n = 9), 29 +/- 0.7 (n = 7), 39 +/- 0.4 (n = 8) weeks gestation, in women with NPP (n = 15) and in non-pregnant women (n = 8). Amiloride-sensitive 22Na uptake was measured in platelets which had been acid loaded, to stimulate NHE, by suspension in isotonic potassium propionate buffer (pH 6.7). Intraplatelet radioactivity was used to calculate the affinity (Km) and the capacity (Vmax) of Na+ uptake. In normotensive women, Vmax (mean +/- s.e.) at 14, 29, 39 weeks gestation and 6 weeks postpartum were 452 +/- 46, 469 +/- 33, 713 +/- 101 and 562 +/- 77 pmolNa+/10(6) cells/min respectively; the third trimester values were higher (P < 0.05) than those in the first and second trimester and were also higher than those of non-pregnant women (415 +/- 20). Vmax of patients with NPP in the third trimester (712 +/- 44) was not different from gestational age-matched controls. Km values were not affected by gestational age or NPP. Plasma cholesterol concentration was positively correlated with Vmax values during normotensive pregnancy (r = 0.493, P < 0.05). In conclusion, the capacity for amiloride-sensitive Na+ uptake by platelets correlates positively with gestational age during normal pregnancy. However, neither the capacity nor affinity for Na+ was altered in NPP platelets suggesting that NHE is not implicated in the pathophysiology of this condition.

Microglial development is altered in immature spinal cord by exposure to radiation.

Previous studies in this laboratory have documented that the microglial environment of the immature spinal cord is altered by exposure to ionizing radiation. As a result, the lumbosacral spinal cord is markedly depleted of both oligodendrocytes and astrocytes, while leaving axons and the overall cytoarchitecture intact. The status of the microglia in the irradiated region is unknown and is of interest given the interactions between microglia and astrocytes recently elucidated by others. This study uses both in vivo and in vitro approaches to examine the microglial population in normal and irradiated immature spinal cord. The lectin, Griffonia (Bandeiraea) simplicifolia, was selected since it marks microglia both in paraffin embedded sections and in cell cultures. Light microscopic examinations of spinal cord sections revealed a reduced microglial population in the irradiated region when compared to littermate controls, and a change in morphology of the remaining microglia to that described by others as "activated". Cultures prepared from lumbosacral spinal cords harvested from 3-day-old rats within 2-4 hr following irradiation were compared with cultures derived from their non-irradiated littermates after 8 days in vitro. Cultures from the irradiated spinal cords revealed trends similar to those observed in vivo, i.e. a reduced microglial population and altered morphology. Although all glial cell types were reduced in cultures from irradiated spinal cords, the few microglia present were usually positioned atop astrocytes. The consistency of reduction in all glial populations in this model shows the microglia to be a novel microenvironment for further studies of roles of microglial within the spinal cord.

Proliferation of astroglia from the adult human cerebrum is inhibited by ethanol in vitro.

Chronic alcoholism is associated with atrophy of the adult brain, while fetal exposure to ethanol can cause microencephaly. Since astroglial pathology is a common feature of ethanol exposure in both humans and animal models, the direct influence of ethanol on proliferation of human astroglia from the gray and white matter of adult temporal lobe was determined and compared. Astroglial cultures were exposed to constant concentrations of ethanol at realistic social and clinical levels (0.1, 0.2 or 0.5%; w/v) for 1 to 5 days. Proliferation was quantified by bromodeoxyuridine labeling and enumeration of replicating cells. Ethanol exposure significantly inhibited proliferation of both gray and white matter astroglia in a dose and duration dependent manner. Gray matter was slightly more sensitive than white matter to inhibition by low to moderate concentrations of ethanol; in contrast, white matter was more sensitive to high ethanol concentrations. Maximum inhibition was 20% in gray matter and 25% in white matter. Human astroglial proliferation was directly inhibited in the absence of neurons, microglia, neuronal degeneration or systemic factors that have confounded in vivo studies. Restricted astroglial proliferation may underlie aspects of the astroglial pathology associated with ethanol exposure.