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B A Bahr

Publications and source records attributed to B A Bahr.

At least 37 records · Page 2Linked to original sources
PubMed1997

A 27-kDa matrix receptor from rat brain synaptosomes: selective recognition of the Arg-Gly-Asp-Ser domain and unique resistance to calcium-dependent proteolysis.

A 27-kDa protein from adult rat brain synaptosomes was purified by matrix-affinity chromatography. The matrix receptor interacted with the Arg-Gly-Asp-Ser sequence recognized by integrin-type adhesion molecules, and was labeled by integrin antibodies. Levels of the 27-kDa species in brain membranes were unaffected by proteolysis, however, conventional integrin subunits exhibited robust degradation. This unique resistance to proteolysis may allow the new matrix receptor to contribute to the stability of synaptic contacts.

Amino Acids↗
PubMed1997

Effect of glycine on prelethal and postlethal increases in calpain activity in rat renal proximal tubules.

The effect of glycine on hypoxia- and ionomycin-induced increases in calpain activity in rat proximal tubules was determined. Calpain activity was determined both in vitro and in the intact cell using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC) and Western blotting for calpain-specific spectrin breakdown products (BDP), respectively. At 7.5 minutes of hypoxia (prelethal injury model) there was a significant (10-fold) increase in in vitro calpain activity that was not inhibited by glycine. At 15 minutes of hypoxia (postlethal injury model) there was a further increase in calpain activity that was inhibited by glycine. Normoxic tubules incubated with the calcium ionophore ionomycin (5 microM) for two minutes and 10 minutes had a significant increase in calpain activity that was not inhibited by glycine. After 15 minutes of hypoxia in the presence of glycine, there was an increase in calpain-specific spectrin breakdown products (BDP) in both Triton X-100 soluble and cytosolic extracts from proximal tubules. Glycine in concentrations up to 10 mM had no direct effect on the in vitro calpain activity of purified calpains. The present study demonstrates that: (1) prelethal increases in calpain activity stimulated by hypoxia and ionomycin treatment are not affected by glycine; (2) calpain-mediated spectrin breakdown during hypoxia occurs in the presence of glycine; (3) glycine does prevent the additional postlethal increase in calpain activity probably by maintaining membrane integrity to calcium influx.

Animals↗
PubMed1996

Effects of heparin on the properties of solubilized and reconstituted rat brain AMPA receptors.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1996

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Amino Acid Sequence↗
PubMed1995

Long-term hippocampal slices: a model system for investigating synaptic mechanisms and pathologic processes.

Organotypic cultures provide a unique strategy with which to examine many aspects of brain physiology and pathology. Long-term slice cultures from the hippocampus, a region involved in memory encoding and one that exhibits early degeneration in Alzheimer's disease and ischemia, are particularly valuable in this regard due to their expression of synaptic plasticity mechanisms (e.g., long-term potentiation) and responsiveness to pathological insults (e.g., excitotoxicity). Long-term slices can be prepared from hippocampi at the second or third postnatal week of development and thus incorporate a number of relatively mature features; further signs of maturation and the preservation of adult-like characteristics occur over succeeding weeks. The stability of the cultured slice renders it an appropriate model for studying 1) prolonged regulation/stabilization events linked to synaptogenesis and certain forms of plasticity, 2) temporal patterns of cellular atrophy associated with pathogenic conditions such as ischemia and epilepsia, and 3) slow processes associated with aging and age-related pathologies.

Animals↗
PubMed1995

Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices.

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 microM calcium. Increases in a single proteolytic fragment of spectrin produced by 10-20 min of NMDA receptor stimulation were substantially (approximately 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that detects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitotoxicity.

Animals↗
PubMed1995

Stable maintenance of glutamate receptors and other synaptic components in long-term hippocampal slices.

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [3H]AMPA and [3H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5-10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10-20 expressed a low to high affinity [3H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e.g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.

Amino Acid Sequence↗
PubMed1995

Induction of calpain-mediated spectrin fragments by pathogenic treatments in long-term hippocampal slices.

The use of cultured hippocampal slices for studies of calpain-mediated pathogenesis was investigated. Breakdown products (BDPs), which result from proteolysis of spectrin by calpain I, were assayed with BDP-specific antibodies developed against peptide sequences on either side of the calpain cleavage site. The antibodies recognized either amino- or carboxy-terminal BDPs (147-kD BDPN and 152-kD BDPC, respectively). Various pathogenic manipulations, including trimethyltin, certain snake venoms and agonists for excitatory amino acid receptors, were found to cause rapid and pronounced increases in the proteolytic fragments. These effects were selective, i.e., chemicals or toxins directed at nonglutamatergic neurons had little effect on BDP concentrations. Transient accumulations of spectrin fragments were obtained with brief applications of N-methyl-D-aspartate; longer infusions resulted in lasting increases. Results similar to these have been observed in vivo with ischemic episodes of varying duration. Agonists of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subclass of glutamate receptors produced significant increases in spectrin proteolysis; however, prolonged exposure of the slices to centrally active drugs that enhance the currents passed by the receptors did not. The sensitivity, selectivity and temporal properties of the proteolytic response support the idea that cultured slices can be used to analyze the events leading to and following from calpain activation in the adult brain.

Amino Acid Sequence↗
PubMed1994

Changes in the concentrations of tau and other structural proteins in the brains of aged mice.

To test whether aging is associated with alterations in the balance of cytoskeletal constituents, the relative concentrations of tau isoforms and seven other structural proteins were compared in the brains of 3-25-month-old mice. A tau species of approx. 63 kDa was substantially increased in the older animals while the levels of ankyrin, talin, spectrin, and actin were differentially decreased. The decrement in ankyrin was evident at earlier ages than that for spectrin. These results suggest that the make-up of the neuronal cytoskeleton changes with age.

Actins↗
PubMed1994

Stimulation of NMDA receptors activates calpain in cultured hippocampal slices.

The hypothesis that intense stimulation of NMDA receptors activates calpain was tested in long-term cultures of hippocampus. Slices prepared from 10-day-old rats were maintained for periods of up to 6 weeks and then assayed for a stable breakdown product that results from the proteolysis of spectrin by calpain. The breakdown product increased dramatically during the first 24 h after tissue preparation and then decreased to a low level that remained unchanged for weeks. NMDA caused a 2- to 3-fold increase in breakdown product that rose linearly with time (5-30 min) and was blocked by the receptor antagonist MK-801. The effect of NMDA was the same throughout the culture period and was dependent upon the concentration of extracellular calcium with no effect at 2 mM and maximal effect at 4 mM calcium. These results indicate that rapid activation of calpain occurs in undamaged hippocampal neurons following stimulation of NMDA receptors.

Animals↗
PubMed1994

Induction of beta-amyloid-containing polypeptides in hippocampus: evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin.

Long-term cultures of brain slices were used to test if the lysosomotropic agent chloroquine induces beta-amyloid-related peptides in hippocampus and if such effects are accompanied by other manifestations of brain aging. Chloroquine administration resulted in the appearance of a carboxyl-terminal fragment of the beta-amyloid precursor protein (APP); the 27-kDa antigen was detectable after 24 h, increased rapidly for 6-10 days, and was eliminated upon drug washout. Immunocytochemical analyses showed that beta-amyloid immunoreactivity accumulated in the perikarya of pyramidal neurons, primarily in the form of punctate bodies. These effects were accompanied by a correlated loss (and recovery) of the presynaptic marker synaptophysin and by a delayed reduction of postsynaptic glutamate receptors, while cytoskeletal proteins were unchanged. Acute administration of chloroquine had no evident effects on synaptic responses but prolonged applications caused a decrease in the maximum amplitude of field potentials. Finally, a brief pretreatment with the excitotoxin kainic acid had little effect with regard to APP fragments or synaptophysin, but altered the events following from a subsequent infusion of chloroquine. Buildup of the 27-kDa APP fragment and loss of synaptophysin were more rapid and, more importantly, did not reverse upon washout of chloroquine. These findings indicate that lysosomal dysfunction in hippocampus results in the accumulation of a particular APP fragment and suggest that this event, or a variable correlated with it, is linked to the loss of synaptic proteins. They also raise the possibility that certain aspects of brain aging reflect a synergism between lysosomal disturbances and excitotoxicity.

Amyloid beta-Peptides↗
PubMed1994

Rapid and stable gene expression in hippocampal slice cultures from a defective HSV-1 vector.

Stable transfer of genetic information into neurons is a powerful strategy to elucidate specific mechanisms of neurophysiology and to develop therapies for neurological disorders. To evaluate the optimal parameters for efficient gene delivery of defective herpes simplex virus type one (HSV-1) vectors into a specific brain region, an HSV-1 vector expressing E. coli beta-galactosidase was used to infect organotypic cultures of hippocampal slices. beta-Galactosidase was expressed as early as 2 h after infection in a dose-dependent manner as measured on immunoblots, and reached a maximum level after approximately 35 h. Expression of the RNA and the antigen was still evident after the longest time sampled (11-12 days), whereas no beta-galactosidase was ever detected in cultured slices infected with a control virus lacking the reporter gene. Hippocampal cells expressing the reporter gene outlined the contour of the neuronal cell body layers in fields CA3 and dentate gyrus; such correspondence was less evident in field CA1. Anatomical, morphological, and immunohistochemical criteria also confirmed that the majority of these infected cells were neurons. beta-Galactosidase was also detected in the somata and processes of infected interneurons. Tests for synaptic pathology associated with virus infection showed no changes in pre- and postsynaptic markers.

Animals↗
PubMed1994

AMPA receptor development in rat telencephalon: [3H]AMPA binding and western blot studies.

Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H] AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM140 (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [3H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component (KD = 400 nM) that dominates the [3H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component (KD = 15 nM) of the [3H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM140 exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Age-related changes in neural cell adhesion molecule (NCAM) isoforms in the mouse telencephalon.

The concentrations of different polypeptide isoforms of the neural cell adhesion molecule NCAM were examined in telencephalic and brainstem-cerebellar tissue from groups of young (3 months) and old (25 months) mice. Antibodies against chick brain NCAM were used in immunoblot analyses to quantify 180 (NCAM180) and 140 (NCAM140) kDa NCAM forms in mouse brain samples containing equal amounts of protein. Telencephalic homogenates from the older group exhibited 37% and 31% less NCAM180 and NCAM140 immunoreactivity, respectively, when compared with homogenates from the younger animals. Brainstem-cerebellar homogenates, however, did not express such age-related changes in the two NCAM isoforms. Age-related changes in isoforms labeled by the anti-NCAM antibodies were not evident in synaptic plasma membranes. NCAM180:NCAM140 ratios were 2- to 3-fold greater in the synaptic membranes vs. homogenates for both age groups. These data suggest that expression levels of NCAM180 and NCAM140 are selectively impaired with aging in the telencephalon, whereas the synaptic contents of these molecules appear to be stably regulated.

Aging↗
PubMed1993

Single channel recordings of reconstituted AMPA receptors reveal low and high conductance states.

Glutamate receptors belonging to the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subclass were partially purified 30- to 60-fold from forebrain of adult rats and incorporated into planar bimolecular lipid membranes. The channel conductance associated with the reconstituted receptors was activated by kainate and AMPA in a manner that suggests cooperative binding of two to three agonist molecules is required to induce channel opening. This conductance was blocked by the specific antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). When the partially purified AMPA receptors were reconstituted by the tip-dipping method in asymmetric saline conditions ('outside-out configuration'), the addition of 300 nM AMPA to the pseudo-extracellular solution elicited single channel current fluctuations that were also inhibited by DNQX. Analyses of the currents revealed that the ion channels of reconstituted AMPA receptors have two distinct conductance levels of 12 and 60 pS with the great majority of receptors belonging to the former variety. These results suggest that reconstitution may be useful in identifying factors that regulate the binding and conductance properties of AMPA receptors.

Animals↗
PubMed1993

Classical noncholinergic neurotransmitters and the vesicular transport system for acetylcholine.

The acetylcholine transporter exhibits such low affinity and specificity for acetylcholine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [3H]serotonin, [3H]noradrenaline, or [3H]glutamate occurred. Serotonin, noradrenaline, and N-acetyl-aspartyl glutamate inhibited active transport of [3H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and gamma-aminobutyric acid were shown here not to inhibit active transport of [3H]-acetylcholine. Noradrenaline was competitive with respect to [3H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [3H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.

Acetylcholine↗
PubMed1992

Mouse telencephalon exhibits an age-related decrease in glutamate (AMPA) receptors but no change in nerve terminal markers.

The central excitatory amino acid receptor selective for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) was examined in brain tissue from mice at 3 and 25 months after birth. Antibodies against the rat GluR-A glutamate receptor subunit (selective for kainate and AMPA) labeled a mouse brain component of about M(r) 100,000. Telencephalic tissue from the older group of mice exhibited 31% less immunoreactivity towards this component as compared with that from the young group. Binding of [3H]AMPA also decreased with age in the telencephalon to an extent which was similar to the loss of receptor immunoreactivity. Scatchard analysis revealed that this reduction is due to a decrease in receptor density and not to a change in binding affinity. In contrast, there were only small age-related changes in AMPA receptor immunoreactivity and binding levels in the brain stem and cerebellum. Binding to dopamine, serotonin, or GABA receptors was not significantly reduced in the older mice. Since the nerve terminal markers synaptophysin and the SV2 glycoprotein were not detectably different in the two groups of mice, the age-related reduction in AMPA receptors is not likely to be due to a general decrease in synaptic density. These data suggest that glutamatergic neurotransmission mediated by AMPA-type receptors is selectively impaired with aging in the telencephalon.

Aging↗