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M V Bennett

Publications and source records attributed to M V Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1994

Spermine potentiation of recombinant N-methyl-D-aspartate receptors is affected by subunit composition.

The present study shows that both the NR1 and NR2 subunits critically affect spermine potentiation of heteromeric recombinant N-methyl-D-aspartate receptors. NR1(011), the most prominent NR1 splice variant in rat forebrain, and NR1(100), prominent in midbrain, were expressed in Xenopus oocytes singly and in combination with NR2A, NR2B, and NR2C subunits. As for NR1(011) homomers, NR1(011)/NR2B receptors exhibited spermine potentiation by two mechanisms: by increasing glycine affinity and by increasing current through receptors with bound N-methyl-D-aspartate and glycine. NR1(011)/NR2A receptors exhibited only the increase in glycine affinity, and NR1(011)/NR2C receptors exhibited neither. As for NR1(100) homomers, NR1(100)/NR2B and NR1(100)/NR2A receptors exhibited spermine potentiation only by increasing the glycine affinity. Spermine produced no potentiation of NR1(100)/NR2C receptors. Thus, the NR2B subunit "permits" both forms of spermine potentiation, the NR2A subunit permits spermine potentiation only by increasing the glycine affinity, and th NR2C subunit permits neither form of potentiation. Spermine actions on NR1/NR2 showed little voltage dependence. These observations are of interest because the NR1 and NR2 subunits are differentially distributed and developmentally regulated. At early postnatal ages, NR2B subunit mRNA was more highly expressed than NR2A and NR2C mRNAs in hippocampus, neocortex, and caudate-putamen. These findings account for many of the observed differences among neurons in polyamine actions and suggest that these actions will vary in a cell-specific and age-related manner.

Alternative Splicing↗
PubMed1994

AMPA/kainate receptor gene expression in normal and Alzheimer's disease hippocampus.

Alzheimer's disease is a progressive dementia characterized by pronounced degeneration of certain populations of neurons in the hippocampus and cerebral cortex of the brain. One theory is that glutamate receptor-mediated toxicity plays a role in cell loss associated with Alzheimer's disease. We used in situ hybridization to examine GluR1, GluR2, and GluR3 messengerRNAs (encoding alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptor subunits) in sections of autopsy samples of Alzheimer's disease brains and age-, sex-, and post-mortem delay-matched brains from non-demented (control) subjects. GluR1 and GluR2 exhibited a heterogeneous distribution in control brain. GluR1 was expressed in granule cells of the dentate gyrus, in pyramidal cells of the CA1 and CA3 hippocampal subfields and in neurons of the subiculum and entorhinal cortex. GluR2 mRNA was at high density in the dentate gyrus and in CA3, but was at low density in CA1, subiculum, and entorhinal cortex. GluR3 hybridization was at very low levels but selectively localized to the dentate gyrus and CA3. In cerebellum, GluR1 was found in granule and Purkinje cell layers. In sections from Alzheimer's disease brain, a high degree of intersubject variability was observed: some samples showed markedly reduced GluR1 mRNA levels in dentate gyrus, CA1 and CA3 relative to controls; others showed no changes. Microscopic observation of emulsion-dipped sections revealed that the reduction of GluR1 seen in the dentate gyrus and CA3 of some Alzheimer's disease subjects was not due to cell loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Mutagenesis rescues spermine and Zn2+ potentiation of recombinant NMDA receptors.

Alternative splicing generates distinct forms of the NMDA receptor subunit NR1. NR1 subunits with an N-terminal insert (termed N1) form receptors in Xenopus oocytes with greatly reduced potentiation by spermine and Zn2+. Oocytes expressing NR1 receptors with N1 exhibited larger NMDA currents than oocytes expressing corresponding receptors without N1. In the present study, we used mutational analysis to investigate structural features of the N1 insert that control current amplitude and spermine and Zn2+ potentiation. Neutralization of positive charges in N1 rescued spermine and Zn2+ potentiation. Positive charges in N1 did not affect spermine or Zn2+ affinity. Neutralization of positive charges in N1 diminished the responses to the level of NR1 receptors lacking N1. The positively charged N1 may increase NMDA currents by causing a conformational change similar to that produced by spermine and Zn2+ in NR1 receptors lacking N1.

Amino Acid Sequence↗
PubMed1994

Characterization of gap junctions between pairs of Leydig cells from mouse testis.

Leydig cells are coupled in vivo by numerous gap junctions. In vivo and in vitro cells were immunolabeled by connexin 43 (Cx43) but not by Cx26 or Cx32 antibodies; immunoblotting confirmed specificity of Cx43 labeling. Pairs of Leydig cells dissociated from mouse testis were studied by dual whole cell voltage clamp, and a high incidence of dye (n = 20) and electrical coupling (n = 60; > 90%) was found. Coupling coefficients were near 1 and junctional conductance (gj) averaged 7.2 +/- 1.2 nS (SE, n = 40). Large transjunctional voltage (Vj) decreased gj; currents decayed exponentially with time constants of seconds that decreased at greater Vj. The residual conductance at large Vj was at least approximately 40% of the initial conductance. Exposure of cell pairs to saline solutions saturated with CO2 (n = 15) or containing 2 mM halothane (n = 15) or 3.5 mM heptanol (n = 15) rapidly and reversibly reduced gj. In eight cell pairs, gating of single junctional channels was observed during halothane-induced reduction in gj. Most gating events at Vj < 40 mV were fit by a Gaussian distribution with a mean of approximately 100 pS. With Vj > 40 mV, smaller transitions of approximately 30 pS were also recorded, and the frequency and duration of the approximately 100-pS transitions decreased. Also, approximately 70-pS transitions between 30- and 100-pS conductances were observed in the absence of 70-pS transitions to or from the baseline, indicating that the 30-pS conductance was a substate induced by large Vj.

Alcohols↗
PubMed1994

The connexins and their family tree.

The connexins, gap junction forming proteins, are encoded by a gene family. Sequence comparisons reveal regions of conservation with functional implications for voltage dependence of junctional conductance, junction formation and regulation by phosphorylation. The best connexin tree shows that most gene duplications giving rise to the family occurred early in or before vertebrate divergence. The topology of most deep branches of the tree is uncertain. Evolutionary rates vary for different paralogous connexin genes.

Amino Acid Sequence↗
PubMed1994

Kainate-induced status epilepticus alters glutamate and GABAA receptor gene expression in adult rat hippocampus: an in situ hybridization study.

In adult rats, intraperitoneal administration of kainic acid, a glutamic acid analog and potent neurotoxin, induces persistent seizure activity that results in electrographic alterations and neuropathology that closely resemble human temporal lobe epilepsy. We used in situ hybridization to identify regions of altered glutamate and GABAA receptor gene expression following kainate-induced status epilepticus. In the CA3/CA4 area, the hippocampal region most vulnerable to neurodegeneration after kainate acid treatment, expression of GluR2 (the AMPA/kainate receptor subunit that limits Ca2+ permeability) and GluR3 was decreased markedly at 12 and 24 hr, times preceding neurodegeneration. These findings raise the possibility that increased formation of Ca(2+)-permeable AMPA/kainate receptors in the CA3/CA4 area may enhance glutamate pathogenicity. Expression of the GABAA alpha 1, subunit was also reduced, indicating a possible decrease in inhibitory transmission, which would also enhance excitotoxicity. GluR1 and NR1 expression was not significantly changed. In the dentate gyrus, a region resistant to neurodegeneration, concomitant increases in GluR2 and GluR3 expression were observed; GluR1, NR1, and GABAA alpha 1 mRNAs were not detectably altered. Analysis of emulsion-dipped sections revealed that the changes in GluR2, GluR3, and GABAA alpha 1 expression represented changes in mRNA content per neuron and were specific to pyramidal cells of the CA3/CA4 area and to granule cells of the dentate gyrus. These findings indicate that kainate seizures modify hippocampal glutamate and GABAA receptor expression in a cell-specific manner. Timing of the changes in glutamate and GABAA receptor mRNAs indicates that these changes may play a causal role in hippocampal neuronal cell loss following kainate-induced seizures.

Animals↗
PubMed1993

Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C.

The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NR1 variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1(111) has all three exons, NR1(000) has none, and NR1(100) has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NR1 receptor expressed in Xenopus oocytes. NR1 receptors that lacked the N-terminal exon (NR1(000), NR1(010), and NR1(011)) exhibited a relatively high affinity for NMDA (EC50 approximately 13 microM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR1(100), NR1(101), and NR1(111)) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. All six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3- to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.

Alternative Splicing↗
PubMed1993

Effects of polyamines on NMDA-induced currents in rat hippocampal neurons: a whole-cell and single-channel study.

Actions of the polyamines spermine and spermidine on NMDA-induced currents were examined in cultured hippocampal neurons from embryonic rat. In whole-cell patch experiments using voltage-clamp, spermine (300 microM) produced about a two-fold potentiation of responses to NMDA (at -70 mV in the presence of saturating glycine); half-maximal potentiation was elicited at 207 microM. The potentiation produced by spermine was somewhat greater at positive potentials. The onset of potentiation was fast (t1/2 < 1 s), indicative of an extracellular site of action. Spermidine was of comparable potency but less efficacious than spermine in potentiating NMDA responses. In excised outside-out patches, spermine exhibited two actions on NMDA-induced single-channel responses. In some patches, it increased the channel open probability; both frequency of channel opening and burst length were increased with no significant change in the mean open duration, which accounted for much of the potentiation seen in whole-cell experiments. In all patches, spermine decreased channel conductance at negative voltages, an effect ascribable to fast channel block (with a possible contribution by charge screening). These results are consistent with opposing actions of polyamines mediated at distinct sites on the NMDA receptor.

Action Potentials↗
PubMed1993

Molecular biology of PCP and NMDA receptors.

The studies described demonstrate that rat brain mRNA directs the synthesis of at least four types of functional EAA receptors in the Xenopus oocyte system, whereas in this system NCB-20 cell mRNA directs the synthesis of only the NMDA type of EAA receptor. The NMDA channel expressed in the oocyte, using either rat brain mRNA or NCB-20 cell mRNA, exhibits the pharmacologic properties of the neuronal receptor, including the functional association with the PCP receptor located within the NMDA-gated channel. The demonstration that mRNA isolated from NCB-20 cells lacking functional NMDA-activated channels, but bearing PCP binding sites, can encode functional NMDA-activated channels in the oocyte indicates some defect or regulating step in posttranslational processing or insertion of the receptors into the plasma membrane in the cell of origin. This is the only cell line known to (1) have PCP receptors that appear to be associated with NMDA receptors and (2) provide a homogeneous, self-replicating population of cells that can be manipulated genetically and by changing the extracellular environment. Consequently, the NCB-20 cell line will be useful for the study of the NMDA receptor and its expression.

Animals↗
PubMed1992

Cultured sympathetic neurons synthesize and release the cytokine interleukin 1 beta.

Autonomic neurons help to regulate immune responses, and there are reciprocal interactions between the nervous and immune systems. This study seeks to define some of the molecular mechanisms that may underlie such interactions. Immunoblot analysis indicated that cultured sympathetic neurons synthesize and release the cytokine interleukin 1 beta (IL-1 beta). In addition, RNA blot analysis of cultured sympathetic neurons demonstrated that the neurons contain mRNA encoding IL-1 beta. It was previously shown that explant cultures of sympathetic ganglia and dissociated cocultures of neurons with ganglionic nonneuronal cells synthesize substance P, whereas in situ levels of substance P and its mRNA are low. An antagonist at the interleukin 1 receptor markedly depressed this increase in substance P in cultures, suggesting that endogenous IL-1 beta mediates the synthetic response, at least in part. Because pure neuronal cultures do not contain substance P and neurons synthesize and release IL-1 beta, the actions of the cytokine require the presence of ganglion nonneuronal cells. These observations suggest a role for autonomic neurons in influencing immune responses by synthesizing and secreting at least two known immunoregulators, the cytokine IL-1 beta and the neuropeptide substance P.

Animals↗
PubMed1992

Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.

Analysis of Variance↗
PubMed1992

Cloning of an apparent splice variant of the rat N-methyl-D-aspartate receptor NMDAR1 with altered sensitivity to polyamines and activators of protein kinase C.

Molecular cloning identified complementary DNA species, from a rat ventral midbrain library, encoding apparent splice variants of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 (which we now term NR1a). Sequencing revealed that one variant, NR1b, differs from NR1a by the presence of a 21-amino acid insert near the amino end of the N-terminal domain and by an alternate C-terminal domain in which the last 75 amino acids are replaced by an unrelated sequence of 22 amino acids. NR1b is virtually identical to NR1a in the remainder of the N- and C-terminal domains, at the 5' and 3' noncoding ends, and within the predicted transmembrane domains and extracellular and cytoplasmic loops. These findings suggest that the two forms of the receptor arise by differential splicing of a transcript from the same gene. Sequencing of other clones indicates the existence of a third variant, NR1c, identical to NR1b in its C terminus but lacking the N-terminal insert. NR1b RNA injected into Xenopus oocytes generated functional homomeric NMDA channels with electrophysiological properties distinct from those of NR1a homomeric channels. NR1b channels exhibited a lower apparent affinity for NMDA and for glutamate. NR1b channels exhibited a lower affinity for D-2-amino-5-phosphonovaleric acid and a higher affinity for Zn2+. The two receptor variants showed nearly identical affinities for glycine, Mg2+, and phencyclidine. Spermine potentiation of NMDA responses, prominent in oocytes injected with rat forebrain message, was also prominent for NR1a receptors, but was greatly reduced or absent for NR1b receptors. Treatment with the protein kinase C activator phorbol 12-myristate 13-acetate potentiated NMDA responses in NR1b-injected oocytes by about 20-fold; potentiation of NMDA responses in NR1a-injected oocytes was much less, about 4-fold. These findings support a role for alternate splicing in generating NMDA channels with different functional properties.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Are Ca(2+)-permeable kainate/AMPA receptors more abundant in immature brain?

The permeability properties of kainate/AMPA receptors are determined by subunit composition. The GluR1 and GluR3 subunits form Ca(2+)-permeable channels and exhibit inward rectification; heteromeric receptors containing the GluR2 subunit are Ca(2+)-impermeable and electrically linear. These observations raise the possibility of a developmental 'switch' in which turning on or off of GluR2 expression regulates the level of Ca2+ permeable kainate/AMPA receptors. We examined the ratio of GluR1 and GluR3 to GluR2 gene expression in developing and adult rat brain by in situ hybridization. A larger value of this ratio is likely to be associated with greater Ca2+ permeability. Our data suggest that in neocortex, striatum and cerebellum the number of Ca(2+)-permeable kainate/AMPA receptors is high at P4 and declines monotonically with age. In hippocampus, the number increases from P7 to P21, after which it declines. These findings provide evidence for a developmental 'switch' in which Ca2+ permeable glutamate receptors are turned off following early developmental events.

Aging↗
PubMed1992

A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32.

We have developed a procedure for the replacement of defined domains with specified domains from other proteins that we used to examine the molecular basis for the differences in voltage-dependent gating between connexins 26 (Cx26) and 32 (Cx32). This technique does not depend on sequence homology between the domains to be exchanged or the presence of restriction endonuclease sites. Rather, it makes use of a PCR strategy to create an adhesive "band-aid" that directs the annealing of the amplified sequence to the correct location in the recipient clone. With this technique we created a series of chimeras involving the replacement of topologically defined protein domains of Cx32 with the corresponding sequences of Cx26. We focused on domains that are predicted to line the gap junction channel as we expect that a component of the voltage-sensing mechanism resides there. Differences between Cx26 and Cx32 in the sequences of their first and second extracellular loops, the cytoplasmic loop, and the third transmembrane domain did not account for the difference in their calculated gating charges. Shifts along the voltage axis in the steady-state conductance-voltage relations of the chimeric connexins were produced by replacement of the first extracellular loop or the cytoplasmic loop and the amino-terminal half of the third transmembrane domain. These data suggest that the voltage-sensing mechanism arises from the interaction of domains lining the aqueous channel and domains deeper in the channel wall.

Amino Acid Sequence↗