Search PubMed⌕ Search

Biomedical subjects

G Lynch

Publications and source records attributed to G Lynch.

At least 19 recordsLinked to original sources
PubMed1995

Proteolysis of spectrin by calpain accompanies theta-burst stimulation in cultured hippocampal slices.

Tests were carried out to determine if repetitive bursts of afferent stimulation activate calpain, a calcium-dependent protease hypothesized to be involved in the production of long-term potentiation. Antibodies against a stable breakdown product that results from proteolysis of spectrin by calpain were used to identify sites of enzyme activation in cultured hippocampal slices. Slices in which theta-burst stimulation was applied to the Schaffer collateral fibers had pronounced accumulations of breakdown product that were restricted to field CA1, the zone innervated by the stimulated axons. Labelling occurred in the form of scattered puncta and was also present in dendritic processes. The extent of these effects was correlated (r = 0.73) with the amount of theta-burst stimulation delivered. Control slices or those receiving low frequency stimulation had variable, but uniformly lower, amounts of breakdown product and were clearly distinguishable from those given theta bursts. Statistical analyses using a six point rating scheme confirmed this point (P < 0.001). These results satisfy an essential prediction of the hypothesis that calpain plays an important role in the induction of long-term potentiation.

Afferent Pathways↗
PubMed1995

Comparison of long-term potentiation in the proximal versus distal stratum radiatum of hippocampal field CA1.

Recent studies indicate that long-term potentiation is accompanied by changes in the waveform of field excitatory postsynaptic potentials in the CA1 field of disinhibited hippocampal slices, suggesting that long-term potentiation alters the kinetics of the glutamate receptor channels that mediate excitatory synaptic responses. The present study examined the effects of stimulating and recording location within stratum radiatum on the magnitude of long-term potentiation and the associated waveform changes. Stimulation of stratum radiatum proximal to the cell body layer evoked field excitatory postsynaptic potentials in proximal stratum radiatum that had decay phases well-fit by single exponentials; long-term potentiation reduced the decay time constant of these potentials, as reported previously. Stimulation of distal stratum radiatum evoked field excitatory postsynaptic potentials in distal stratum radiatum that were contaminated by positive after-potentials; meaningful estimates of decay time constants for these responses could not be obtained. Long-term potentiation of distal responses tended to be smaller than that obtained proximally. Comparisons were also made between responses recorded distally and proximally for either distal or proximal stimulation. For both stimulation loci, the distally-recorded responses had positive after-potentials and the proximally-recorded responses did not. The decay time constants for proximally-recorded responses to distal stimulation decreased significantly after long-term potentiation. Proximal and distal stimulation both produced greater long-term potentiation recorded proximally than distally and the difference was significantly greater for proximal stimulation. When long-term potentiation was induced by stimulation of proximal and distal sites simultaneously, the difference between proximally- and distally-recorded long-term potentiation was significantly reduced. Paired-pulse facilitation was greater when recorded proximally but the variation in facilitation with stimulation and recording position was different from that obtained for long-term potentiation. Paired-pulse facilitation of response amplitude was slightly reduced after long-term potentiation, but the change did not depend on the stimulation-recording arrangement; facilitation of response slope was not reliably affected by long-term potentiation. These results indicate that the shape of the dendritic field excitatory postsynaptic potential is influenced by the relative position of recording electrodes in stratum radiatum; when the position is such that after-potentials are minimized, long-term potentiation produces a decrease in the decay time constant of the synaptic field potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Effect of thiocyanate on AMPA receptor mediated responses in excised patches and hippocampal slices.

The binding affinity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for [3H]AMPA is increased 10-30-fold by the chaotropic anion thiocyanate. The present experiments tested if thiocyanate alters AMPA receptor mediated current fluxes and if any such effects are reflected in the waveform of synaptic responses. Currents were measured after a step application of glutamate or AMPA to patches excised from pyramidal cells of hippocampal slice cultures. Application of 1 mM AMPA produced responses with an average peak amplitude of 86 pA at -50 mV and a 10-90% rise time of 1.7 +/- 0.1 ms; the responses desensitized to a steady-state level below 10% of the peak current with a time constant of 11.1 +/- 0.7 ms. Glutamate in presence of D-amino-phosphonopentanoate produced similar responses which were inhibited by 6-cyano-7-nitro-quinoxaline-dione and enhanced by aniracetam or cyclothiazide and thus are characteristic for AMPA receptors. Thiocyanate accelerated the decay of AMPA responses two-fold and reduced the peak current by 30-50% with an EC50 of 3.2 mM which is comparable to its EC50 for enhancing binding. Effects on the desensitization of glutamate induced responses were much smaller and only evident at the highest thiocyanate concentration; no effect was seen on response amplitude. Binding and physiological effects can be adequately explained by assuming that thiocyanate enhances conversion from the sensitive to the desensitized state of the receptor and reduces ligand dissociation from the desensitized state. Synaptic responses were measured in disinhibited hippocampal slices. Perfusion with 20 mM sodium thiocyanate increased the slope of the field excitatory postsynaptic potential by 44.9 +/- 4.2% and reduced its decay time by 10.4 +/- 4.3%. The former effect appears to result at least in part from an increase in transmitter release since it was accompanied by a decrease in paired-pulse facilitation and was reduced in magnitude after enhancing transmitter release. The decrease in the decay time constant points to an effect of thiocyanate on AMPA receptors in situ which is similar to that seen in excised patches. These results demonstrate that an increase in binding affinity may be indicative of reduced rather than enhanced current flow through AMPA receptors. In addition, the results provide further evidence that the kinetics of the AMPA receptor channel contribute significantly to at least the decay phase of fast excitatory synaptic responses.

Animals↗
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

Sulfated polyanions prevent HIV infection of lymphocytes by disruption of the CD4-gp120 interaction, but do not inhibit monocyte infection.

Sulfated polyanions (SPs) bind variably to lymphocyte-expressed CD4 and inhibit binding of monoclonal antibodies to the first two domains of CD4. To further define this interaction, soluble recombinant CD4 (sCD4; four extracellular domains), its truncated amino-terminal two-domain derivative, and three linear peptide analogues spanning residues 6-60 (6-24, 20-40, 41-60) in the first domain were investigated for SP binding. Dextran sulfate (DXS) (500 kDa), polyvinyl sulfate, fucoidan, and carrageenan-kappa, each immobilized on carboxymethyl cellulose fibers, bound strongly to both the two-domain and four-domain recombinant CD4 molecules (similar to that observed with native CD4), whereas dextran sulfate (5 kDa), chondroitin 6-sulfate, and pentosan sulfate bound relatively poorly. No peptide binding to SPs was observed. Recombinant gp120 bound poorly (< 10%) to all of the immobilized polyanions, except pentosan sulfate (17%), for which some binding was noted. Binding of radiolabeled V3 loop peptide to SPs was slightly greater, with 20-30% binding to polyvinyl sulfate, dextran sulfate (500 kDa), and pentosan sulfate. Competitive binding studies demonstrated the predominance of sCD4 rather than rgp120 binding to SPs and supported previous data demonstrating a binding site for DXS (500 kDa) on the first domain of CD4 adjacent to the gp120 binding site and recognized by OKT4C and E monoclonal antibodies. Hence disruption of the CD4-gp120 interaction is probably responsible for most of the observed antiviral activity of SPs toward HIV infection of lymphocytes. However, HIV infection and gp120 binding to monocytes was unaffected by SPs, probably because SPs were unable to block the CD4-gp 120 interaction in monocytes.

Amino Acid Sequence↗
PubMed1994

Kainate binding to the AMPA receptor in rat brain.

Displacement of [3H]AMPA and [3H]CNQX by kainate was measured in membranes and solubilized fractions from rat brain. In soluble fractions, plots of [3H]AMPA and [3H]CNQX binding displaced by kainate resulted in one-site fits with Ki values in the range of 1-3 microM. In membranes, plots of [3H]AMPA binding displaced by kainate resulted in graphs which were better fit by two-site regression analysis than by a one-site fit. The Ki value for the high-affinity component of these two-site fits was 3-9 microM and the low-affinity component Ki was in the range of 70-120 microM; similar values were determined for kainate displacement of [3H]CNQX. The presence of thiocyanate ions had no effect on kainate displacement of [3H]CNQX. Since the affinity for kainate of the presumed synaptic AMPA receptor is in the range of EC50 values for kainate determined from physiological studies, these data contribute further evidence for the idea that kainate binding to synaptic AMPA receptors may be responsible for many of kainate's physiological effects.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
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↗
PubMed1993

Cyclothiazide decreases [3H]AMPA binding to rat brain membranes: evidence that AMPA receptor desensitization increases agonist affinity.

The effects of cyclothiazide, a drug which blocks AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor desensitization, were tested on binding of [3H]AMPA to rat brain membranes. Cyclothiazide reduced [3H]AMPA binding by lowering the apparent affinity of the AMPA receptor. The magnitude of the decrease was temperature dependent and greater for membrane-bound than for solubilized receptors. These data provide evidence that desensitization increases the affinity of the AMPA receptor for agonists and indicate that a significant percentage of AMPA receptors in conventional equilibrium binding assays are in a desensitized state.

Animals↗
PubMed1992

Factors regulating the magnitude of long-term potentiation induced by theta pattern stimulation.

Electrical stimulation patterned after the hippocampal theta rhythm produces a robust and stable long-term potentiation (LTP) effect. Pharmacological manipulations were used in the present studies in an effort to relate characteristics of the responses occurring during theta stimulation to the magnitude of potentiation which follows it. Comparisons were made using five or ten bursts of stimulation which respectively induce sub-maximal or near maximal degrees of LTP. DPCPX, a drug that increases release by blocking adenosine A1 receptors, was used to enhance the depolarization produced by individual theta bursts. This resulted in a marked increase in the amount of stable LTP induced by five theta bursts but did not affect that resulting from ten bursts. This finding suggested that depolarization occurring during a burst response influences per burst potentiation but not the ceiling on maximum LTP. Aniracetam, a nootropic drug that enhances responses via an action on glutamate (AMPA) receptors, was used to test this conclusion. Like DPCPX, aniracetam increased the size of the burst response and enhanced the degree of LTP caused by five but not ten theta bursts. Forskolin, an activator of adenylate cyclase, was used to test the effects of blocking the hyperpolarization normally present between theta bursts on the induction of LTP. The drug augmented the degree of LTP resulting from five theta bursts and, in contrast to DPCPX and aniracetam, nearly doubled that obtained with ten bursts. Thus the drug affected both per burst potentiation and the ceiling on LTP. These results are discussed in terms of an hypothesis in which the magnitude of NMDA receptor mediated currents affects the degree of potentiation produced by individual theta bursts while the duration of the currents is related to the limit on the maximum LTP induced by a series of bursts. The possible implications of the findings for learning are also considered.

Animals↗
PubMed1992

Seizure activity-induced changes in polyamine metabolism and neuronal pathology during the postnatal period in rat brain.

Systemic injection of kainic acid (KA) does not cause neuronal pathology in limbic structures in rat brain prior to postnatal day (PND) 21. The present study tested if the development of the pathogenic response is associated with the maturation of a link between seizure activity and polyamine metabolism. Pathology was assessed with histological techniques and with the binding of [3H]Ro5-4864, a ligand for the peripheral type benzodiazepine binding sites (PTBBS), a marker of glial cell proliferation. In agreement with previous results, peripherally administered kainate at doses sufficient to induce intense behavioral seizures produced a loss of Nissl staining in hippocampus after PND 21 but not at earlier ages. The pattern of neuronal damage observed after PND 21 resembled that found in adult animals: extensive losses of Nissl staining in area CA3 of hippocampus and in piriform cortex, more modest effects in CA1 and sparing of the granule cells of the dentate gyrus. Similarly, no increase in [3H]Ro5-4864 binding as a result of KA administration was observed in hippocampus and piriform cortex until PND 21. Ornithine decarboxylase (ODC) activity and putrescine levels were high in the neonatal brain and decreased to reach adult values by PND 21. KA-induced seizure activity did not significantly alter both variables until PND 21. After PND 21, ODC activity and putrescine levels markedly increased 16 h after KA-induced seizure activity in hippocampus and piriform cortex. The magnitude of the effects increased between PND 21 and PND 30, at which point the changes in both parameters were comparable to those found in adults. Polyamines stimulate the activity of the calcium-dependent proteases calpain in brain fractions and may increase calpain-mediated proteolysis in situ. In accord with this, kainate-induced breakdown of spectrin, a preferred substrate of calpain, measured 16 h after KA injection followed a developmental curve parallel to that for kainate-induced increases in putrescine levels. These results indicate that the onset of vulnerability to seizure activity triggered by kainic acid is correlated with the development of an ODC/polyamine response to the seizures and further support a critical role for the ODC/polyamine pathway in neuronal pathology following a variety of insults.

Animals↗
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↗
PubMed1992

Purification of an Arg-Gly-Asp selective matrix receptor from brain synaptic plasma membranes.

Brain synaptic plasma membranes specifically associated with matrix protein monolayers containing the Arg-Gly-Asp sequence recognized by integrin-type adhesion receptors. Experiments using fibronectin affinity chromatography to identify the synaptosomal receptors responsible for this interaction led to the purification of a 55 kDa Arg-Gly-Asp recognition protein that is labelled by antibodies against the alpha 5 beta 1 integrin.

Amino Acid Sequence↗
PubMed1992

Factors governing the potentiation of NMDA receptor-mediated responses in hippocampus.

A modified medium containing an AMPA receptor antagonist and low concentrations of magnesium was used to investigate the factors governing the potentiation of synaptic responses mediated by NMDA receptors. When long-term potentiation (LTP) was induced in standard medium and NMDA responses were analyzed by changing to the modified medium, no statistically significant differences were observed between potentiated and control pathways. Returning the slices to the standard medium showed that LTP was still present, indicating that the potentiation effect was not reversed by the modified medium. High-frequency stimulation applied in the modified medium produced an enhancement of synaptic responses, but this was not occluded by prior potentiation in standard medium. The degree of potentiation induced in the modified medium and expressed by NMDA responses was larger in the presence than in the absence of inhibition and, unlike LTP, was proportionately larger when recorded in the stratum pyramidale than in the stratum radiatum. These results indicate that the potentiation of NMDA receptor-mediated responses triggered by high-frequency stimulation applied in modified medium differs in several respects from the LTP induced in standard conditions. They confirm that LTP is expressed to a markedly different degree by NMDA and non-NMDA receptors and suggest that events that do not necessarily accompany LTP affect the potentiation of NMDA receptor-dependent synaptic responses.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1992

Receptor changes and LTP: an analysis using aniracetam, a drug that reversibly modifies glutamate (AMPA) receptors.

The hypothesis that long-term potentiation (LTP) involves receptor modifications was tested with aniracetam, a nootropic drug that selectively increases currents mediated by the AMPA subclass of glutamate receptors. Aniracetam had different effects on the waveform of synaptic potentials in hippocampus before and after induction of LTP: (1) the drug caused a slight reduction (or delay) of the initial segment of the response after LTP; and (2) the facilitatory effects of aniracetam occurred at a later time point in the response after LTP than before. The interactions between LTP and aniracetam were still present when synaptic responses were greatly reduced by partial blockade of postsynaptic receptors and were not reproduced by increasing release or the number of stimulated synapses. A mathematical treatment of synaptic currents produced the following results: (1) if aniracetam facilitates AMPA receptor currents simply by reducing desensitization, then its complex interaction with LTP emerges when potentiation changes the kinetic and conductance properties of receptor channels; (2) if aniracetam also significantly increases conductance, then the experimental data can be reproduced by modeling LTP as an increase in channel conductance alone.

Animals↗
PubMed1992

Changes in polyamine levels and spectrin degradation following kainate-induced seizure activity: effect of difluoromethylornithine.

The induction of ornithine decarboxylase (ODC) in adult CNS and the resulting changes in polyamine levels are often observed under conditions associated with activation of NMDA receptors, calpain stimulation and spectrin degradation. The present study was directed at evaluating the links between these two sets of events. We measured the effects of an acute treatment of adult rats with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, on biochemical alterations following kainate-induced seizure activity. Beside ODC activity and polyamine levels, we assayed the in situ spectrin degradation and the in vitro binding of 3H-Ro5-4864, a ligand for the peripheral benzodiazepine binding sites which is a good marker of glial proliferation, at various time intervals following systemic kainic acid (KA) injection. Kainate-induced seizure activity was followed by a transient increase in ODC activity, a long-lasting increase in putrescine levels and spectrin degradation, and a delayed increase in 3H-Ro5-4864 binding, mainly in hippocampus and piriform cortex. Treatment of the animals with DFMO markedly reduced the increase in putrescine levels up to 7 days after KA injection. It also reduced the increase in spectrin breakdown observed at 16 h but not at 4 and 7 days after KA injection. Finally, it did not modify the increase in 3H-Ro5-4864 binding measured 4 and 7 days after KA injection. The levels of putrescine were positively correlated with the extent of spectrin proteolysis in KA-treated animals whether or not they were treated with DFMO, at 16 h but not at 7 days after KA injection. The results indicate that the extent of spectrin breakdown observed shortly after KA-induced seizure activity is causally related to the changes in ODC activity and putrescine levels. Although the data are consistent with the idea that putrescine could be a marker for acute pathology, they do not support a role for polyamines in delayed neurotoxicity.

Animals↗
PubMed1992

Benzodiazepines block long-term potentiation in slices of hippocampus and piriform cortex.

The effects of two benzodiazepines, diazepam and triazolam, on long-term potentiation were tested in slices of hippocampus and piriform cortex. The drugs had little influence on baseline synaptic responses but both were very effective in blocking LTP elicited by theta pattern stimulation. The effects were fully reversible upon washout. Diazepam reduced the increase in burst responses that occurs during theta stimulation and thus appears to interfere with the initial triggering events for long-term potentiation. This may reflect the enhancing action of the drug on GABA-mediated inhibitory potentials. Triazolam did not detectably change the burst responses elicited by theta pattern stimulation. Experiments with slices of piriform cortex indicated that triazolam also failed to disrupt the development of long-term potentiation but instead caused the potentiation to decay back to baseline in 15-30 min. Triazolam thus seems to act on the mechanisms that stabilize long-term potentiation. These results provide a possible explanation for the amnestic effects of benzodiazepines in humans and animals and support the hypothesis that long-term potentiation contributes to memory encoding.

Analysis of Variance↗
PubMed1992

Evidence that high- and low-affinity DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites reflect membrane-dependent states of a single receptor.

Binding of DL-alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([3H]AMPA) to lysed rat brain membranes in the presence of potassium thiocyanate resulted in curvilinear Scatchard plots that could be resolved by regression analysis into a large low-affinity component and a small high-affinity component. Solubilization with Triton X-100 resulted in solubilized and nonsolubilized fractions that were considerably enriched in the high-affinity component and correspondingly reduced in the low-affinity component. It thus appears that solubilization converts low-affinity AMPA receptors into high-affinity receptors. Also, synaptic plasma membranes were found to be greatly enriched in the low-affinity form and deficient in the high-affinity form of the AMPA receptor. These experiments provide evidence for the hypothesis that the high- and low-affinity components of AMPA binding are interconvertible states of the same receptor rather than separate binding sites and that the conversion of these receptors from their native high-affinity state to the low-affinity state occurs on insertion of the receptors into synapses.

Animals↗