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J A Connor

Publications and source records attributed to J A Connor.

At least 19 recordsLinked to original sources
PubMed1995

Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice.

The magnitude and dynamics of [Ca2+] changes in spines and dendrites of hippocampal CA1 pyramidal neurons have been characterized using a low affinity fluorescent indicator, mag-Fura 5, that is sensitive to Ca2+ in the micromolar range. During tetanic stimulation (1 s), we observed progressive [Ca2+] increases in distal CA1 spines to as much as 20-40 microM, both in organotypic slice culture and acute slice. Similar accumulations were reached during continuous depolarization (+10 mV, 1 s) when K+ channels had been blocked, but not with spike trains driven by postsynaptic current injection. The large [Ca2+] increases due to tetanic stimulation were blocked by APV, indicating that NMDA receptor-dependent influx was critical for the large responses. These findings have significant implications for low affinity Ca(2+)-dependent biochemical processes and show a new upper limit for [Ca2+] changes measured in these neurons during stimulation.

2-Amino-5-phosphonovalerate↗
PubMed1995

Long-term depression of naïve synapses in adult hippocampus induced by asynchronous synaptic activity.

1. Two independent Schaffer collateral pathways converging to the same pyramidal cell were alternately stimulated by 2-Hz trains (900 pulses) offset by a 150-ms interval in adult rat hippocampal slices. The second input underwent an immediate and persistent long-term depression (LTD). Depression in the first input was smaller than the second input. A narrower interpulse interval (20 ms) failed to induce LTD in either input. 2. Neither the N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid nor the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxylphenyl-glycine blocked this associative LTD. However, coapplication of these two antagonists blocked LTD. 3. Associative LTD was blocked by prior injection of the Ca2+ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid into the postsynaptic cell and by bath-applied L-NG-nitroarginine, a nitric oxide synthesis inhibitor. 4. We propose that temporally confined, asynchronous synaptic activity weakens the efficacy of naïve synapses in slices from the adult hippocampus.

Animals↗
PubMed1994

Dendritic Ca2+ accumulations and metabotropic glutamate receptor activation associated with an N-methyl-D-aspartate receptor-independent long-term potentiation in hippocampal CA1 neurons.

Bathing hippocampal slices in the potassium channel blocker tetraethylammonium (TEA), while stimulating the Schaffer collaterals at a low frequency, induces Ca(2+)-dependent, N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation of synaptic transmission (LTPk) in CA1 neurons. We have combined ratio imaging of fura-2 and mag-fura-5 in hippocampal CA1 neurons with intracellular and field recordings to evaluate postsynaptic Ca2+ changes that occur in the induction of LTPk. Test stimuli were applied at 0.05 Hz to stratum radiatum in the presence of the NMDA receptor antagonists D,L-2-amino-5-phosphonovaleric acid (100 microM) or MK-801 (10 microM). During TEA exposure (15-25 mM; 10 min), cells fired prolonged action potentials both spontaneously and in response to test stimuli resulting in transient, micromolar Ca2+ accumulations in both somata and dendrites. The initial EPSP slope, measured 60 min after TEA wash-out, was potentiated to approximately 200% of control. The Ca2+ channel blocker nimodipine (10 microM) greatly reduced Ca2+ transients in both magnitude and duration and prevented LTPk induction. Pretreatment of slices with compounds that block metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis, L-2-amino-3-phosphonopropionic acid (L-AP3, 50-200 microM) or L-aspartate-beta-hydroxamate (50-100 microM), as well as protein kinase C (PKC) inhibitors (sphingosine, 20 microM; RO-31-8220, 0.2 microM; or calphostin C, 2 microM) also blocked LTPk. Ca2+ transients were unaffected by L-AP3 or RO-31-8220. These findings suggest that Ca2+ influx through voltage-gated channels and co-activation of PKC by mGluRs are both necessary for induction of LTPk. Activation of mGluRs must also occur in NMDA receptor-dependent induction paradigms, but is possibly of lesser importance owing to the much greater gating of Ca2+ directly into the dendritic spines.

2-Amino-5-phosphonovalerate↗
PubMed1994

Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists.

A predictable pattern of selective neuronal cell death occurs in organotypic slice cultures of neonatal rat hippocampus during the second and third weeks in vitro. We serially examined organotypic cultures at three, four, seven, 14, 21 and 28 days in vitro, using uptake of the fluorescent dye propidium iodide to identify degenerating cells. After seven days in vitro, the cell degeneration that accompanies the slicing procedure appears to have ended. However, at 14 days in vitro, degenerating neurons could be identified in area CA3. When many degenerating cells were present in a slice, they were distributed in the dentate hilus (CA4) and proximal portions of CA1 as well. Neuronal degeneration involving mainly CA1 pyramidal cells was still apparent at 21 days in vitro, but was much less marked than at 14 days. Study of fixed cultures with light and electron microscopy methods confirmed the presence of degenerating neurons with a pyknotic or vacuolated appearance. Spontaneous neuronal degeneration at 14 and at 21 days in vitro was almost entirely prevented by the addition of 10.5 mM Mg2+ or 3 mM kynurenic acid (a glutamate receptor antagonist), beginning at seven days in vitro. Cell death was markedly decreased by treatment with 100 microM DL-2-amino-5-phosphonovaleric acid (a selective antagonist of N-methyl-D-aspartate glutamate receptors). Removal of the blocking agents by returning cultures to control media at 28 days in vitro induced widespread neuronal degeneration, involving all the regions of the hippocampal slice cultures. The inhibition of spontaneous neuronal cell death by glutamate receptor antagonists and by blockade of glutamate release at synapses suggests that the mechanism of cell death involves glutamate receptors. The time course of degeneration suggests that the vulnerability to glutamate excitotoxicity is an aspect of developmentally regulated components of glutamatergic synapses acquired in the hippocampal organotypic cultures after the first week in vitro.

2-Amino-5-phosphonovalerate↗
PubMed1994

Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death.

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

Animals↗
PubMed1994

Weak excitation and simultaneous inhibition induce long-term depression in hippocampal CA1 neurons.

1. Weak excitation to rat hippocampal CA1 neurons via Schaffer collaterals at a frequency of 0.1 or 0.2 Hz accompanied by repeated brief exposures to the inhibitory transmitter gamma-amino-butyric acid (GABA) causes a long-term depression (LTD, up to 90% of the control) of the stimulated pathway. This depression can be reversed by high-frequency stimulation. 2. Although inhibition is necessary for the induction of this LTD, the depression can be produced with either the GABAA or the GABAB receptor agonists. 3. This conjunctive LTD could not be blocked by the N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovaleric acid. 4. It was, however, blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonopropionic acid and (RS)-alpha-methyl-4-carboxyphenylglycine, indicating that activation of a metabotropic glutamate receptor is necessary for the LTD. Induction also appeared to require an intracellular Ca2+ increase. 5. Because GABAergic inhibition often modulates glutamatergic transmission in the brain, we propose that this form of synaptic modification is of potential importance for neural plasticity.

Animals↗
PubMed1994

Trans-ACPD, a metabotropic receptor agonist, produces calcium mobilization and an inward current in cultured cerebellar Purkinje neurons.

1. 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a racemic mixture of 1-aminocyclopentane-1S,3R-dicarboxylic acid and 1-aminocyclopentane-1R,3S-dicarboxylic acid, a selective agonist of the metabotropic glutamate receptor, was applied to mouse Purkinje neurons (PNs) in culture. Measurements of free intracellular Ca2+ were made using fura-2 microfluorimetric imaging and of membrane current using perforated-patch voltage-clamp recording in separate experiments. 2. Brief pulses of t-ACPD (< or = 100 microM, 1-5 s) consistently produced a large (200-600 nM) increase in dendritic Ca2+ that was sometimes followed by a somatic increase. The dendrites typically returned to basal Ca2+ levels within 10-30 s. 3. Ca2+ increases produced by t-ACPD were measured in Ca(2+)-free external saline [0.5 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], suggesting that they result from intracellular mobilization rather than influx. In addition, Ca2+ increases were not attenuated by a mixture of DL-AP5 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [antagonists of N-methyl-D-aspartate (NMDA) and AMPA/kainate receptors, respectively], but were almost entirely eliminated by L-AP3 (100 microM), a putative metabotropic receptor antagonist or by preincubation of the cultures in pertussis toxin. 4. Brief pulses of t-ACPD (10 microM) produced a small inward current that was associated with an increase in membrane conductance. This current was reversibly blocked by L-AP3 but not by treatments that attenuate some voltage-gated K+ currents. Thus this current is unlikely to underlie the depolarization that is produced by metabotropic agonists in hippocampal pyramidal cells by K(+)-channel closure. 5. The t-ACPD induced inward current was attenuated by substitution of external Na+ with Li+ or choline, or by application of the membrane-permeable Ca2+ chelator, bis-(2-aminophenoxy)-N,N,N',N'- tetraacetic acid (BAPTA)/AM. One mechanism that could mediate this current is electrogenic Nao/Cai exchange, triggered by Ca2+ mobilization.

Animals↗
PubMed1993

The role of Ca2+ entry via synaptically activated NMDA receptors in the induction of long-term potentiation.

Influx of Ca2+ through the NMDA subtype of glutamate receptor is widely accepted as a trigger for many forms of neural plasticity. However, direct support for this model has been elusive, since indirect activation of dendritic voltage-sensitive Ca2+ channels is difficult to exclude. We have optically measured synaptically induced changes in cytoplasmic free Ca2+ concentration in pyramidal cell dendrites in hippocampal slices. Steady postsynaptic depolarization to the synaptic reversal potential eliminated the effect of voltage-sensitive Ca2+ channels. Under these conditions, synaptically induced Ca2+ transients were observed, which were blocked by the NMDA receptor antagonist APV. In addition, the magnitude of LTP was diminished when induced with the postsynaptic membrane held at progressively more positive potentials. LTP could be completely suppressed at potentials near +100 mV. These results provide important experimental support for a role for Ca2+ influx through NMDA receptors in synaptic plasticity.

2-Amino-5-phosphonovalerate↗
PubMed1993

Induction of cerebellar long-term depression in culture requires postsynaptic action of sodium ions.

Cerebellar long-term depression (LTD) is a persistent attenuation of the parallel fiber-Purkinje neuron (PF-PN) synapse induced by conjunctive stimulation of PF and climbing fiber (CF) inputs. A similar phenomenon is seen in the voltage-clamped PN in tissue culture when iontophoretic quisqualate application and PN depolarization are substituted for PF and CF stimulation, respectively. In this model, LTD induction requires activation of both AMPA and metabotropic receptors, together with PN depolarization. We have sought to determine the role of the AMPA receptor in LTD induction. The AMPA receptor does not appear to exert its effect by directly gating Ca2+ influx. Replacement of external Na+ during quisqualate/depolarization conjunction with permeant ions caused a blockade of LTD induction, suggesting that Na+ influx through the AMPA-associated channel is necessary for this process. Similarly, pairing quisqualate pulses with depolarizing steps near ENa also failed to induce LTD. The present results indicate that postsynaptic Na+ influx is necessary for LTD induction. While a portion of the relevant Na+ influx is provided by voltage-gated channels, the AMPA-associated ion channel is most important in this regard.

Animals↗
PubMed1992

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons.

The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20-30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.

Action Potentials↗
PubMed1992

Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated Ca1 neurons.

Spatiotemporal changes of the intracellular calcium ion (Ca2+) were recorded by digital ratio imaging of fura-2 in pyramidal neurons acutely isolated from the adult guinea-pig hippocampus. Increases in calcium were evoked in tetrodotoxin (2 microM) containing saline either by stimulation with the excitatory amino acids, glutamate or N-methyl-D-aspartate, or by depolarization with high potassium (50 mM). Local stimulation with excitatory amino acids, applied from a microelectrode with 1-2-s iontophoretic pulses at the dendrite, induced a rapid increase in intracellular Ca2+ predominantly supported by a Ca2+ influx at the site of stimulation (primary response). Ca2+ levels recovered within 1-2 min in about one-half of the neurons examined. In the remaining neurons the initial exposure to excitatory amino acids induced a non-recovering gradient of Ca2+, highest at the site of stimulation, that lasted for periods of minutes (secondary response). Within the population that showed recovery from the initial agonist exposure, a second, or in some cases, a third application triggered the sustained, secondary response. Pretreatment of neurons with the protein kinase inhibitor sphingosine (10 microM) blocked development of the secondary response but had no effect on the primary response to the excitatory amino acids. There were no Ca2+ increases in Ca(2+)-free medium with either agonist, and responses to N-methyl-D-aspartate were blocked by 2-amino-4-phosphovaleric acid and significantly reduced at physiological concentrations of Mg2+ (1.8 mM). The maintained gradient of Ca2+ was supported by a continuous influx of calcium from outside the cell. In contrast, dendritic gradients of Ca2+ induced by short exposures to high potassium (50 mM, 5 s) collapsed immediately at the end of the stimulus and could be repeatedly evoked. Minute-long exposures to high K, induced large, repeatable changes in Ca2+ but there was always rapid recovery in normal saline. K depolarization applied after excitatory amino acid stimulation produced larger Ca2+ changes than the same K stimulus applied before the cell was stimulated with the excitatory amino acid. Bath application of GABA (10-100 microM) reduced the magnitude of the maintained Ca2+ gradients. The functional significance of the extended, secondary response cannot be directly established from these measurements on isolated neurons, but its properties could give rise, in part, to mechanisms involved in neural plasticity, in kindling epileptogenesis or in glutamate-induced toxicity.

Animals↗
PubMed1992

Recruitment of Ca2+ channels by protein kinase C during rapid formation of putative neuropeptide release sites in isolated Aplysia neurons.

Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.

Action Potentials↗
PubMed1992

Ca2+ signalling in postsynaptic dendrites and spines of mammalian neurons in brain slice.

Postsynaptic Ca2+ changes are involved in control of cellular excitability and induction of synaptic long-term changes. We monitored Ca2+ changes in dendrites and spines during synaptic and direct stimulation using high resolution microfluorometry of fura-2 injected into CA3 pyramidal neurons in guinea pig hippocampal slice. When driven by current injection from an intracellular electrode or with synaptic stimulation, postsynaptic Ca2+ accumulations were highest in the proximal dendrites with a pronounced fall-off towards the soma and some fall-off towards more distal dendrites. Muscarinic activation by low concentrations of carbachol strongly increased intradendritic Ca2+ accumulation during directly-evoked repetitive firing. This enhancement occurred in large part because muscarinic activation suppressed the normal Ca(2+)-dependent activation of K-channels that mediates adaptation of firing. Repetitive firing of cholinergic fibers in the slice reproduced the effects of carbachol. Inhibition of acetylcholine-esterase activity by eserine enhanced the effects of repetitive stimulation of chlolinergic fibers. All effects were reversible and were blocked by the muscarinic antagonist atropine. Ca2+ accumulations in postsynaptic spines might be the basis of specificity of synaptic plasticity. With selective stimulation of few associative/comissural fibers, Ca2+ accumulated in single postsynaptic spines but not in the parent dendrite. With strong stimulation, dendrite levels also increased but spine levels were considerably higher. The NMDA-receptor antagonist AP-5 blocked Ca(2+)-peaks in spines, but left Ca2+ changes in dendrite shafts largely unaffected. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, developed with repeated stimulation. Our results demonstrate a spine entity that can act independent from the dendrite with respect to Ca(2+)-dependent processes. Muscarinic augmentation of dendritic Ca2+ levels might reduce diffusional loss of Ca2+ from hot spines into the parent dendrite, thus supporting cooperativity and associativity of synaptic plasticity.

Animals↗
PubMed1992

Specific involvement of Ca(2+)-calmodulin kinase II in cholinergic modulation of neuronal responsiveness.

1. Muscarinic agonists when applied in the hippocampus at low concentrations suppress intrinsic controls on neuronal excitability through the block of Ca(2+)-activated K conductance(s), gK (Ca), underlying the adaptation of firing and slow afterhyperpolarization (sAHP) in CA1 and CA3 neurons. Carbachol, for example, is effective at 0.1-0.3 microM suggesting activation of a relatively high-affinity receptor. 2. We have examined the mechanism of this action by using a new, highly specific, peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as other kinase inhibitors and show that the muscarinic block of gK (Ca) relies on CaMKII activation in both CA1 and CA3 neurons. Thus phosphorylation of these channels or of an intermediary protein causes the channels to remain closed in the presence of Ca2+ and depolarization. 3. The very similar electrophysiological effects of serotonergic and glutamatergic agonists are mediated either through other kinases or by entirely different processes. 4. Block of intrinsic phosphatase activity by okadaic acid also reduced adaptation and sAHP, and muscarinic agonists had no further effect on these quantities. 5. The removal of presynaptic cholinergic inputs to the hippocampus in animals has a deleterious effect on the performance of tasks requiring spatial memory and is also implicated as a cause of cognitive disorders in humans. By increasing Ca2+ accumulation during electrical activity and promoting CaMKII activity, muscarinic input provides parallel reinforcing pathways for the induction of long-term potentiation, an important cellular memory mechanism. This suggests a possible link between behavioral and cellular approaches to the analysis of learning and memory.

Adaptation, Physiological↗
PubMed1992

An electrophysiological correlate of protein kinase C isozyme distribution in cultured cerebellar neurons.

Protein kinase C (PKC) is a family of at least seven closely related molecules (isozymes) that vary in terms of their requirements for activation and their distribution among cells of the brain. A striking example of this differential distribution is seen in the cerebellum, where Purkinje cells express PKC-I, an isozyme that is strongly activated by both phorbol ester (PE), and low doses of cis-unsaturated fatty acid (c-UFA), while granule cells predominantly express PKC-II, an isozyme that is strongly activated by PE but not c-UFA. Both Purkinje and granule cells have large, easily recorded voltage-gated K currents. These currents are attenuated by PKC activators in several other varieties of neuron. We hypothesized that the effects of these two PKC activators would be predicted by the distribution of the relevant PKC isozyme, and that the delayed outward rectifier current, IK, would be attenuated by both PE and c-UFA in Purkinje cells, but only by PE in granule cells. This hypothesis was confirmed in perforated-patch recordings. The attenuation produced by both activators could be blocked by application of a specific PKC inhibitor, RO-31-8220, and could not be mimicked by inert forms of PE or c-UFA. To our knowledge, this study represents the first report of an electrophysiological correlate of PKC isozyme distribution.

Animals↗
PubMed1991

Participation of postsynaptic PKC in cerebellar long-term depression in culture.

Long-term depression (LTD) in the intact cerebellum is a decrease in the efficacy of the parallel fiber-Purkinje neuron synapse induced by coactivation of climbing fiber and parallel fiber inputs. In cultured Purkinje neurons, a similar depression can be induced by iontophoretic glutamate pulses and Purkinje neuron depolarization. This form of LTD is expressed as a depression of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA)-mediated current, and its induction is dependent on activation of metabotropic quisqualate receptors. The effect of inhibitors of protein kinase C (PKC) on LTD induction was studied. Inhibitors of PKC blocked LTD induction, while phorbol-12,13-diacetate (PDA), a PKC activator, mimicked LTD. These results suggest that PKC activation is necessary for the induction of cerebellar LTD.

Animals↗
PubMed1991

Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses.

The possibility that postsynaptic spines on neuronal dendrites are discrete biochemical compartments for Ca(2+)-activated processes involved in synaptic plasticity is a widely proposed concept that has eluded experimental demonstration. Using microfluorometry on CA3 neurons in hippocampal slices, we show here that with weak presynaptic stimulation of associative/commissural fibres, Ca2+ accumulates in single postsynaptic spines but not in the parent dendrite. Stronger stimulation also promotes changes in dendrites. The NMDA-receptor antagonist AP-5 blocks changes in Ca2+ in spines. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, develop with repeated stimulation. The observed compartmentalization allows for the specificity, cooperativity and associativity displayed by memory models such as long-term potentiation.

2-Amino-5-phosphonovalerate↗