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M Constantine-Paton

Publications and source records attributed to M Constantine-Paton.

At least 19 recordsLinked to original sources
PubMed2001

Developmental depression of glutamate neurotransmission by chronic low-level activation of NMDA receptors.

Slabs of slow-release plastic (Elvax) containing NMDA or solvent were implanted over the rat colliculus beginning on postnatal day 8 (P8). Whole-cell patch clamping in the superficial superior collicular layers (sSCs) from P10 to P21 demonstrated a severe decrease in spontaneous EPSC frequency after chronic NMDA treatment. The decrease was not attributable to an increase in GABA(A) receptor-mediated inhibition and was present only when NMDA receptor (NMDAR) current was blocked by Mg(2+). Analysis of miniature EPSCs indicated that many active sites on NMDA-treated neurons lacked functional AMPA and kainate receptor (AMPA/KAR) currents, and AMPA/KAR:NMDAR current ratios of evoked EPSCs were also significantly reduced. In addition, the normal downregulation of NMDAR decay time in sSC neurons at P11 was absent after NMDA treatment. Nevertheless, neither AMPA nor NMDA receptor subunit expression was altered by NMDA treatment, and experiments with the NMDAR antagonist ifenprodil suggested that incorporation of NR2A-containing NMDARs at the sSC synapses was unperturbed. Thus, disrupting but not blocking NMDARs suppresses the development of AMPA/KAR currents. The absence of the P11 NMDAR current downregulation is likely a secondary effect resulting from the reduction of AMPA/KAR function. Chronic agonist application reduces but does not eliminate NMDAR conductances. Therefore these data support an active role for NMDAR currents in synaptic development. Prolonged NMDA treatment in vivo, which couples reduced postsynaptic Ca(2+) responses with normally developing afferent activity, produces a long-lasting synaptic depression and stalls glutamatergic synaptogenesis, suggesting that the correlation between robust NMDAR activation and afferent activity is an essential component during normal development.

Aging↗
PubMed2001

Chronic NMDA receptor blockade from birth increases the sprouting capacity of ipsilateral retinocollicular axons without disrupting their early segregation.

We have investigated the role of the NMDA glutamate receptor (NMDAR) in the genesis and regulation of structural plasticity during synaptogenesis in the visual layers of the rat superior colliculus (sSC). In this neuropil, three projections compete for synaptic space during development. By fluorescently labeling the projections of both eyes and imaging them with confocal microscopy, we can quantify the sprouting of the ipsilateral retinal projection that follows removal of a portion of the contralateral retinal and/or corticocollicular projection. Using these techniques we have studied the effects of NMDAR blockade under different levels of competition. NMDARs were chronically blocked from birth [postnatal day 0 (P0)] by suspending the competitive antagonist 2-amino-5-phosphonopentanoic acid in the slow release plastic Elvax, a slab of which was implanted over the sSC. Such treatment alone does not impair the normal segregation of the retinal projections. However, if sprouting of the ipsilateral projection is initiated with a small contralateral retinal lesion at P6, this sprouting can be further increased by blocking NMDARs from birth. Sprouting of the ipsilateral retinal projection is also induced by retinal lesions made at P10/P11, but NMDAR blockade does not augment the sprouting induced by this later lesion. However, when combined with simultaneous ablation of the ipsilateral visual cortex, P10/P11 lesions show increased sprouting after NMDAR blockade. These data indicate that P0 NMDAR blockade does not eliminate synaptic competition in the sSC. Instead, early elimination of NMDAR function appears to facilitate sprouting that is gated in a stepwise manner by the other visual afferents.

2-Amino-5-phosphonovalerate↗
PubMed2000

Activity-dependent induction of tonic calcineurin activity mediates a rapid developmental downregulation of NMDA receptor currents.

Whole-cell recording in the superficial layers of the developing superior colliculus (sSC) reveals a large drop in NMDA receptor (NMDAR) current decay time synchronized across all neurons and occurring consistently between P10 and P11. We show that blocking the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN) in the postsynaptic neuron can abolish this drop. The regulation is induced prematurely by 1-2 hr of electrical stimulation in P10 collicular slices only if CaN and NMDAR currents can be activated in the neuron. These data suggest that a long-lasting, CaN-mediated control of NMDAR kinetics is rapidly initiated by heightened activity of the NMDAR itself and demonstrate a novel developmental and tonic function of CaN that can play an important role in modulating the plasticity of the developing CNS.

Action Potentials↗
PubMed2000

NMDA receptor-mediated control of protein synthesis at developing synapses.

We demonstrate a rapid and complex effect of N-methyl-d-aspartate receptor (NMDAR) activation on synaptic protein synthesis in the superior colliculi of young rats. Within minutes of receptor activation, translation of alpha Ca2+/calmodulin dependent kinase II (alphaCamK II) was increased, whereas total protein synthesis was reduced. NMDAR activation also increased phosphorylation of eukaryotic elongation factor 2 (eEF2), a process known to inhibit protein translation by reducing peptide chain elongation. Low doses of cycloheximide, which reduce elongation rate independently of eEF2 phosphorylation, decreased overall protein synthesis but increased alphaCaMK II synthesis. These observations suggest that regulation of peptide elongation via eEF2 phosphorylation can link NMDAR activation to local increases in the synthesis of specific proteins during activity-dependent synaptic change.

2-Amino-5-phosphonovalerate↗
PubMed2000

Chronic NMDA exposure accelerates development of GABAergic inhibition in the superior colliculus.

Maturation of excitatory synaptic connections depends on the amount and pattern of their activity, and activity can affect development of inhibitory synapses as well. In the superficial visual layers of the superior colliculus (sSC), developmental increases in the effectiveness of gamma-aminobutyric acid (GABA(A)) receptor-mediated inhibition may be driven by the maturation of visual inputs. In the rat sSC, GABA(A) receptor currents significantly jump in amplitude between postnatal days 17 and 18 (P17 and P18), approximately when the effects of cortical inputs are first detected in collicular neurons. We manipulated the development of these currents in vivo by implanting a drug-infused slice of the ethylene-vinyl acetate copolymer Elvax over the superior colliculus of P8 rats to chronically release from this plastic low levels of N-methyl-D-aspartate (NMDA). Sham-treated control animals received a similar implant containing only the solvent for NMDA. To examine the effects of this treatment on the development of GABA-mediated neurotransmission, we used whole cell voltage-clamp recording of spontaneous synaptic currents (sPSCs) from sSC neurons in untreated, NMDA-treated, and sham-treated superior colliculus slices ranging in age from 10 to 20 days postnatal. Both amplitude and frequency of sPSCs were studied at holding potentials of +50 mV in the presence and absence of the GABA(A) receptor antagonist, bicuculline methiodide (BMI). The normal developmental increase in GABA(A) receptor currents occurred on schedule (P18) in sham-treated sSC, but NMDA treatment caused premature up-regulation (P12). The average sPSCs in early NMDA-treated neurons were significantly larger than in age-matched sham controls or in age-matched, untreated neurons. No differences in average sPSC amplitudes across treatments or ages were present in BMI-insensitive, predominantly glutamatergic synaptic currents of the same neurons. NMDA treatment also significantly increased levels of glutamate decarboxylase (GAD), measured by quantitative western blotting with staining at P13 and P19. Cell counting using the dissector method for MAP 2 and GAD(67) at P13 and P19 indicated that the differences in GABAergic transmission were not due to increases in the proportion of inhibitory to excitatory neurons after NMDA treatment. However, chronic treatments begun at P8 with Elvax containing both NMDA and BMI significantly decreased total neuron density at P19 ( approximately 15%), suggesting that the NMDA-induced increase in GABA(A) receptor currents may protect against excitotoxicity.

Aging↗
PubMed1999

Nitric oxide in the retinotectal system: a signal but not a retrograde messenger during map refinement and segregation.

The role of nitric oxide (NO) as a mediator of synaptic plasticity is controversial in both the adult and developing brain. NO generation appears to be necessary for some types of NMDA receptor-dependent synaptic plasticity during development but not for others. Our previous work using several NO donors revealed that Xenopus laevis retinal ganglion cell axons stop growing in response to NO exposure. We demonstrate here that the same response occurs in tectal neuron processes bathed in the NO donor S-nitrosocysteine (SNOC) and in RGC growth cones to which SNOC is very locally applied. We show that NO synthase (NOS) activity is present in the Rana pipiens optic tectum throughout development in a dispersed subpopulation of tectal neurons, although effects of NO on synaptic function in a Rana pipiens tectal slice were varied. We chronically inhibited NOS in doubly innervated Rana tadpole optic tecta using L-N(G)-nitroarginine methyl ester in Elvax. Despite significant NOS inhibition as measured biochemically, eye-specific stripes remained normally segregated. This suggests that NOS activity is not downstream of NMDA receptor activation during retinotectal synaptic competition because NMDA receptor activation is necessary for segregation of retinal afferents into ocular dominance stripes in the doubly innervated tadpole optic tectum. We conclude that NO has some signaling function in the retinotectal pathway, but this function is not critical to the mechanism that refines the projection and causes eye-specific stripes.

Animals↗
PubMed1999

The role of neural activity in synaptic development and its implications for adult brain function.

In much of the developing nervous system, electrical activity guides the formation of neural connections, with lasting effects on adult brain function. Epilepsy, a defect in neuronal excitability, might result from abnormal patterns of activity in the young brain. Many connections are organized by selective stabilization of synapses when they are activated simultaneously on the same postsynaptic cell during a sensitive period in early life. This process often involves calcium entry through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. The magnitude of the current passed by this receptor depends on its subunit composition, which varies with age and brain region. Although receptor configurations that admit large calcium currents are permissive of synaptic plasticity, they also increase neural vulnerability to excitotoxic cell death. In most regions of developing brain, activity that can drive NMDA receptors initially is low and increases with maturation. Thus, the replacement of NMDA receptors that flux large calcium currents during early periods of synaptic organization with NMDA receptor subtypes that flux less calcium as synapses become more active, more effective, and less plastic allows maturing neurons to maintain optimal levels of intracellular calcium in the face of drastic developmental changes in their inputs. We have proposed that this transition in NMDA receptors from high to low calcium permeabilities is itself activity dependent. This idea is supported by data showing that many synaptic proteins, including receptor subunits, can be regulated by activity. Cultured cerebellar granule neurons require NMDA receptor stimulation for survival and differentiation, which may replicate the activation provided by the arrival of mossy fiber innervation in vivo. In these cultures, chronic depolarization and glutamate or NMDA treatment induces more mature NMDA receptor subunit expression patterns and function and also increases the expression of several gamma-aminobutyric acid type A (GABAA) receptor subunits, changing that receptor's function. In addition, evidence from in vivo studies indicates that synaptic maturation itself may depend on NMDA receptor activity. During the formation of topographic connections between the retina and superior colliculus (SC) of young rats, chronic local application of the competitive NMDA receptor antagonist +2-amino-5-phosphonovalerate (D-APV) blocks the normal developmental up-regulation of NMDA receptor subunit 1 (NR1) mRNA and nitric oxide synthase activity, as well as maturation of calcium and calmodulin-dependent kinase distribution, activity, and substrate phosphorylation. Together, these recent molecular findings suggest that chronic seizure disorders could result from any of a variety of early developmental events. Any disturbance that locally perturbs regulation of NMDA receptors or the temporal correlations in synaptic activity that drive these receptors has the potential to alter the normal development of local circuitry and the critical balance of inhibition and excitation required to contain seizure activity.

Aging↗
PubMed1998

Suppression of sprouting: An early function of NMDA receptors in the absence of AMPA/kainate receptor activity.

Electrophysiological studies have documented the existence of synapses showing only NMDA ionotropic glutamate receptor function that are therefore presumably "silent" at resting membrane potentials. Silent synapses are more prevalent in young than in older neurons, and NMDA receptor activity at such contacts may facilitate the appearance of functional AMPA receptors. However, it is uncertain whether such silent synapses actually have a function in young neurons independent of AMPA receptor induction. Using a newly characterized culture system for neurons from larval Xenopus tecta, we show that blocking NMDA receptors or preventing changes in intracellular free Ca2+ concentration with BAPTA AM significantly increases neurite sprouting and elongation in contacted but not in isolated neurons. Blocking AMPA/KA receptors or Na+-dependent action potentials does not mimic this effect. Moreover, in these young neurons, NMDA receptor-dependent Ca2+ responses to glutamate measured with confocal fluo-3 imaging are retained during AMPA/KA receptor blockade. The data suggest that many of the young contacts in these cultures are active even though they use only NMDA ionotropic glutamate receptors. Calcium influx through the NMDA receptor at these contacts seems to reduce neurite motility. This effect should lead to the accumulation of glutamatergic inputs on NMDA receptor-expressing dendrites, which could facilitate the onset of AMPA/KA receptor function and the action potential-dependent phase of synaptogenesis.

2-Amino-5-phosphonovalerate↗
PubMed1998

LTP and activity-dependent synaptogenesis: the more alike they are, the more different they become.

Recent data suggest that long-term potentiation and activity-dependent synaptogenesis share the same mechanism at the initiation stage during which NMDA receptor activity is necessary to increase the postsynaptic response via AMPA receptor currents. However, several fundamental differences between the environments of young and mature synapses and the neurons that support them suggest that the same cellular mechanism is facilitated by very different parameters in the young versus the mature brain.

Animals↗
PubMed1997

N-methyl-D-aspartate receptor activation and visual activity induce elongation factor-2 phosphorylation in amphibian tecta: a role for N-methyl-D-aspartate receptors in controlling protein synthesis.

N-methyl-D-aspartate receptor (NMDAR) activation has been implicated in forms of synaptic plasticity involving long-term changes in neuronal structure, function, or protein expression. Transcriptional alterations have been correlated with NMDAR-mediated synaptic plasticity, but the problem of rapidly targeting new proteins to particular synapses is unsolved. One potential solution is synapse-specific protein translation, which is suggested by dendritic localization of numerous transcripts and subsynaptic polyribosomes. We report here a mechanism by which NMDAR activation at synapses may control this protein synthetic machinery. In intact tadpole tecta, NMDAR activation leads to phosphorylation of a subset of proteins, one of which we now identify as the eukaryotic translation elongation factor 2 (eEF2). Phosphorylation of eEF2 halts protein synthesis and may prepare cells to translate a new set of mRNAs. We show that NMDAR activation-induced eEF2 phosphorylation is widespread in tadpole tecta. In contrast, in adult tecta, where synaptic plasticity is reduced, this phosphorylation is restricted to short dendritic regions that process binocular information. Biochemical and anatomical evidence shows that this NMDAR activation-induced eEF2 phosphorylation is localized to subsynaptic sites. Moreover, eEF2 phosphorylation is induced by visual stimulation, and NMDAR blockade before stimulation eliminates this effect. Thus, NMDAR activation, which is known to mediate synaptic changes in the developing frog, could produce local postsynaptic alterations in protein synthesis by inducing eEF2 phosphorylation.

Amphibians↗
PubMed1997

Temporal correlations between functional and molecular changes in NMDA receptors and GABA neurotransmission in the superior colliculus.

Activation of the NMDA subtype of glutamate receptor is required for activity-dependent structural plasticity in many areas of the young brain. Previous work has shown that NMDA receptor currents decline approximately at the time that developmental synaptic plasticity ends, and in situ hybridization studies have suggested that receptor subunit changes may be occurring during the same developmental interval. To establish a system in which the relationship between these properties of developing synapses can be explored, we have combined patch-clamp recordings with mRNA- and protein-level biochemical analyses to study the developmental regulation of NMDA receptors in the superficial layers of the rat superior colliculus. These experiments document an abrupt decrease in the NMDA receptor contribution to synaptic currents that occurs before eye opening and is closely associated with changes in NR1 protein, rapidly rising levels of the NMDA receptor subunit NR2A, and decreasing levels of NR2B. The functional and molecular changes also are correlated with the developmental decline in structural plasticity in these layers. In addition, both physiological and biochemical methods show evidence of GABA-mediated inhibition in the superficial collicular layers beginning after eye opening. This may provide an additional heterosynaptic mechanism for controlling excitation and plasticity in this neuropil by pattern vision. Thus our findings lend support to the idea that high levels of NMDA receptor function are associated with the potential for structural rearrangement in CNS neuropil and that the functional downregulation of this molecule results, at least partially, from changes in its subunit composition.

Age Factors↗
PubMed1997

Experimental down-regulation of the NMDA channel associated with synapse pruning.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in activity-dependent synapse stabilization, but its role as a detector of correlated activity during development is debated. In the amphibian retinotectal system, synaptic sorting and stabilization occur throughout larval life, and map refinement is dependent on continuous NMDA receptor function. Moreover, tadpole tecta chronically treated with NMDA selectively fail to maintain retinal synapses wherever their activity correlations are lowest. To determine whether this synapse elimination is associated with a specific down-regulation of NMDA receptor function, whole cell voltage-clamp recordings were made from single neurons in tectal slices. After chronic NMDA treatment, decreases in the magnitude of NMDA currents were detected in glutamatergic synaptic currents, in agonist-evoked currents, and in single-channel currents activated by NMDA. The results suggest that the efficacy of NMDA receptors on tectal neurons determines the amount of correlation required to stabilize sets of tectal inputs during formation of the retinotectal projection.

Animals↗
PubMed1996

NMDA receptor activation-responsive phosphoproteins in the developing optic tectum.

A front phosphorylation assay followed by two-dimensional gel electrophoresis was used to detect proteins in the tadpole optic tectum, the phosphorylation state of which is regulated by NMDA receptor activation. Five proteins with isoelectric points between 4 and 7 displayed marked increases in their phosphorylation state in response to application of 10 microM glutamate and 50 microM NMDA. This response was inhibited by 60 microM 2-amino-5-phosphopentanoic acid. These proteins are termed NMDA receptor activation-responsive phosphoproteins (NARPPs). Two NARPPs were identified as both in vitro and in vivo substrates for protein kinase C. Of these two NARPPs, one was located in the postsynaptic density (NARPP-50), and one was located in the nuclear fraction (NARPP-21). Phosphorylation of NARPP-21 was also induced by application of the metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (100 microM). Phosphorylation of all NARPPs was eliminated by dantrolene, which inhibits release of calcium from intracellular stores. In adult tecta, only NARPP-21 and -50 were phosphorylation. Thus the phosphorylation state of most NARPPs is regulated differently when synaptic plasticity is low. Further characterization of NARPPs should lead to identification of second messenger systems involved in NMDA receptor signaling and developmental synaptic plasticity.

Animals↗
PubMed1996

Exogenous nitric oxide causes collapse of retinal ganglion cell axonal growth cones in vitro.

We show that nitric oxide (NO) from applied NO-donating chemicals induces collapse of ganglion cell axonal growth cones extending from explants of tadpole retina in culture. Peroxynitrite, a neurotoxic product of NO and superoxide reaction, did not induce collapse, and oxyhemoglobin, which binds NO, blocked the highly effective collapsing activity of the NO donor S-nitrosocysteine. Membrane-permeable analogs of cyclic guanosine monophosphate had no collapsing activity. Inhibitors of NO synthase did not induce collapse. NO is a potential retrograde messenger through which postsynaptic neurons signal to their inputs to modify synaptic efficacy following NMDA receptor activation. Our results suggest a role for NO as such a messenger during development of the retinotectal projection.

Animals↗
PubMed1996

Chronic NMDA receptor antagonism during retinotopic map formation depresses CaM kinase II differentiation in rat superior colliculus.

We examined the effects of chronic NMDA receptor antagonism on the normal postnatal differentiation of calcium- and calmodulin-dependent kinase II (CaM kinase II) in the rat superior colliculus. At postnatal day (P) zero, most CaM kinase II protein, as well as CaM kinase II activity, was detected in the soluble fraction. In vitro phosphorylation of P0 superior colliculus revealed several prominent substrates in both the particulate and soluble fractions. At P19 there was more particulate enzyme than soluble enzyme, and CaM kinase II activity in the particulate fraction was higher than in P0 particulate tissue. Additionally, in vitro phosphorylation of P19 superior colliculus revealed many more CaM kinase II substrates. Chronic NMDA receptor antagonism with 2-amino-5-phosphonovalerate (DL-AP5) caused CaM kinase II to retain many of the characteristics of the enzyme found in P0 untreated superior colliculus. In P19 superior colliculus treated with LD-AP5 from birth, most of the protein was in the soluble fraction, CaM kinase II activity was largely restricted to the soluble fraction, and only a few substrates were observed by in vitro phosphorylation. These effects were not observed in tissue treated with the inactive isomer, L-AP5. These results suggest that synaptic maturation is slowed by antagonism of NMDA receptors during retinotopic map formation.

2-Amino-5-phosphonovalerate↗
PubMed1995

Analysis of synaptic distribution within single retinal axonal arbors after chronic NMDA treatment.

Activation of the NMDA subtype of glutamate receptor has been implicated in structural synaptic plasticity in many developing sensory systems. In the frog retinotectal system, chronic exposure of the optic tectum to NMDA, which decreases the effectiveness of NMDA receptors (Debski et al., 1991), results in the pruning of the branches of retinal terminal arbors (Cline and Constantine-Paton, 1990). However, it is difficult from these studies to relate the involvement of NMDA receptors to changes in synapse distribution. In this study, we have developed an EM sampling procedure to quantitatively compare the amount and the distribution of synaptic contact within single retinal arbors. We report that within each retinal arbor, synaptic contact gradually increases from the main branches to the end branches of the arbor. Chronic NMDA treatment, however, significantly reduces the total amount of synaptic contact within each arbor. This reduced synaptic contact appears to be due to the pruning of the end branches, and the synapses these branches bear. The results are consistent with the hypothesis that NMDA receptor is an integral part of the mechanism that stabilizes coactive synapses, and that maintenance of an axonal branch requires a minimum density of synapses that are correlated with converging neighbors.

Animals↗
PubMed1994

Dynamics of retinotectal synaptogenesis in normal and 3-eyed frogs: evidence for the postsynaptic regulation of synapse number.

Quantitative stereological methods were used to determine if the number, density, and types of synaptic connections formed during development are regulated by presynaptic input or by postsynaptic target cells in the optic tectum of normal and 3-eyed Rana pipiens tadpoles and frogs. Our analysis indicates that the number and size of synapses is approximately the same in both tecta of 3-eyed tadpoles and frogs, even though one tectal lobe is receiving input from twice the normal complement of retinal ganglion cells. Moreover, the number and size of synapses in the tectal lobes of 3-eyed animals did not differ significantly from values determined for normal tadpoles and frogs of the same developmental stage. These data suggest strongly that developing tectal cells regulate the number of synaptic contacts they will form. Differences in several morphological features between singly and doubly innervated tecta, however, including synapse density, distribution and complexity, amount of extracellular space, and number of myelin figures, suggest that the presence of supernumerary input retards tectal maturation. We propose that the noncorrelated activity of retinal ganglion cell terminals in the doubly innervated tectum results in fewer stabilized synapses per unit volume of neuropil and in the delayed maturation of the tectal neuropil. Taken together, our data suggest a complex dynamic interaction between retina and tectum during development.

Animals↗