Biomedical subjects

Martha Constantine-Paton

Publications and source records attributed to Martha Constantine-Paton.

14 recordsLinked to original sources

PubMed2006

Long-term potentiation in the juvenile superior colliculus requires simultaneous activation of NMDA receptors and L-type Ca2+ channels and reflects addition of newly functional synapses.

The visual layers of the rodent superficial superior colliculus (sSC) have been the focus of many development studies of the molecular bases of retinotopic map formation, the role of early retinal waves in this process, and the development of glutamate synapses. Previous studies have documented long-term potentiation (LTP), believed to be critical to developmental synapse refinement, in the rodent sSC. However, the means of induction and the preparations used have varied widely, and thus cellular changes underlying this LTP remain ambiguous. Whole-cell and perforated patch clamping were used in this study to elucidate the cellular mechanism of electrically evoked LTP in the juvenile rat sSC. This LTP required relatively low-frequency stimulation (20 Hz) and simultaneous activation of NMDA receptors and L-type Ca2+ channels. Experiments focused on narrow-field vertical neurons, a documented excitatory cell type in the stratum griseum superficiale using bipolar stimulation in the stratum opticum. Strontium (Sr2+) replacement of calcium (Ca2+) was applied to study evoked quantal events before and after LTP induction at the same synapses. Paired-pulse ratio and coefficient of variance analyses examined presynaptic release. Increases in quantal frequency were invariably found in the absence of increases in quantal amplitude and probability of release. These data suggest that electrically stimulated LTP, in the juvenile sSC after eye opening, selectively involves the addition or stabilization of AMPA receptors at the large number of silent synapses known to appear in the sSC after eye opening.

Age Factors↗

PubMed2006

Shining light on spike timing-dependent plasticity.

Although spike timing-dependent plasticity has been well-characterized in vitro, it is less clear to what degree spike timing-dependent plasticity contributes to shaping visual system properties in vivo. In this issue of Neuron, two papers by Vislay-Meltzer et al. and Mu and Poo provide evidence that STDP contributes to the effects of sensory stimuli in refinement of the retinotectal system in Xenopus.

Action Potentials↗

PubMed2006

Developmental period for N-methyl-D-aspartate (NMDA) receptor-dependent synapse elimination correlated with visuotopic map refinement.

During a short perinatal interval, N-methyl-D-aspartate receptor (NMDAR) function is essential to a process in which spontaneous retinal waves focus retinal axon arbors in the superficial layers of the rodent superior colliculus (sSC). Here we provide evidence that this NMDAR-dependent axonal refinement occurs through elimination of uncorrelated retinal synapses arising from disparate loci, rather than stabilization of topographically appropriate inputs. The density of synaptic release sites within fluorescently labeled retinal terminals was counted in double-labeling experiments using confocal microscopy and antibodies against synaptophysin or synapsin-1. Chronic NMDAR blockade from birth increased retinal axon synapse density at postnatal days (P) 6, 8, and 10, suggesting that NMDAR currents reduce synapse density during the refinement period. With assay at P14, after focal arborization has been established, the effect disappeared. Conversely, chronic NMDA treatment, known to induce functional synaptic depression in the sSC, decreased retinocollicular synapse density at P14, but not earlier, during the refinement period (P8). Thus during the development of retinocollicular topographic order, there is a period when NMDAR activity predominantly eliminates retinal axon synapses. We were able to extend this period by using retinal lesions to reduce synaptic density in a defined zone. Synapse density on intact retinocollicular axons sprouting into this zone was increased by NMDAR blockade, even when examined at P14. Thus, the period of NMDAR-dependent synaptic destabilization is terminated by a factor related to the density and refinement of retinal arbors.

Animals↗

PubMed2005

RNA interference of Xenopus NMDAR NR1 in vitro and in vivo.

Here, we present a short interfering RNA (siRNA) application that reduces the expression of NR1 the obligate subunit of the NMDA receptor (NMDAR) and virtually eliminates NMDAR function in a small subset of neurons within otherwise normally developing Xenopus laevis tadpoles. We designed two plasmids each containing a CMV promoter driving a dsRed "reporter" cDNA and DNA coding for one short hairpin RNA (shRNA) under the control of the U6 promoter. The shRNA was cleaved to produce a siRNA against NR1 transcript (iNR1). NR1 transcript and protein, differentiation and survival of NR1 knockdown neurons, were assayed in vivo. iNR1 effects on NMDAR function used Xenopus tectal neuron cultures, Ca2+ imaging and patch-clamp electrophysiology. NR1 transcript, protein and NMDAR function was significantly reduced or eliminated in iNR1-expressing neurons. Protein, transcript and function for the closely related AMPA receptor (AMPAR) subunit GluR1 was unperturbed. In vivo imaging of small groups of iNR1 neurons at two different ages revealed that siRNA knockdown of the NMDAR does not affect differentiation or survival of young neurons. Thus, with siRNA, molecular hypotheses about synaptic mechanisms of circuit formation can be rapidly tested in intact developing vertebrates using normal neurons in the same animals as controls.

Animals↗

PubMed2005

NMDA receptor currents suppress synapse formation on sprouting axons in vivo.

NMDA receptors (NMDARs) play an important role in the structural maintenance and functional strength of synapses. The causal relationship between these anatomical and functional roles is poorly defined. Using quantitative confocal microscopy, synaptic vesicle immunoreactivity, and differential label of retinal projections, we measured axon volume and synapse density along ipsilateral retinal axons (ipsi axons) sprouting into the superficial visual layers of the superior colliculus (sSC) deafferented by a contralateral retinal lesion (a scotoma) 8 d earlier. When retinal lesions were made at postnatal day 6 (P6), glutamatergic synaptic currents on neurons within the scotoma were significantly reduced. Both ipsi axon sprouting and synapse density were increased by chronic d-AP-5 antagonism of NMDARs. Conversely, ipsi axon sprouting and synapse density were reduced by chronic exposure to the agonist, NMDA, known to functionally depress glutamate transmission in this system. After P11 lesions, however, NMDAR blockade had no effect on sprouting or synapse density. Developmental changes in NMDAR current kinetics could not account for this difference in the structural effects of NMDAR function. Also, synaptic current frequencies within the scotoma were not affected after the P11 lesions. The corticocollicular projection matures during the P11 survival interval and, as indicated by previous work, it is a source of competition for synaptic space and probably of maintained activity in the older sSC. Thus, our results suggest that during early development, NMDAR currents predominantly destabilize nascent synapses. As the neuropil matures, however, competition for synaptic space suppresses axon sprouting and synapse formation regardless of NMDAR function.

Animals↗

PubMed2004

Retina-driven dephosphorylation of the NR2A subunit correlates with faster NMDA receptor kinetics at developing retinocollicular synapses.

We describe a homeostatic mechanism that limits NMDA receptor currents in response to early light activation of a developing visual pathway. During the second postnatal week of rodent retinocollicular development, the Ca2+-activated phosphatase calcineurin (CaN) mediates a rapid, activity-induced shortening in the decay time of NMDA receptor (NMDAR) currents. We show that protein kinase A acts in opposition to CaN to maintain NMDAR currents with long decay times. The CaN-mediated change is coincident with the initial expression of the NMDAR subunit NR2A. Using NR2A knock-out mice and dialyzing neurons with a constitutively active CaN, we demonstrate that NR2A subunits are necessary for the effect of CaN on NMDAR current kinetics. In wild-type mice, Ser900 of NR2A, previously implicated in CaN-mediated glycine-independent desensitization, becomes chronically dephosphorylated by postnatal day 11 as NMDAR current decay times become faster. Pharmacologically disrupting early photoreceptor-driven activity in the retina eliminates the dephosphorylation of NR2A and prevents the shortening in NMDAR current decay time. These data suggest that the developmental onset of retinal activity increases CaN-mediated dephosphorylation of NR2A subunits newly incorporated into synaptic NMDARs of the superior colliculus, thereby providing a mechanism for the early and rapid reduction of NMDAR current decay time in visual neurons.

Animals↗

PubMed2004

Microarray analysis of microRNA expression in the developing mammalian brain.

BACKGROUND: MicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals. RESULTS: We isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain. CONCLUSION: We describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.

Animals↗

PubMed2004

Eye opening rapidly induces synaptic potentiation and refinement.

NMDA receptor (NMDAR)-mediated increases in AMPA receptor (AMPAR) currents are associated with long-term synaptic potentiation (LTP). Here, we provide evidence that similar changes occur in response to normal increases in sensory stimulation during development. Experiments discriminated between eye opening-induced and age-dependent changes in synaptic currents. At 6 hr after eye opening (AEO), a transient population of currents mediated by NR2B-rich NMDARs increase significantly, and silent synapses peak. Sustained increases in evoked and miniature AMPAR currents occur at 12 hr AEO. Significant changes in AMPAR:NMDAR evoked current ratios, contacts per axon, and inputs per cell are present at 24 hr AEO. The AMPAR current changes are those seen in vitro during NMDAR-dependent LTP. Here, they are a consequence of eye opening and are associated with a new wave of synaptic refinement. These data also suggest that new NR2B-rich NMDAR currents precede and may initiate this developmental synaptic potentiation and functional tuning.

Animals↗

PubMed2004

Post-transcriptional gene silencing in neurons.

The techniques evolving from the rapidly developing field of small RNAs promise accessible approaches to dissecting cellular and molecular mechanisms of higher brain function. Here, a current overview of the technology is presented, along with an outline of how these approaches might help neuroscientists to more rapidly uncover the cellular and molecular bases of behavior.

Animals↗

PubMed2004

Receptor compartmentalization and trafficking at glutamate synapses: a developmental proposal.

This article focuses on NMDA receptor subunit changes that occur in the forebrain and midbrain during development, namely the switch from predominance of NMDA receptors rich in NR2B subunits to that of NMDA receptors rich in NR2A subunits. We review the potential roles in brain plasticity of two membrane-associated guanylate kinases (MAGUKs), SAP102 and PSD95, which form a scaffold for the ion-passing glutamate receptors at the postsynaptic density, and we consider the known functional significance of these molecules in subunit switching. In addition, based on recent analyses of the synaptic location of glutamate receptors, activity-dependent changes in developing visual neurons, and extensive data on MAGUKs, we propose a model of glutamatergic synaptic differentiation. In this model, different NMDA receptor scaffolding and signaling complexes effect the trafficking and synaptic localization of NR2A-rich and NR2B-rich receptors, leading to tangential compartmentalization of these receptors and their movement between synaptic and extrasynaptic compartments.

Animals↗

PubMed2003

Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons.

The membrane-associated guanylate kinase PSD-95 scaffolds N-methyl-d-aspartate receptors to cytoplasmic signaling molecules, and associates with other glutamate receptors at central synapses. However, regulation of PSD-95 in vivo is poorly understood. We provide evidence of an activity-dependent redistribution of PSD-95 to dendrites in central visual neurons that is tied to eye opening. Six hours after eye opening, increased dendritic PSD-95 coimmunoprecipitates with the same proportions of stargazin, increased proportions of the N-methyl-d-aspartate receptor subunit NR2A, and decreased proportions of NR2B. Sustained high levels of PSD-95 in dendrites are dependent on continued pattern vision in juvenile but not mature animals, suggesting that the stabilization of PSD-95 at synapses may be involved in the control of developmental plasticity.

Animals↗

PubMed2003

Developmental loss of miniature N-methyl-D-aspartate receptor currents in NR2A knockout mice.

The N-methyl-d-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.

Animals↗

PubMed2003

Chronic NMDA receptor blockade from birth delays the maturation of NMDA currents, but does not affect AMPA/kainate currents.

The activity of the N-methyl-D-aspartate receptor (NR) regulates the composition of excitatory synapses and mediates multiple forms of synaptic and structural plasticity. In the superficial superior colliculus (sSC) of the rat, NR activity is essential for the full refinement of retinotopy during development. We have examined the NR's role in synaptic development by chronically treating the sSC from birth with the competitive antagonist (+/-)-2-amino-5-phosphonopentanoic acid (AP5) released by the slow-release polymer Elvax. Whole-cell voltage-clamp recordings were used to characterize excitatory postsynaptic potentials (EPSCs) in slices from postnatal day (P)12-20 sSC. Chronic NR blockade reduced the ratio of AMPA/kainate receptor (AMPAR) to NR peak current amplitudes of both spontaneous (s)EPSCs and evoked EPSCs. Spontaneous NR current amplitude was increased following treatment, while spontaneous AMPAR currents were identical to those of controls, indicating that the ratio change was due to an increased NR current. Comparison of sEPSC frequency, AMPAR current rectification, and quantitative Western blots indicated that the characteristics of AMPARs at the synapse are normal following AP5 treatment. In the sSC, NR currents show a rapid decrease in decay time on P11 and previous studies in slices indicate this change results from a NR-mediated activation of the phosphatase calcineurin. Consistent with this in vitro finding, the down-regulation failed to occur in sSC chronically treated with AP5 in vivo. Together the present data show that NR function is necessary for subsequent NR current regulation in vivo, but it is not essential for the developmental expression of normal AMPAR currents.

Adaptation, Physiological↗

PubMed2002

Interaction between metabotropic and NMDA subtypes of glutamate receptors in sprout suppression at young synapses.

Recently, NMDA receptors (NMDARs) have been implicated in a cell contact-dependent suppression of sprouting in cultured Xenopus tectal neurons during an early period when neither AMPA/kainate (KA) receptors nor action potentials play a prominent role in cell-cell communication. We asked how the NMDA receptors function in the absence of the depolarizing effect of AMPA/KA receptor activity. We show that type II metabotropic glutamate receptors (mGluRs) can operate synergistically with NMDA receptors in the absence of AMPA/KA receptor function to suppress an early neurite sprouting response of the tectal neurons. Calcium imaging with fluo-3 AM and morphological analyses after exposure to glutamate receptor antagonists show that a combination of AMPA/KA receptor and type II mGluR blockers mimics the decrease in intracellular free calcium in response to glutamate and the structural effects produced by NMDA receptor antagonists in these cultures. Patch-clamp analyses confirmed a decrease in NMDA receptor-mediated currents with type II mGluR blockade, and 8-bromo cAMP application produced a decrease in NMDA receptor-mediated calcium influx. These data suggest that type II mGluRs potentiate NMDA receptor function by decreasing cAMP levels in tectal neurons. We also show that NMDARs exhibit low magnesium sensitivity in tectal neurons during the first few days in culture. Thus both metabotropic and ionotropic glutamate receptors can play a role in the contact-mediated suppression of ongoing sprouting at early neuron-neuron contacts before action potential activity.

Animals↗