Activity-Dependent PSD Formation and Stabilization of Newly Formed Spines in Hippocampal Slice Cultures

Development and remodeling of synaptic networks occurs through a continuous turnover of dendritic spines. However, the mechanisms that regulate the formation and stabilization of newly formed spines remain poorly understood. Here, we applied repetitive confocal imaging to hippocampal slice cultures to address these issues. We find that, although the turnover rate of protrusions progressively decreased during development, the process of stabilization of new spines remained comparable both in terms of time course and low level of efficacy. Irrespective of the developmental stage, most new protrusions were quickly eliminated, in particular filopodia, which only occasionally lead to the formation of stable dendritic spines. We also found that the stabilization of new protrusions was determined within a critical period of 24 h and that this coincided with an enlargement of the spine head and the expression of tagged PSD-95. Blockade of postsynaptic AMPA and NMDA receptors significantly reduced the capacity of new spines to express tagged PSD-95 and decreased their probability to be stabilized. These results suggest a model in which synaptic development is associated with an extensive, nonspecific growth of protrusions followed by stabilization of a few of them through a mechanism that involves activity-driven formation of a postsynaptic densit

Anesthetics Rapidly Promote Synaptogenesis during a Critical Period of Brain Development

Experience-driven activity plays an essential role in the development of brain circuitry during critical periods of early postnatal life, a process that depends upon a dynamic balance between excitatory and inhibitory signals. Since general anesthetics are powerful pharmacological modulators of neuronal activity, an important question is whether and how these drugs can affect the development of synaptic networks. To address this issue, we examined here the impact of anesthetics on synapse growth and dynamics. We show that exposure of young rodents to anesthetics that either enhance GABAergic inhibition or block NMDA receptors rapidly induce a significant increase in dendritic spine density in the somatosensory cortex and hippocampus. This effect is developmentally regulated; it is transient but lasts for several days and is also reproduced by selective antagonists of excitatory receptors. Analyses of spine dynamics in hippocampal slice cultures reveals that this effect is mediated through an increased rate of protrusions formation, a better stabilization of newly formed spines, and leads to the formation of functional synapses. Altogether, these findings point to anesthesia as an important modulator of spine dynamics in the developing brain and suggest the existence of a homeostatic process regulating spine formation as a function of neural activity. Importantly, they also raise concern about the potential impact of these drugs on human practice, when applied during critical periods of development in infants

Modulating effects of neurosteroids on ionotropic GABA and ATP receptors in rat primary sensory neurones

Les neurones du ganglion rachidien véhiculant les informations nociceptives effectuent leur premier relais synaptique dans la corne dorsale de la moelle épinière. Ces synapses utilisent comme neurotransmetteur rapide le glutamate. Présynaptiquement, cette transmission peut être respectivement inhibée ou facilitée par l'activation de récepteurs ionotropiques du GABA (GABAA), ou de l'ATP (P2X). Les neurones du ganglion rachidien, de la corne dorsale de la moelle épinière et les cellules gliales environnantes possèdent l'équipement enzymatique leur permettant de synthétiser des neurostéroïdes. Notre objectif était de déterminer si les neurostéroïdes peuvent moduler les récepteurs GABAA et P2X exprimés par les neurones sensoriels primaires. Notre travail a permis de mettre en évidence pour la première fois une modulation des récepteurs P2X par des neurostéroïdes. Nos résultats nous permettent de supposer l'existence d'une modulation différente du message nociceptif par des récepteurs GABAA et P2X, selon le type de neurostéroïde synthétisé localement et le type de récepteur P2X exprimé sur les terminaisons intraspinales des nocicepteurs. Cette modulation par les neurostéroïdes pourrait être accentuée lors d'une acidification du milieu extracellulaire observée dans des conditions inflammatoires, étant donné que le pH acide potentialise également les récepteurs P2X incluant la sous-unité P2X2. Nos résultats suggèrent également que la DHEA et la progestérone pourraient s'avérer être de nouveaux outils pharmacologiques très utiles pour identifier dans les cellules natives la présence de récepteurs hétéromériques et homomériques exprimant la sous-unité P2X2. Enfin, il est connu que les récepteurs GABAA et P2X jouent un rôle important dans la modulation du message nociceptif dans la corne dorsale de la moelle épinière. Par conséquent, la DHEA ou la progestérone, ou la manipulation de leur biosynthèse pourraient constituer des cibles intéressantes dans le traitement de la douleur.Primary sensory neurones conveying nociceptive information make their first synaptic relay in the dorsal horn of the spinal cord. These synapses are excitatory and use glutamate for rapid transmitter. Presynaptically, this transmission can be respectively inhibited or facilitated by ionotropic GABA (GABAA) or ATP (P2X) receptors. Primary sensory neurones, dorsal horn neurons, and glial cells can synthesise neurosteroids. Our aim was to determine if neurosteroids can modulate GABAA and P2X receptors in primary sensory neurones. Our work shows for the first time that P2X receptors are modulated by neurosteroids. Our results indicate that nociceptive information may be modulated differentially by GABAA and P2X receptors depending on the neurosteroids locally synthesised and on the type of P2X receptors expressed by intraspinal endings of nociceptors. This modulation by neurosteroids could be increased in inflammatory conditions, when extracellular pH is becoming acid, because low pH increase P2X receptors that contain the P2X2 subunit. Our results also suggest that DHEA and progesterone could prove to be useful pharmacological tools to identify homomeric or heteromeric P2X receptors containing the P2X2 subunit in native cells. As GABAA and P2X are playing an important role in modulating nociceptive information in the dorsal horn of the spinal cord, DHEA and progesterone, or the manipulation of their biosynthesis, could be interesting targets in pain therapy

Synaptic remodeling at the heart of memory processes

At the turn of the 21st century, the development of laser scanning microscopy combined with the methods of neuronal transfection with fluorescent proteins permitted the first ever real-time visualization of excitatory synapses following the induction of learning- related patterns of activity in living neurons in vitro, and later following sensory experience in living animals. These techniques, that are still improving today, led to the discovery of a causal link between learning and memory and structural plasticity of synapses, leading to long-term neuronal network remodeling. It has opened a new era in the study of the mechanisms that underlie learning and memory, and provided new therapeutic leads for brain disorders. This thesis describes my scientific contribution in this context

Modulating effects of neurosteroids on ionotropic GABA and ATP receptors in rat primary sensory neurones

Les neurones du ganglion rachidien véhiculant les informations nociceptives effectuent leur premier relais synaptique dans la corne dorsale de la moelle épinière. Ces synapses utilisent comme neurotransmetteur rapide le glutamate. Présynaptiquement, cettePrimary sensory neurones conveying nociceptive information make their first synaptic relay in the dorsal horn of the spinal cord. These synapses are excitatory and use glutamate for rapid transmitter. Presynaptically, this transmission can be respectivel

Activity-dependent PSD formation and stabilization of newly formed spines in hippocampal slice cultures

Development and remodeling of synaptic networks occurs through a continuous turnover of dendritic spines. However, the mechanisms that regulate the formation and stabilization of newly formed spines remain poorly understood. Here, we applied repetitive confocal imaging to hippocampal slice cultures to address these issues. We find that, although the turnover rate of protrusions progressively decreased during development, the process of stabilization of new spines remained comparable both in terms of time course and low level of efficacy. Irrespective of the developmental stage, most new protrusions were quickly eliminated, in particular filopodia, which only occasionally lead to the formation of stable dendritic spines. We also found that the stabilization of new protrusions was determined within a critical period of 24 h and that this coincided with an enlargement of the spine head and the expression of tagged PSD-95. Blockade of postsynaptic AMPA and NMDA receptors significantly reduced the capacity of new spines to express tagged PSD-95 and decreased their probability to be stabilized. These results suggest a model in which synaptic development is associated with an extensive, nonspecific growth of protrusions followed by stabilization of a few of them through a mechanism that involves activitydriven formation of a postsynaptic density

LTP promotes a selective long-term stabilization and clustering of dendritic spines.

Dendritic spines are the main postsynaptic site of excitatory contacts between neurons in the central nervous system. On cortical neurons, spines undergo a continuous turnover regulated by development and sensory activity. However, the functional implications of this synaptic remodeling for network properties remain currently unknown. Using repetitive confocal imaging on hippocampal organotypic cultures, we find that learning-related patterns of activity that induce long-term potentiation act as a selection mechanism for the stabilization and localization of spines. Through a lasting N-methyl-D-aspartate receptor and protein synthesis-dependent increase in protrusion growth and turnover, induction of plasticity promotes a pruning and replacement of nonactivated spines by new ones together with a selective stabilization of activated synapses. Furthermore, most newly formed spines preferentially grow in close proximity to activated synapses and become functional within 24 h, leading to a clustering of functional synapses. Our results indicate that synaptic remodeling associated with induction of long-term potentiation favors the selection of inputs showing spatiotemporal interactions on a given neuron

Dendritic spine formation and stabilization

Formation, elimination and remodeling of excitatory synapses on dendritic spines represent a continuous process that shapes the organization of synaptic networks during development. The molecular mechanisms controlling dendritic spine formation and stabilization therefore critically determine the rules of network selectivity. Recent studies have identified new molecules, such as Ephrins and Telencephalin that regulate filopodia motility and their transformation into dendritic spines. Trans-synaptic signaling involving nitric oxide, protease, adhesion molecules and Rho GTPases further controls contact formation or the structural remodeling of spines and their stability. Evidence also suggests that activity and induction of plasticity participate to the selection of persistent spines. Together these new data provide a better understanding of the mechanisms, speed and steps leading to the establishment of a stable excitatory synapse