Mathias De Roo

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.

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.

Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis.

Background:Recent experimental observations suggest that, in addition to induce neuroapoptosis, anesthetics can also interfere with synaptogenesis during brain development. The aim of this study was to pursue this issue by evaluating the exposure time-dependent effects of volatile anesthetics on neuronal cytoarchitecture in 16-day-old rats, a developmental stage characterized by intense synaptogenesis in the cerebral cortex. Methods:Whistar rats underwent isoflurane (1.5%), sevoflurane (2.5%), or desflurane (7%) anesthesia for 30, 60, and 120 min at postnatal day 16, and the effect of these treatments on neuronal cytoarchitecture was evaluated 6 h after the initiation of anesthesia. Cell death was assessed using Fluoro-Jade B staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Ionotophoretic injections into layer 5 pyramidal neurons in the medial prefrontal cortex allowed visualization of dendritic arbor. Tracing of dendritic tree was carried out using the Neurolucida station (Microbrightfield, Williston, VT), whereas dendritic spines were analyzed using confocal microscopy. Results:Up to a 2-h-long exposure, none of the volatile drugs induced neuronal cell death or significant changes in gross dendritic arbor pattern of layer 5 pyramidal neurons in pups at postnatal day 16. In contrast, these drugs significantly increased dendritic spine density on dendritic shafts of these cells. Importantly, considerable differences were found between these three volatile agents in terms of exposure time-dependent effects on dendritic spine density. Conclusion:These new results suggest that volatile anesthetics, with different potencies and without inducing cell death, could rapidly interfere with physiologic patterns of synaptogenesis and thus might impair appropriate circuit assembly in the developing cerebral cortex.

Cocaine Disinhibits Dopamine Neurons by Potentiation of GABA Transmission in the Ventral Tegmental Area

Drugs, Dopamine, and Disinhibition Drugs often change the neuronal circuitry in the brain and thereby cause a long-lasting change in behavior. Using a wide range of in vivo and in vitro techniques in mice, Bocklisch et al. (p. 1521) observed that cocaine profoundly altered dopamine neuron function and that drug-evoked synaptic plasticity in a specific set of neurons represents a crucial step in circuit remodeling. Changes in specific neuronal circuits suggest that drug-evoked synaptic plasticity facilitates drug-adaptive behavior. Drug-evoked synaptic plasticity in the mesolimbic system reshapes circuit function and drives drug-adaptive behavior. Much research has focused on excitatory transmission in the ventral tegmental area (VTA) and the nucleus accumbens (NAc). How drug-evoked synaptic plasticity of inhibitory transmission affects circuit adaptations remains unknown. We found that medium spiny neurons expressing dopamine (DA) receptor type 1 (D1R-MSNs) of the NAc project to the VTA, strongly preferring the GABA neurons of the VTA. Repeated in vivo exposure to cocaine evoked synaptic potentiation at this synapse, occluding homosynaptic inhibitory long-term potentiation. The activity of the VTA GABA neurons was thus reduced and DA neurons were disinhibited. Cocaine-evoked potentiation of GABA release from D1R-MSNs affected drug-adaptive behavior, which identifies these neurons as a promising target for novel addiction treatments.

Developmental Stage-dependent persistent impact of propofol anesthesia on dendritic spines in the rat medial prefrontal cortex

Background:Recent observations demonstrate that anesthetics rapidly impair synaptogenesis during neuronal circuitry development. Whether these effects are lasting and depend on the developmental stage at which these drugs are administered remains, however, to be explored. Methods:Wistar rats received propofol anesthesia at defined developmental stages during early postnatal life. The acute and long-term effects of these treatments on neuronal cytoarchitecture were evaluated by Neurolucida and confocal microscopy analysis after iontophoretic injections of Lucifer Yellow into layer 5 pyramidal neurons in the medial prefrontal cortex. Quantitative electron microscopy was applied to investigate synapse density. Results:Layer 5 pyramidal neurons of the medial prefrontal cortex displayed intense dendritic growth and spinogenesis during the first postnatal month. Exposure of rat pups to propofol at postnatal days 5 and 10 significantly decreased dendritic spine density, whereas this drug induced a significant increase in spine density when administered at postnatal days 15, 20, or 30. Quantitative electron microscopy revealed that the propofol-induced increase in spine density was accompanied by a significant increase in the number of synapses. Importantly, the propofol-induced modifications in dendritic spine densities persisted up to postnatal day 90. Conclusion:These new results demonstrate that propofol anesthesia can rapidly induce significant changes in dendritic spine density and that these effects are developmental stage-dependent, persist into adulthood, and are accompanied by alterations in synapse number. These data suggest that anesthesia in the early postnatal period might permanently impair circuit assembly in the developing brain.

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.

N-cadherin mediates plasticity-induced long-term spine stabilization

Synaptic persistence is enhanced by N-cadherin, which clusters together in response to neural activity and long-term potentiation induction in dendritic spines.

GABA Regulates Dendritic Growth by Stabilizing Lamellipodia in Newly Generated Interneurons of the Olfactory Bulb

The initial formation and growth of dendrites is a critical step leading to the integration of newly generated neurons into postnatal functional networks. However, the cellular mechanisms and extracellular signals regulating this process remain mostly unknown. By directly observing newborn neurons derived from the subventricular zone in culture as well as in olfactory bulb slices, we show that ambient GABA acting through GABAA receptors is essential for the temporal stability of lamellipodial protrusions in dendritic growth cones but did not interfere with filopodia dynamics. Furthermore, we provide direct evidence that ambient GABA is required for the proper initiation and elongation of dendrites by promoting the rapid stabilization of new dendritic segments after their extension. The effects of GABA on the initial formation of dendrites depend on depolarization and Ca2+ influx and are associated with a higher stability of microtubules. Together, our results indicate that ambient GABA is a key regulator of dendritic initiation in postnatally generated olfactory interneurons and offer a mechanism by which this neurotransmitter drives early dendritic growth.

Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS‐specific GDH‐null mice (CnsGlud1−/−); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns‐Glud1−/− brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns‐Glud1−/− mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns‐Glud1−/− mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up‐regulation of astrocyte‐type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.

Bilateral whisker trimming during early postnatal life impairs dendritic spine development in the mouse somatosensory barrel cortex

The rodent somatosensory barrel cortex is an ideal model for studying the impact of sensory experience on developing brain circuitry. To examine whether and how interference with sensory perception in the early postnatal period can affect the development of synaptic networks in this system, we took advantage of a transgenic mouse strain expressing the yellow fluorescent protein in layer 5B pyramidal neurons of the somatosensory cortex. By using ex vivo confocal imaging, we first demonstrate a cortical‐layer‐specific increase in the number of dendritic spines during postnatal development on apical dendritic shafts of these cells extending up to cortical layer 1. Next, by performing bilateral whisker trimming at distinct developmental stages, we show that disruption of sensory perception before postnatal day 20 impairs dendritic spine development in apical dendritic segments within layers 1 and 2/3 but not in layer 4. The whisker trimming‐induced decrease in dendritic spine density during this period is accompanied by a highly significant decrease in dendritic spine head diameter. Finally, we also show that these whisker trimming‐induced morphological alterations of dendritic spines during the early postnatal period are no longer detectable in adult animals. Altogether, these findings further emphasize the important role of sensory activity in synaptic network assembly in the developing barrel cortex. They also support an as yet unidentified structural mechanism that might contribute to the layer‐ and cell‐type‐specific physiological effects of whisker trimming during the early postnatal period. J. Comp. Neurol. 518:1711–1723, 2010.