Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors.

Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4-8, corresponding to 4-8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4-8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)-CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral-CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 µm), but not by orexin-1 receptor inhibition (SB334867, 1 µm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg(-1)), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep-wake cycle.

Intracortical connectivity of layer VI pyramidal neurons in the somatosensory cortex of normal and barrelless mice.

In mice, barrels in layer IV of the somatosensory cortex correspond to the columnar representations of whisker follicles. In barrelless (BRL) mice, barrels are absent, but functionally, a columnar organization persists. Previously we characterized the aberrant geometry of thalamic projection of BRL mice using axonal reconstructions of individual neurons. Here we proceeded with the analysis of the intracortical projections from layer VI pyramidal neurons, to assess their contribution to the columnar organization. From series of tangential sections we reconstructed the axon collaterals of individual layer VI pyramidal neurons in the C2 barrel column that were labelled with biocytin [controls from normal (NOR) strain, 19 cells; BRL strain, nine cells]. Using six morphological parameters in a cluster analysis, we showed that layer VI neurons in NOR mice are distributed into four clusters distinguished by the radial and tangential extent of their intracortical projections. These clusters correlated with the cortical or subcortical projection of the main axon. In BRL mice, neurons were distributed within the same four clusters, but their projections to the granular and supragranular layers were significantly smaller and their tangential projection was less columnar than in NOR mice. However, in both strains the intracortical projections had a preference for the appropriate barrel column (C2), indicating that layer VI pyramidal cells could participate in the functional columnar organization of the barrel cortex. Correlative light and electron microscopy analyses provided morphometric data on the intracortical synaptic boutons and synapses of layer VI pyramidal neurons and revealed that projections to layer IV preferentially target excitatory dendritic spines and shafts.

Experience-dependent and cell-type-specific spine growth in the neocortex.

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size and the number of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.

Characterization of the HeCo mutant mouse: a new model of subcortical band heterotopia associated with seizures and behavioral deficits.

In human, neuronal migration disorders are commonly associated with developmental delay, mental retardation, and epilepsy. We describe here a new mouse mutant that develops a heterotopic cortex (HeCo) lying in the dorsolateral hemispheric region, between the homotopic cortex (HoCo) and subcortical white matter. Cross-breeding demonstrated an autosomal recessive transmission. Birthdating studies and immunochemistry for layer-specific markers revealed that HeCo formation was due to a transit problem in the intermediate zone affecting both radially and tangentially migrating neurons. The scaffold of radial glial fibers, as well as the expression of doublecortin is not altered in the mutant. Neurons within the HeCo are generated at a late embryonic age (E18) and the superficial layers of the HoCo have a correspondingly lower cell density and layer thickness. Parvalbumin immunohistochemistry showed the presence of gamma-aminobutyric acidergic cells in the HeCo and the mutant mice have a lowered threshold for the induction of epileptic seizures. The mutant showed a developmental delay but, in contrast, memory function was relatively spared. Therefore, this unique mouse model resembles subcortical band heterotopia observed in human. This model represents a new and rare tool to better understand cortical development and to investigate future therapeutic strategies for refractory epilepsy.

Spine growth precedes synapse formation in the adult neocortex in vivo.

Dendritic spines appear and disappear in an experience-dependent manner. Although some new spines have been shown to contain synapses, little is known about the relationship between spine addition and synapse formation, the relative time course of these events, or whether they are coupled to de novo growth of axonal boutons. We imaged dendrites in barrel cortex of adult mice over 1 month, tracking gains and losses of spines. Using serial section electron microscopy, we analyzed the ultrastructure of spines and associated boutons. Spines reconstructed shortly after they appeared often lacked synapses, whereas spines that persisted for 4 d or more always had synapses. New spines had a large surface-to-volume ratio and preferentially contacted boutons with other synapses. In some instances, two new spines contacted the same axon. Our data show that spine growth precedes synapse formation and that new synapses form preferentially onto existing boutons.

The barrel cortex as a model to study dynamic neuroglial interaction.

There is increasing evidence that glial cells, in particular astrocytes, interact dynamically with neurons. The well-known anatomofunctional organization of neurons in the barrel cortex offers a suitable and promising model to study such neuroglial interaction. This review summarizes and discusses recent in vitro as well as in vivo works demonstrating that astrocytes receive, integrate, and respond to neuronal signals. In addition, they are active elements of brain metabolism and exhibit a certain degree of plasticity that affects neuronal activity. Altogether these findings indicate that the barrel cortex presents glial compartments overlapping and interacting with neuronal compartments and that these properties help define barrels as functional and independent units. Finally, this review outlines how the use of the barrel cortex as a model might in the future help to address important questions related to dynamic neuroglia interaction.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex.

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton-spine interface and an increase in glutamate transporter expression in astrocytic processes.

Formation of dendritic spines with GABAergic synapses induced by whisker stimulation in adult mice.

During development, alterations in sensory experience modify the structure of cortical neurons, particularly at the level of the dendritic spine. Are similar adaptations involved in plasticity of the adult cortex? Here we show that a 24 hr period of single whisker stimulation in freely moving adult mice increases, by 36%, the total synaptic density in the corresponding cortical barrel. This is due to an increase in both excitatory and inhibitory synapses found on spines. Four days after stimulation, the inhibitory inputs to the spines remain despite total synaptic density returning to pre-stimulation levels. Functional analysis of layer IV cells demonstrated altered response properties, immediately after stimulation, as well as four days later. These results indicate activity-dependent alterations in synaptic circuitry in adulthood, modifying the flow of sensory information into the cerebral cortex.

Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex.

Neuron-glia interactions are essential for synaptic function, and glial glutamate (re)uptake plays a key role at glutamatergic synapses. In knockout mice, for either glial glutamate transporters, GLAST or GLT-1, a classical metabolic response to synaptic activation (i.e., enhancement of glucose utilization) is decreased at an early functional stage in the somatosensory barrel cortex following activation of whiskers. Investigation in vitro demonstrates that glial glutamate transport represents a critical step for triggering enhanced glucose utilization, but also lactate release from astrocytes through a mechanism involving changes in intracellular Na(+) concentration. These data suggest that a metabolic crosstalk takes place between neurons and astrocytes in the developing cortex, which would be regulated by synaptic activity and mediated by glial glutamate transporters.

Subcellular domain-restricted GABAergic innervation in primary visual cortex in the absence of sensory and thalamic inputs.

Distinct classes of GABAergic synapses target restricted subcellular domains, thereby differentially regulating the input, integration and output of principal neurons, but the underlying mechanism for such synapse segregation is unclear. Here we show that the distributions of two major classes of GABAergic synapses along the perisomatic and dendritic domains of pyramidal neurons were indistinguishable between primary visual cortex in vivo and cortical organotypic cultures. Therefore, subcellular synapse targeting is independent of thalamic input and probably involves molecular labels and experience-independent forms of activity.

Structural basis for map formation in the thalamocortical pathway of the barrelless mouse.

Barrelless mice (BRL) homozygous for the BRL mutation that disrupts the gene coding for adenylyl cyclase type I on chromosome 11 lack spatial segregation of layer IV cortical cells and of the thalamocortical axons (TCAs) into barrel domains. Despite these morphological perturbations, a functional topographic map has been demonstrated. We reconstructed individual biocytin-injected TCAs from thalamus to barrel cortex in NOR (normal) and BRL mice to analyze to what extent the TCA arborization pattern and bouton distribution could explain the topographic representation of the whisker follicles. In BRL, the geometry of TCA is modified within layer IV as well as in infragranular layers. However, in both strains, the spatial distribution of TCA in layer IV reflects the spatial relationship of their cell bodies in the ventrobasal nucleus of the thalamus. The morphometric analysis revealed that TCAs of both strains have the same length, branch number, and number of axonal boutons in layer IV. However, in barrelless, the boutons are distributed within a larger tangential extent. Analysis of the distribution of boutons from neighboring thalamic neurons demonstrated the existence in layer IV of domains of high bouton density that in both strains equal the size and shape of individual barrels. We propose that the domains of high bouton density are at the basis of the whisker map in barrelless mice.

Altered synapse formation in the adult somatosensory cortex of brain-derived neurotrophic factor heterozygote mice.

Increased sensory stimulation in the adult whisker-to-barrel pathway induces the expression of BDNF as well as synapse formation in cortical layer IV. Here, we investigated whether BDNF plays a role in the alterations of connectivity between neurons by analyzing the ultrastructure of the BDNF heterozygote mouse, characterized by a reduced level of BDNF expression. Using serial section electron microscopy, we measured synapse density, spine morphology, and synaptic vesicle distribution to show that mice with a reduced level of BDNF have a barrel neuropil that is indistinguishable from wild-type controls. After 24 hr of whisker stimulation, however, there is no indication of synapse formation in the heterozygous mouse. Whereas the balance between excitatory and inhibitory synapses is modified in the controls, it remains constant in the heterozygotes. The distribution of synaptic vesicles in excitatory synapses is the same in heterozygous and wild-type mice and is not influenced by the stimulation paradigm. Spine volume, however, is unchanged by stimulation in the wild-type animals, but does increase significantly in the heterozygous animal. These results provide evidence that, in vivo, BDNF plays an important role in the structural rearrangement of adult cortical circuitry as a consequence of an increased sensory input.

Adenylate cyclase 1 as a key actor in the refinement of retinal projection maps.

cAMP occupies a strategic position to control neuronal responses to a large variety of developmental cues. We have analyzed the role of calcium-stimulated adenylate cyclase 1 (AC1) in the development of retinal topographic maps. AC1 is expressed in retinal ganglion cells (RGCs) from embryonic day 15 to adulthood with a peak during the first postnatal week. At that time, the other calcium-stimulated AC, AC8, is expressed in the superior colliculus (SC) but not in the RGCs. In mice of the barrelless strain, which carry an inactivating mutation of the AC1 gene, calcium-stimulated AC activity is reduced by 40-60% in the SC and retina. RGC projection maps were analyzed with a variety of anterograde and retrograde tracers. After an initially normal development until postnatal day 3, retinal fibers from the ipsilateral and contralateral eye fail to segregate into eye-specific domains in the lateral geniculate nucleus and the SC. Topographic defects in the fine tuning of the retinotectal and retinogeniculate maps are also observed with abnormalities in the confinement of the retinal axon arbors in the anteroposterior and mediolateral dimensions. This is attributable to the lack of elimination of misplaced axon collaterals and to the maintenance of a transient ipsilateral projection. These results establish an essential role of AC1 in the fine patterning of the retinal map. Calcium-modulated cAMP production in the RGCs could constitute an important link between activity-dependent changes and the anatomical restructuring of the retinal terminal arbors within central targets.

Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period.

The neocortical GABAergic network consists of diverse interneuron cell types that display distinct physiological properties and target their innervations to subcellular compartments of principal neurons. Inhibition directed toward the soma and proximal dendrites is crucial in regulating the output of pyramidal neurons, but the development of perisomatic innervation is poorly understood because of the lack of specific synaptic markers. In the primary visual cortex, for example, it is unknown whether, and to what extent, the formation and maturation of perisomatic synapses are intrinsic to cortical circuits or are regulated by sensory experience. Using bacterial artificial chromosome transgenic mice that label a defined class of perisomatic synapses with green fluorescent protein, here we show that perisomatic innervation developed during a protracted postnatal period after eye opening. Maturation of perisomatic innervation was significantly retarded by visual deprivation during the third, but not the fifth, postnatal week, implicating an important role for sensory input. To examine the role of cortical intrinsic mechanisms, we developed a method to visualize perisomatic synapses from single basket interneurons in cortical organotypic cultures. Characteristic perisomatic synapses formed through a stereotyped process, involving the extension of distinct terminal branches and proliferation of perisomatic boutons. Neuronal spiking in organotypic cultures was necessary for the proliferation of boutons and the extension, but not the maintenance, of terminal branches. Together, our results suggest that although the formation of perisomatic synapses is intrinsic to the cortex, visual experience can influence the maturation and pattern of perisomatic innervation during a postnatal critical period by modulating the level of neural activity within cortical circuits.

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human.

Membrane potential dynamics of neocortical projection neurons driving target-specific signals.

Primary sensory cortex discriminates incoming sensory information and generates multiple processing streams toward other cortical areas. However, the underlying cellular mechanisms remain unknown. Here, by making whole-cell recordings in primary somatosensory barrel cortex (S1) of behaving mice, we show that S1 neurons projecting to primary motor cortex (M1) and those projecting to secondary somatosensory cortex (S2) have distinct intrinsic membrane properties and exhibit markedly different membrane potential dynamics during behavior. Passive tactile stimulation evoked faster and larger postsynaptic potentials (PSPs) in M1-projecting neurons, rapidly driving phasic action potential firing, well-suited for stimulus detection. Repetitive active touch evoked strongly depressing PSPs and only transient firing in M1-projecting neurons. In contrast, PSP summation allowed S2-projecting neurons to robustly signal sensory information accumulated during repetitive touch, useful for encoding object features. Thus, target-specific transformation of sensory-evoked synaptic potentials by S1 projection neurons generates functionally distinct output signals for sensorimotor coordination and sensory perception.

A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.