Egbert Welker

Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex

Do new synapses form in the adult cortex to support experience-dependent plasticity? To address this question, we repeatedly imaged individual pyramidal neurons in the mouse barrel cortex over periods of weeks. We found that, although dendritic structure is stable, some spines appear and disappear. Spine lifetimes vary greatly: stable spines, about 50% of the population, persist for at least a month, whereas the remainder are present for a few days or less. Serial-section electron microscopy of imaged dendritic segments revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. Experience-dependent plasticity of cortical receptive fields was accompanied by increased synapse turnover. Our measurements suggest that sensory experience drives the formation and elimination of synapses and that these changes might underlie adaptive remodelling of neural circuits.

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 α-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.

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.

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.

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.

Organization of the projections from barrel cortex to thalamus in mice studied with Phaseolus vulgaris-leucoagglutinin and HRP

SummaryIn order to elucidate the geometric organization of projections from the barrel cortex to the thalamus, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were made. The injections were confined to one barrel column (i.e. barrel in layer IV + cortical tissue above and below it). Axonal terminations could be demonstrated in three thalamic nuclei: reticularis (RT), ventrobasalis (VB) and posterior (PO). Anterograde terminal labelling was obtained in RT + VB; in PO only; or in RT + VB + PO. The terminals labelled in PO were much larger than those in RT and VB. The termination areas in RT, VB and PO were shaped like rods which have a rostro-caudal orientation. These cortico-thalamic projections are discretely and topographically organized. The clearest such arrangement was found in VB. Here, projections from the A row of barrels in BF terminate dorsally, whereas those from the C row end ventrally. Barrel A1 projects to the lateral part of VB, whereas A4, to more medial parts; other rows are arranged similarly. These results were compared with the distribution of thalamo-cortical projection neurons that were labelled after iontophoretic HRP injections in individual barrels. We concluded that the corticothalamic projections originating from one barrel column contact an arc of barreloids in VB.

Organization of feedback and feedforward projections of the barrel cortex: a PHA-L study in the mouse.

SummaryIn order to analyze the organization of the efferent projections of single barrel columns (BC, i.e. a barrel in layer IV of parietal cortex plus the cortical tissue above and below it), we made small iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the barrel cortex of 20 adult mice. On the basis of reconstructions of the sites of terminal labelling, the brain regions receiving projections from the barrel cortex could be identified and classified in five groups. Each group is characterized by the topography of the distribution of efferents arising from a single BC. The projections to the trigeminal sensory complex are point to point: i.e. one BC projects only to the site of termination of the primary sensory neurons innervating the corresponding whisker follicle. In the ventrobasal thalamic nucleus BC projections are not restricted to the corresponding barreloid; instead they contact parts of barreloids belonging to one arc. In the reticular and posterior thalamic nuclei the projections from a row of BCs converge to a collective termination site, whereas in the superior colliculus the projections from an arc of BCs converge to a common termination site. There is a complete overlap of BC projections in restricted zones within SII, motor cortex, perirhinal cortex, contralateral barrelfield, caudoputamen and pons. The organization of the efferents from the barrel cortex demonstrates a contrast between feedback and feedforward projections from this important area of neocortex.

Plasticity in the barrel cortex of the adult mouse: effects of peripheral deprivation on GAD-immunoreactivity.

SummaryThe whisker-to-barrel pathway of the adult mouse was used in a study on the effects of peripheral sensory deprivation on GAD-immunoreactivity in the somatosensory cortex. At varying periods of time after removal of a set of vibrissal follicles, mice were processed for immunohistochemistry using an antibody against GAD. In sections tangential to the cortical surface we observed, in the barrels whose follicles were removed, decreased immunoreactivity as early as three days after surgery. The decrease was due to a lesser numerical density of stained puncta and to less intense staining of those remaining. GAD-positive somata were also less intensely stained, whereas their number did not seem to be changed. The changes, apparent at 3 days after the surgery, were restricted to the barrels corresponding to the removed follicles and were maximal at 2–4 weeks. At longer survival times (until 7 months) the immunoreactivity returned to normal, coincident with the regeneration of peripheral nerve fibres in the absence of their follicles. We conclude that GAD-immunoreactivity in the barrel cortex swiftly reacts to modifications of neuronal activity evoked in the periphery.

A comparative analysis of the morphology of corticothalamic projections in mammals

Recent anatomical tracing methods have revealed new principles underlying the organization of corticothalamic connections in the mammalian nervous system. These data demonstrated the distribution of two types of synaptic contacts in the corticothalamic projection: small (<1 microm) and giant (2-10 microm) axon terminals. We compare the organization of corticothalamic projections in the auditory, somatosensory, visual, and motor systems of a variety of mammalian species, including the monkey. In all these systems and species, both types of corticothalamic terminals have been observed. Small endings formed the major corticothalamic terminal field, whereas giant terminals were less numerous and formed additional terminal fields together with small terminals. After comparing their spatial distribution, as well as the degree of reciprocity between the corticothalamic and thalamocortical projections, different roles are proposed for small and giant endings. Small terminals are typically present in the projection serving the feed-back control of the cerebral cortex on the thalamic nucleus from which it receives its main projection. In contrast, giant terminals are involved in feed-forward projections by which activity from a cortical area is distributed, via the thalamus, to other parts of the cerebral cortex. The cross-species and cross-systems comparison reveals differences in the mode of feed-forward projection, which may be involved in the activation of other parts of the same cortical area or form part of a projection that activates other cortical areas.