Connectivity in neurodevelopmental disorders

Neural connectivity in autism spectrum disorders

Immunohistochemical analysis of microglia (green) interacting with dendrites of layer 2/3 pyramidal neurons (red) in the medial prefrontal cortex of p56 mice.
Immunohistochemical analysis of microglia (green) interacting with dendrites of layer 2/3 pyramidal neurons (red) in the medial prefrontal cortex of p56 mice.
ASD is a neurodevelopmental disorder characterized by three core features, shortcomings in social interactions and communication, as well as repetitive behaviours.

One approach to better understand the aetiology of ASD is to take candidate autism genes identified from human patients, generate mice conditionally mutant for these genes, and analyse functionally and structurally the neural circuits controlling these behaviours. A prevailing hypothesis for ASD occurrence is an early imbalance between excitation and inhibition in circuits associated with these behaviours, leading to an eventual underdevelopment of long-range connectivity paired with an overdevelopment of local circuits and disruption of cortical oscillations. The role of autophagy linked to de-regulated synaptic pruning in the aetiology of ASD is currently under intensive investigation.

We are working on an understanding of structural changes in neural connectivity in mice mutant for the high confident ASD gene cntnap2, a member of the neurexin family. Cntnap2 has been shown to function in the stabilization of synapses, while other members of the neurexin family have generally synaptogenic activities, that is the capacity to induce synapse formation.

We focus our analyses on parts of the medial prefrontal cortex, and the somatosensory cortex, that is regions believed to function as hubs to integrate neural circuits controlling social behaviours.

We are studying the development of circuits in these areas from early postnatal times to adult mice which enables us to detect (also) transient defects in neural circuit development.

To facilitate our analysis, we have generated cntnap2 mutant mice in which a small fraction of pyramidal neurons from layers 2/3 and 5 are specifically labelled, allowing high-resolution analyses of excitatory neural circuits, projection patterns, and synaptic connectivities.

We find transient disturbances in the density and form of dendritic spines of layer 2/3 and layer 5 pyramidal neurons which will affect the balance between excitation and inhibition in these areas. We are studying now a disruption of long-range projection patterns of these pyramidal neurons.

To understand the defects on a molecular level, we investigate the hypothesis that spine pruning is disrupted due to a disturbance of microglia function and/or neuronal autophagy in cntnap2 mutant mice. We investigate in parallel possible changes in the inhibitory circuitry of these mice.

Mechanisms of development of the retino-collicular projection

Topographic targeting errors of temporal retinal axons (arrow) occur only if both the retinal and collicular expression of ephrinA5 is abolished (G-H), but not if either the collicular (C-D) or the retinal (E-F) ephori A5 expression is abolished
Topographic targeting errors of temporal retinal axons (arrow) occur only if both the retinal and collicular expression of ephrinA5 is abolished (G-H), but not if either the collicular (C-D) or the retinal (E-F) ephori A5 expression is abolished
The retino-collicular/tectal projection has been extensively studied as a model system to understand the development of topographic connections. Here, axons from the temporal retina project to the anterior superior colliculus (SC, optic tectum in lower vertebrates) while axons from the nasal retina project to the posterior SC. Similarly dorsal and ventral retina are connected to lateral and medial tectum. Fundamental work of F. Bonhoeffer and colleagues in the 1980s (Walter et al., 1987) laid the foundation for cloning of the key molecules controlling the development of this projection that is members of the Eph family of receptor tyrosine kinases and their ephrin ligands (Cheng et al., 1995; Drescher et al., 1995). Knock-out studies in mice have demonstrated that these molecules are the key players in the development of this map. However, while the “text-book” view states that map development is dependent on axon-target interactions mediated by interactions between EphAs on retinal axons and ephrinAs in the colliculus, our recent work has shown that there is a substantial mapping role for EphA/ephrinA interactions between RGC axons themselves that is independent of their expression in the colliculus (Suetterlin and Drescher, Neuron 2014). It remains unclear how general these axon-axon interactions are for map development, as we have investigated only the function of one out of three ephrinAs expressed on retinal axons. To address this, we aim to generate new mouse models by deleting all ephrinAs either in the colliculus to abolish all axon-target interactions, or in the retina, to abolish all axon-axon interactions. Overall, we hypothesize that retinal axons can self-organise to form a topographic map in a target area devoid of guidance information. The axon-axon interactions used in the olfactory system provide possibly the most impressive example of the power of target-independent axon-axon self-organization (for review, Sakano Neuron 2014).

The role of autophagy in controlling branching of retinal ganglion cell axons

Analysis of expression patterns in retinal ganglion cells axons using super resolution SIM shows the co-localization of Arl8 (A) with LAMP1, a marker for lysosomes (B).
Analysis of expression patterns in retinal ganglion cells axons using super resolution SIM shows the co-localization of Arl8 (A) with LAMP1, a marker for lysosomes (B).
Axon branching is an essential step during the establishment of neural connectivity in the vertebrate CNS. While numerous molecules and mechanisms have been proposed to control axon branching, one long standing - though little understood - concept is given in the synaptotropic hypothesis which proposes a tight link between the formation of presynapses and axonal branches, such that – for example - axon branches emerge preferentially from presynaptic structures. In C. elegans, presynapse formation in selected axons is controlled by the small GTP binding protein Arl8. We have now cloned chick Arl8B and characterised its role during (interstitial) axon branching of retinal ganglion cells (RGCs). We found that a loss-of-function approach of Arl8B results in a decrease, while a gain-of-function approach led to an increase in the number of RGC axon branches. In addition, knockdown and overexpression of Arl8B shifts axon branches to more proximal or distal parts of RGC axons, respectively. Arl8 is located on lysosomes, and might contribute to a regulation of autophagy, a regulated intracellular degradation process crucial for cellular homeostasis. We are at present studying directly the role of autophagy in axonal morphology, either genetically or by a pharmacological inactivation of autophagy. Our data indicate that a disruption of autophagy results in a disturbance of axon branching of RGCs. Furthermore, super resolution SIM shows a (partial) co-localisation between presynaptic structures and autophagosomes and lysosomes. Our current approaches aim to further understand how autophagy regulates axon branching. For this we will analyse the function of the autophagic machinery in retinal ganglion cell axons using time-lapse microscopy in vitro, and by using conditional knockout approaches to study the development of neural circuits after selectively inactivating autophagy in vivo.