Mechanisms of plasticity specification during an embryonic critical period.

Transient experiences during formative periods of development, called 'critical periods', have lasting impact on how our brains function. As indicated by the name, critical periods are of fundamental importance to the development of nervous systems; during these developmental phases functional properties of nerve cells are specified, which determine how networks perform. Importantly, errors that occur during a critical period often remain locked in, unable to be corrected. Many neurodevelopmental disorders, including epilepsy, schizophrenia and autism spectrum disorder, are now thought of as network mis-adjustments that arise during critical periods (of childhood or adolescence). Conversely, clinical interventions during the critical period could prove highly effective; for example, transient re-balancing of neuronal activity during the critical period can permanently rescue the effects of mutations that would otherwise cause epilepsy. Because critical periods are so pivotal in whether or not a nervous system gains normal function, it is important that we understand the underlying mechanisms.

These questions are exceedingly difficult to investigate when working with complex nervous systems, such as mammalian sensory systems, which thus far have been the go-to model systems. Excitingly, recent work by our collaborators, Prof. Richard Baines, University of Manchester, has shown that critical periods are probably universal phenomena, common to all nervous systems, including insects. Building on their findings, we have discovered an explicit critical period at the neuromuscular junctions (nerve-muscle connection) in the fruitfly, Drosophila. This has many advantages, including being comparatively large, as well as easy to access and manipulate. Having been extremely well characterised (in other contexts) through the work of many laboratories, we can now build on these solid foundations and make rapid progress. For example, we have already discovered that several forms of previously studied neuronal plasticity (mechanisms that allow nervous systems to adjust and learn) are regulated by transient critical period experience in late embryogenesis, and that some are disabled following brief embryonic experiences.

To illustrate, we have chosen to work with temperature as a stimulus this animal would normally encounter in the wild, ranging from 18-29 degrees centigrade. Excitingly, we find that if the 'extremes' of either 18 or 29 degrees are transiently experienced for a few hours as late embryos, this markedly changes how nerve cells develop (e.g., their growth) and how they behave (e.g., no longer able to change in ways thought necessary for learning). This is mirrored at the level of animal behaviour, animals being 'stuck' and unable to adapt to changes in their environment. Scientifically, this is exciting; though in view of climate change, observing the dramatic, lasting effects that a difference of a few degrees can have on nervous system development and animal behaviour, is quite alarming.

We now plan to use the advantages of this highly tractable experimental system to identify the mechanisms by which transient critical periods specify important nerve cell properties (Objective 1).

Second, we will investigate how it is that transient experiences can have lasting effects. This is currently not understood, though our preliminary data point to epigenetic mechanisms as a means of changing and maintaining changes in gene expression.

In summary, we have identified a simplified, but powerful experimental model system with which to investigate fundamental questions that important to our general understanding of nervous system development. The mechanisms involved are almost certainly conserved. The output from this work is therefore likely to have impact in both clinical as well as ecological settings.

1. Determining mechanisms by which critical period (CP) experience specifies neuronal plasticity.
a) Postsynaptic sensitivity: We will investigate how lasting changes in DGluRIIB expression is achieved:
-transcriptionally; assayed by RT-qPCR and smiFISH;
-post-transcriptionally; test by replacement of 3' and 5' UTRs;
-post-translationally: monitor available DGluRIIA and -B domain swap chimeras.

b) Presynaptic: We will screen PHP-linked genes as candidates for CP regulation by genetic interaction (50% dosage) in a sensitised background of sub-threshold ROS generators or scavengers. PHP induced by temperature shift (establ. in lab) will be recorded by sharp electrode e-phys. As a second tier, targeted RNAi knock-down (pre- or postsynaptically) will validate and refine.

c) Structural: We will transiently modulate known pre-synaptic (CamKII, PKA, synapsin) and post-synaptic (Wnt, BMP) structural plasticity pathways during the CP, by expression of phospho-mimetic, constitutive forms or inhibitory peptides during the CP; backed up by systemic modulation using pharmacological agents.

2: Investigating how transient events effect lasting changes in gene expression. We find changes in chromatin modification linked to CP experience. To investigate this furher we will:
a) use Chromatin Accessibility profiling using Targeted DamID (CATaDa), targeted to motorneurons or muscles to determine chromatin accessibility following (18 vs 25 vs 29oC) CP experience.
b) To corroborate CATaDa data by targeting DamID specifically to His3K9Ac, we will generate a Dam-fusion with the His3K9Ac binding domain from ENL/AF9-related. This will confirm hits from 2.a and provide new candidates for 1.a., 1.b. and 1.c.
c) CP-regulated Chromatin modifications: We will test if HDAC1 and SAGA complex mediate CP-induced chromatin modifications, using mutants and RNAi-knockdown, assayed by anti-His3K9Ac staining and AF9-DamID; then if CP experience alters HDAC1 or SAGA complex expression.