The three main phases of neural development 1. Genesis of neurons (and migration). 2. Outgrowth of axons and dendrites, and synaptogenesis. 3. Refinement of synaptic connections.

2. Outgrowth of axons and dendrites, and synaptogenesis. In class you heard about the x/y axis: What about the Z axis? y x

The Z axis: the brain is laminated (layered) How is the correct lamina/layer found?

Brief reminder: the retina to LGN - Input: photoreceptors - Output: RGCs - Processes of retinal interneurons (amacrine and bipolar cells) form synapses on dendrites of RGCs in the inner plexiform layer (IPL). - Subsets of interneurons and RGCs arborize and form synapses in just one or a few of the IPL lamina. - These lamina-specific circuits determine the visual features to which RGC subtypes respond.

CAMs (cell adhesion molecules): Dscam and Sidekicks - Part of the Immunoglobulin super family (IgSF). - Promote adhesion and/or repulsion. - Dscam was isolated from the chromosome band which is critical for many of the neurological phenotypes of Down Syndrome. - Implicated in correct brain wiring (axon guidance, dendritic arborization, etc.). Blue: immunoglobulin domains Pink: fibronectin type III domains Grey: membrane Green: carboxy-terminal sequences predicted and shown to bind PDZ domains

Dscam, DscamL, Sidekick-1 and Sidekick-2 are expressed by non-overlapping subsets of retinal neurons. mRNA (in-situ hybridization): no overlap protein (immunostaining): no overlap + lamination RGCs laminae

Dscam, DscamL, Sidekick-1 and Sidekick-2 are expressed by non-overlapping subsets of retinal neurons. * sites of lower expression Thus, Dscam, DscamL, Sidekick-1 and Sidekick-2 mark four distinct synaptic pathways in the retina.

Dscams and Sidekicks mediate homophilic adhesion only no. of aggregates

Depletion of Dscam or Sidekicks (with RNAi) selectively perturbs arbors of neurons in specific IPL sublaminae Independent markers (RGCs): Dscam → R-cadherin Sidekick-1 → Cadherin-7 Sidekick-2 → Calbindin Expected result: Dscam and Sdks are necessary for correct arbor lamination

depletion of Dscam or Sidekicks selectively perturbs arbors of neurons in specific IPL sublaminae (continued). The effect is specific: 1. No disruptions were seen in uninfected areas of the same retinae. 2. Depletion of each IgSF member affected only the RGC subset marked by the coexpressed gene. 3. No displacement of processes formed by cells, which do not express Dscam or either Sidekick (choline acetyltransferase- and substance P-positive). ChAT RNAi-Dscam

Ectopic expression of a Dscam or Sidekick (PiggyBac) reroutes arbors of the expressing cell to normally Dscam-positive or Sidekick-positive IPL sublaminae. Control: GFP=all cells +Dscam: GFP=Dscam- expressing cells +Sdk2: GFP=Sdk2- expressing cells ChAT-expressing cells are also affected Dscam and Sdks are sufficient for correct arbor lamination

Ectopic expression of a Dscam or Sidekick reroutes arbors of the expressing cell to normally Dscam- positive or Sidekick-positive IPL sublaminae. Dscam and Sdks are sufficient for correct arbor lamination

Drosophila Dscams act as repulsive factors, BUT these results suggest that Dscams and Sidekicks act as attractive (adhesive) cues Dscams and Sidekicks indeed act as attractive (adhesive) cues SV2-synaptic vesicle protein In-vitro

Conclusions: 1. Dscams and Sidekicks specify 4 parallel pathways in the inner retina. 2. Dscams and Sidekicks are necessary for sub-lamina specific arborization. 3. Dscams and Sidekicks are sufficient for sub-lamina specific arborization. 4. This is mediated through homophilic adhesion (attraction).

Suggested mechanisms: 1.Regulation of the arborization of pre- and postsynaptic processes. 2.Target recognition. 3.Stabilization of synaptic contacts.

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