Nervous System Development

• The human brain has approximately 10¹¹ neurons, each forming over 10³ synapses, resulting in over 10¹⁴ connections that enable sensing, thinking, memory, and action.

• Brain wiring is determined by nature (genes) and nurture (experience)

• This chapter focuses on the mechanisms of nervous system development, specifically wiring in the visual system

Retinal Ganglion Cell Axon Targets

• Retinal ganglion cells (RGCs) transmit visual information to the brain
• For example, a midget RGC located in the fovea of the left eye relays information about long-wavelength light
• Dendrites must connect to bipolar and amacrine cells to construct a receptive field
• Axons must exit the eye, travel through the optic nerve, decide to cross at the optic chiasm, travel along the optic tract, and branch to the lateral geniculate nucleus (LGN) and superior colliculus
• Retinotopic maps in the brain preserve the spatial relationships of the visual image from the retina
• Axons must terminate at retinotopic positions in the superior colliculus and LGN based on retinal location
• It is important to choose correct synaptic partners according to cell identity as a red-ON center midget cell
• RGC axons terminate at appropriate target locations to ensure accurate retinotopic map formation

Optic Nerve Regeneration and Axon Predetermination

• Two concepts explaining the connection between neurons and their targets exist:
  • Axons connect to many targets, and functions select some connections, pruning others
  • Axons are predetermined to directly choose their targets
• In the early 20th century, scientists believed functional selection was a factor in establishing neuronal connections
• Roger Sperry's experiments on optic nerve regeneration in amphibians suggested neurons are predetermined to choose targets
• Amphibians can regenerate nerve connections after damage
• Sperry rotated newt eyes by 180°, severed the optic nerve, and allowed RGC axons to regrow into the brain
• Behavior tests assessed what the newt saw with the manipulated eye
• Three outcomes were possible: restored vision, blurred vision, or inverted vision
• Vision was restored, but inverted
• This indicated RGC axons have information related to their eye positions, and the brain has information relating to eye positions
• Despite the eye rotation altering spatial receptive fields, positional information enabled RGC axons to connect with original target neurons, restoring vision in an inverted way

Chemoaffinity and Retinotectal Connections

• Tectum is the main RGC target in lower vertebrates
• Retina forms a 2D image in external world which is reconstructed in the tectum using point-to-point topographic projections of retinal ganglion cell axons
• Sperry and colleagues studied regenerating RGC axon growth into the tectum.
• Optic nerves were transected, half of RGCs were ablated, and remaining axon terminations were examined
• Findings showed ventral RGCs project to the medial tectum, dorsal RGCs to the lateral tectum
• Anterior (nasal) RGCs project to the posterior tectum, posterior (temporal) RGCs project to the anterior tectum
• Here RGC axons passed through the empty tectum and homed in on their original targets
• These observations provided conclusive evidence for RGC axons being predetermined to connect to specific brain targets following regeneration
• The chemoaffinity hypothesis (1963): brain cells and fibers have individual identification tags, presumably cytochemical, distinguishing them at the single-neuron level
• Growing fibers are selective and link with specific neurons based on chemical affinity

Challenges and Refinements of Chemoaffinity

• Connection mechanisms during normal development may differ from regenerative mechanisms.
• Regeneration has stricter requirements for molecular recognition because it occurs outside the typical developmental milieu
• It is impossible for Our genome to encode tags or recognition proteins for all neurons and connections
• Protein gradients could provide positional information in retinal and tectal fields.
• Different levels of the same protein could enforce targeting of multiple neurons

Posterior Tectum and Temporal Retinal Axon Repulsion

• The chemoaffinity hypothesis inspired scientists to find cytochemical tags that guide axons toward targets.
• The retinotectal mapping served as a model system
• This predicted molecular differences between tectum cells for retinal axons to select targets, and molecular differences between retinal axons for them to react differently to tectum cues
• Biochemical studies showed differences between chick posterior and anterior tectum membrane proteins
• In vitro assay: membranes from anterior or posterior tectum were laid down in alternating stripes
• Nasal retinal axons grew without selectivity, but temporal retinal axons grew preferentially on anterior tectum
• Temporal retinal axons' preference for the anterior tectum could be due to attraction or repulsion
• Protein activities were abolished to identify if proteins were attractants and repellents by selectively heat-inactivating anterior or posterior tectal membrane proteins
• Temporal RGC axons lost selectivity when posterior tectal membrane proteins were heat inactivated
• Temporal axons respond to activity from posterior tectal membranes, normally targeting the anterior tectum because they are repelled by protein components there

Ephrins, Eph Receptors, and Retinotectal Mapping

• The specific protein responsible for repulsion in posterior tectal membrane was identified as ephrin-A5, part of the ephrin family
• Ephrins are extracellular proteins attached to the plasma membrane
• Ephrins bind to Eph receptors, transmembrane proteins
• Purified ephrin-A5 repelled temporal retinal axons in the stripe assay, mimicking posterior tectal membrane
• Ephrin-A5 mRNA is expressed in a posterior > anterior gradient in the tectum
• EphA3, a receptor for ephrin-A5, is expressed in a temporal > nasal gradient in the retina
• RGC axons from the most temporal retina express the most EphA3, making them sensitive to posterior > anterior ephrin-A5 gradient

Ephrins and Axon Targeting Validation

• Function of ephrins in retinal axon targeting in vivo was confirmed by viral misexpression of ephrins in chicks and ephrin knockout experiments in mice
• In mice, ephrin-A5 and a related ligand ephrin-A2 are expressed in posterior > anterior gradients in the superior colliculus
• Temporal axons did not exhibit selective targeting to the posterior superior colliculus in EphrinA5/A2 double-knockout mice; axons were scattered along the anterior-posterior axis
• Nasal axons were also affected and mistargeted axons formed clusters
• This indicates that ephrin-A2 and ephrin-A5 are essential for RGC axon targeting along the anterior-posterior axis of the superior colliculus
• Targeting along the medial-lateral axis was intact, indicating that ephrin-A/EphA signaling is important for only one axis

Single Gradients and Axonal Guidance

• Nasal axons also express EphA, albeit at low levels; it is unclear as to why they aren't repelled by ephrin-A, instead targeting the posterior tectum highly enriched in the repellent activity
• One possibility: Competition between RGC axons fills the target space
• Nasal axons are "pushed" more posteriorly given they have a lower repellent receptor expression that those occupying the anterior tectum
• Constant EphA overexpression in a subset of RGCs in mice provided evidence to support this model
• 40% of RGCs express the Islet 2 (Isl2) transcription factor
• Additional EphA receptor expression is driven by IsI2 promoter, creating two gradients with different EphA expression levels
• ki/ki are the homozygous EphA3 knock-in mice that displayed a duplicated map in the posterior colliculus: Dye injections at a single small spot in the retina labeled two separate target areas along the anterior-posterior axis due to their corresponding genotypes

EphA/Ephrin-A interactions, Mechanisms, and Signaling

• RGC axon target positions in the superior colliculus are determined by relative rather than absolute levels of EphA
• E.g., an IsI2-negative temporal RGC (number 7) axon targets the anterior superior colliculus due to its high EphA level
• Ki/ki mice axon targeted to the middle of the superior colliculus, even with the expression level unchanged, as its EphA level was surpassed by IsI2-positive RGCs overexpressing EphA3
• Besides axon-axon competition, bidirectional ephrin-A/EphA signaling contributes to RGC axon targeting:
  • RGC axons express a nasal > temporal ephrin-A countergradient
  • Tectum expresses an anterior > posterior EphA countergradient
• Besides ephrin-A → EphA signaling: EphA in the superior colliculus acts as a repulsive ligand for retinal axons expressing ephrin-A, called reverse signaling
• Reverse signaling explains why nasal axons prefer the posterior superior colliculus since they express the highest ephrin-A levels and choose target regions with the lowest EphA levels
• Loss of all ephrin-As: abolishes receptors in nasal RGC to detect the EphA gradient in the superior colliculus, removing their targeting selectivity
• Additionally, Ephrin-A from RGC axons can repel EphA-expressed as retinal and contributes, with axon-axon repulsion contributing, to retinocollicular mapping

Graded Patterns and Axon Guidance

• FGF mRNA expression is graded En2 expression is due to be in turn regulated by members of the fibroblast growth factor (FGF) family of secreted proteins from the the midbrain-hindbrain junction and concides with the posterior edge of tectum
• secreted FGF proteins form a posterior > anterior gradient in the tectum
• FGFs can upregulate En2 and ephrin-A expression and downregulate EphA expression in tectum explant cultures
• Developmental patterning molecules such as FGFs help set up graded expression of axon guidance molecules via transcriptional regulation
• These and subsequent studies suggested similar mechanisms could operate in the wiring of the nervous system in general due to the expression of ephrins and Ephs as complimentary ligand-receptor pairs

Molecular Biology of Axon Guidance

• Axons are guided away from repellents (repulsive molecular cues) and toward attractants (attractive molecular cues).
  • long-range cues - secreted proteins that act at a distance from their cells or origin
  • short-range cues - usually cell-surface-bound proteins that require contact between the cells that produce them and the axons they guide to exert their effects:can also be secreted proteins bound to the extracellular matrix.
• Ligands activate receptors expressed on the surface of axonal growth cones. Ephrin-As are contact-mediated repulsive ligands, since these proteins are bound to membranes by a GPI anchor and repel RGC axons that express the EphA receptors. Some families of axon guidance cues contain both secreted and membrane-bound proteins; this is the case for the semaphorins. Some guidance cues can act as an attractant in one context and a repellant in a different context. An important class of contact-mediated attractive molecules is the cell adhesion molecules, named for their ability to cause cells to adhere to other cells or to the extracellular matrix. These include immunoglobulin superfamily cell adhesion molecules (Ig CAMs) and Ca2+-dependent cell adhesion proteins (cadherins). Some Ig CAMs and cadherins are homophilic cell adhesion proteins, which facilitate adhesion between cells via direct binding of the same proteins from apposing cells, whereas others are heterophilic, binding to different members of these protein families or proteins from other families. Cell adhesion proteins can promote axon guidance by providing a permissive environment for axons to extend or by stabilizing transient contact between axons and their targets. Remarkably, many of these axon guidance cues are evolutionarily conserved across worms, flies, chickens, and mammals. Netrin/Unc6 In the vertebrate spinal cord, commissural neurons of the dorsal spinal cord send their axons ventrally toward the floor plate, a structure at the ventral midline of the spinal cord. Floor plate explants promoted axon outgrowth from commissural neurons in dorsal spinal cord explants and caused these axons to turn toward floor plate explants, leading to: the floor plate must contain (a) chemoattractant(s) for commissural axons that are also outgrowth promoting. In the mouse, only one netrin is present in the spinal cord; it is expressed in the floor plate and the ventricular zone (an. In netrin knockout mice, commissural axons exhibit severe defects in their guidance toward the floor plate.

To cross, or not to cross: that is the question

• In all vertebrates, crossing RGC axons form a optic chiasm which is likely an ancestral state (fish, tadpoles, and birds)
  • The eyes are typically lateral and tow eyes sample mutually exclusive regions of visual space, left - right brain and vise-versa
• In animals which eyes face forward to view partially overlapping regions of visual space their information is sent to both sides of the brain, and converges in the primary visual cortex for binocular depth perception
• Different animal species results from different %'s of RG axons crossing the midline at the optic chiasm (including eye positions
• In humans: nasal retina (60% of total RGCS) contralateral projection temporal retina (~40% of total RGCS) ipsilateral projection Projections of temporal RGCs from the ipsilateral eye temporal RGCs and nasal RGCs from the contralateral eye that survey the converge in the same brain hemisphere. Carnivores: 15-30% and mice is 3-5:

Growth Cone Behavior

• Growth cones from both ipsilateral and contralateral retinal axons slow down considerably at the chiasm.
• Thin projections (filopodia) surrounded by sheet-like webbing (lamellipodia)-undergo cycles of extension and retraction driven by the polymerization and depolymerization of actin and by the motor protein myosin
• Contralateral axons then quickly move forward, whereas ipsilateral axons continue to extend and retract until ipsilaterally directed filopodia are consolidated, giving rise to new growth cones.
• The molecular basis for the differing behaviors of ipsilaterally and contralaterally projecting axons has been identified in mice

Cell Biology and Signaling at the Growth Cone

• Growth cone - growing tip of the axon contains two prominent structures: thin filopodia and the veil looking sheet (lamellipodia)
• F-Actin and microtubules -F-actin fibers- branched networks supporting lamellipodia and bundles enriched in filopodia -Like F-actins, the the microtubules are highly dynamic and are supported by structural forces, and also a stabilizing force
• Cell turning: - attractive cues stabalize and negative cues destabilize.
• Rho Family of small GTPases - Axon guidance that transmit signals from receptors vai Rho GTPases are transferred to downstream effectors that modulate the cytoskeleton activity. RHO GEFs convert RHo to the active(GTP-bound) and GAPS facilitate the intrinsic GTPase activity of Rho to the -active GDP-bound

How do experience and neuronal activity contribute to wiring?

• Hubel + Wiesel Series -Visual cortex neurons experiments found differing guidance via vision in which they discovered monocular deprivation

Monocular Deprivation and Visual Cortex Development

• Cortical neurons manifest binocular responses by combining inputs from neurons of the lateral geniculate nucleus (LGN).
• Signals from the left and right eyes converge. The cortex creates binocular vision (4-37B).
• Hubel and Wiesel mapped in primary visual cortex: stimuli from the ipsilateral eye + from contralateral eye or from same side -This experiment: tested if visual input had anything to do with the receptive field of the cortical neuron .

Visual responses under deprivation

• The cat's eyelid is sutured at birth, therefore depriving the eye of sight!
• Little change is found when comparing the retinal neurons of both eyes and two LGNS
• A profound difference was found in V1 neurons! the majority of cortical neurons were driven by input from the normal eye; very few were from the deprived eye
• Behavioral: Animals lost visual functions when comparing visual
• Hubel + Wiesel Time Course - tested when significant effect tookplace? * Beginning Fourth Weak after birth - 12W gradually
• 1* Monocular deprivation after the critical period is nothing like the large effect - during the critical period.
• 2* Blocking one eye just for short days (4TH -/ 5TH) has big effects on vision as with the cortical the responses V1 then has injected RAA - transneuronal tracer was used was use that then the V1 could visualize that the ocular dominance then the columns layer will do the visual.
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Two Important Tests performed:

• 1* Blocking all vision = the animals had visual, with that the responses, there weren't, to be affected with the resulting , from deprivation.
• 2* Eye is not the atrophy disuse

V1 vs Molecular, in Deprevation

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Three important types-experiments in the process

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