How do we know that functional synapses are eliminated during maturation? A: Measurement of PSP amplitude after presynaptic stimulation in developing vs. mature synapses

The postsynaptic potential generated by multiple afferents is additive Fig 9.2 p251: Increasing electrical stimulus intensity in a presynaptic field recruits increasing numbers of afferent cells, leading to quantal increases in postsynaptic potential, as measured in mV amplitude.

Fewer inputs generate less PSP in mature synapses Field stimulation of presynaptic neuronal afferents; intracellular recording of evoked PSP amplitude in response to excitatory input. In mature synapse, fewer afferents are present per postsynaptic cell.

Blocking synaptic activity with TTX prevents synapse elimination at the rat neuromuscular junction Fig 9.13 p260: TTX= tetrodotoxin, inhibits firing of action potentials via binding voltage-gated Na channels (pufferfish), potent neurotoxin Normally in rat, polyinnervation declines betw. P10-P15. If cuff is placed around motor nerve root from P9-P19, action potentials are eliminated & multiple nerves innervate muscle.

Rat soleus muscle (leg)/ sciatic nerve Converse experiment: excess stimulation  precocious synapse elimination Fig 9.14 p260: Stimulation of action potentials in the sciatic nerve via electrical activity in muscle (postsynaptic terminals) leads to presynaptic pruning of synapses Rat soleus muscle (leg)/ sciatic nerve

Greater distance between nerve terminals: less elimination Fig 9.15 p 261: If synapses are close together on a single muscle fiber, one will be eliminated; the further they are apart, the less likely one will be lost **Antagonism between synapses mediated by postsynaptic cell** > 2-3 mm: both synapses may survive 1-2 mm: one synapse will be removed

With dual innervation, strong synaptic activity of the 1st depresses activation of 2nd synapse 2 motor nerve roots: Fig 9.26 p 271: At the lumbrical foot muscle, there is dual innervation of 2 motor neuron roots, LP & SN. Strong stimulation of LP  weakened ability of SN to activate foot muscle contraction. In muscle-neuron cocultures, stimulating neuron 2 (blue) leads to reduced synaptic current evoked from neuron 1, which persists >40 min  less current

Does synaptic depression lead to synapse elimination?

Yes: the AChR blockade experiment Pharmacological blockade Normal development See Fig 9.27 p 272: as NMJs mature, alpha MNs refine their connections so that MN: muscle fiber = 1:1 ratio. If drug (eg. Bungarotoxin from krait snake) is applied to NMJ synapses, AChRs are lost & then axon withdraws from muscle fiber.

Loss of AChRs precedes synapse elimination (live imaging) Fig 9.28 p 272: Stimulating 1 MN causes reduced AChRs Red/yellow: Rhodamine-bungarotoxin

Four mechanisms of synapse elimination during development Find fig– AChRs, synapse elimination

Protein kinases A & C (PKA/PKC) mediate activity-dependent synaptic loss Fig 9.27 p 272: PKA increases synaptic strength locally, & PKC is probably activated by Ca2+; PKC phosphorylates AChRs on inactive terminal & initiates their removal from the synapse

Postsynaptic Ca2+ depresses synaptic activity, unless presynaptic cell is stimulated Fig 9.29 p 273: UV uncaging of calcium in muscle causes evoked synaptic currents from the presynaptic neuron to be reduced (red lines, top) Black = baseline current in muscle. If neuron is stimulated before Ca2+ release, synaptic strength is not diminished by postsynaptic calcium caged Ca2+ in muscle: UV-stimulated

During development, synapses can be rearranged on target cells

Mammalian retinogeniculocortical (visual) pathway for binocular vision RGCs from each eye converge (separately) at the LGN of the thalamus, then LGN neurons project to the visual cortex (layer IV) in discrete locations (which will resolve to ocular dominance columns), which then synapse onto the same neuron in layer II/III.

In the visual cortex layer IV, LGN terminals are initially mixed, then resolve into columns From Neuroscience textbook by Purves et al??

In the LGN, layers form to segregate RGC inputs, then ocular dominance columns are generated in cortex

Tracing the cat visual pathway during the first few months Fig 9.4 p 253: Radiolabeled tracer (tritiated Pro) is delivered to the lateral geniculate nucleus (LGN) of the thalamus, then transsynaptically delivered to layer IV of the visual cortex in the brain. The innervation begins before the cat can see, but is slowly refined after the eyes are opened. 1st segregation occurs at P14

Excessive innervation in the cortex is removed between P22-P92 Fig 9.4 p 253: In B, surface imaging shows activity patterns on surface of Cx; in cross-section, cortex shows eye-specific stripes, demonstrating gradual refinement of the axonal arborization within Layer IV of the cortex.

Covering one eye from 2 wks – 22 mo leads to expanded eye-specific cortical domains in layer IV Control Radiolabeled tracer injected into non-deprived eye shows expanded ocular dominance columns in cortical layer IV (visual cx). Monocular- deprived Cortical domains= Ocular dominance columns

Single-neuron recordings in vivo: the majority of neurons are binocular (respond to input from both eyes) Extracellular electrode Fig 9.7 p 256: Wiesel and Hubel examined ocular deprivation & visual coding Layer IV neurons: monocular Layers I-III, V-VI: neurons respond to both eyes

*increased loss (closed) + failure to eliminate (open) synapses If one eye is closed throughout life, it will lose its synaptic connectivity Fig.9-8 p257: Closed eye from early age causes loss of stabilized connections to the brain, & other eye “takes over” the locations in the cortex in place of the closed eye neurons *increased loss (closed) + failure to eliminate (open) synapses

# responsive neurons is much lower than non-deprived controls No visual input during development does not eradicate ocular dominance columns or response to visual stimuli Fig 9.9 p257: to the surprise of Wiesel & Hubel, this total deprivation did not remove ocular dominance columns, or ability to respond to visual input. However, a large # neurons are non-responsive to stimulation. # responsive neurons is much lower than non-deprived controls *Competition hypothesis: Difference in activity level determines strength of projection

Deflection of one eye strongly suppresses binocular innervation Fig 9.10 p258: In contrast to controls, which have mostly binocular vision and innervation, cats with a surgically-deflected eye have neurons which only respond to one eye or the other, not both *timing of activation must be critical to maintain connectivity

Strabismus also changes intrinsic local cortical projections (see Fig 9.11 p258): in non-strabismic animals, dye labeled neurons from both columns in layer IV; in strabismic animals, dye labeled only neurons in the same vertical column in all cortical layers ** activity influences not only sensory projections to brain, but also intracortical projections Retrograde dye labeling: in strabismic cortex, local projections to other layers are confined to monocular columns *activity-dep refinement within the cortex

Uncorrelated inputs segregate Synchronous stimulation of optic nerves blurs segregation Asynchronous stimulation of optic nerves sharpens segregation

In development of the visual pathway, synaptic refinement occurs first in the LGN, then in the cortex (IV)

Before light input, RGCs begin firing in waves across retina Fig 9.21 p267: recordings of RGC spontaneous, synchronous bursts of activity across early postnatal retina

Spontaneous waves of RGC activity are lost by P15 Page 267: Arrays showing where on the retina the neurons are activated at the given times; wave patterns are lost by P15.

Only blockade of neuronal activity ablates formation of columns Fig 9.23 p269: Visual light input into eyes is not critical for formation of the ocular dominance columns, only neuronal firing. Injection of TTX will prevent column formation *TTX: voltage-gated Na+ channel blocker

Donald Hebb, psychologist (1904-1985) - The Hebbian theory: synaptic strength is derived from repeated and persistent stimulation of a postsynaptic cell by a presynaptic neuron Carla Shatz, neurobiologist “Cells that fire together, wire together”: The presynaptic cell must fire first, and in turn cause the postsynaptic cell to fire synchronously with other activating inputs Shatz studies development of the visual system and synaptic plasticity;

Synapses are strengthened by synchronous firing on shared target