Developmental Neuroscience · Unit 9: Experience refines the wiring

Activity Before Experience: Spontaneous Retinal Waves

Before the eyes can see, the retina fires on its own, in correlated bursts that sweep across it as waves. This lesson asks what drives those waves at each stage of development, why a brain would use activity at all to finish its wiring, and what blocking or shifting the waves does to the maps they feed. Afterwards you can read a blockade experiment and say what it can and cannot show about competition between the two eyes.

Watch first Inputs That Fire in Step Are Kept · 2:42

In the retina of a newborn ferret, whose eyes have not yet opened, ganglion cells fire bursts lasting a few seconds that sweep across the tissue at about 100 micrometres per second, separated by a minute or two of silence.

Nothing is being seen. Ganglion cells are the retina’s output neurons; their axons make up the optic nerve. Here they fire on their own, in groups, and the group moves. Why would a retina that cannot yet sense an image bother to send this pattern to the brain? The answer is that the brain is still wiring itself, and this pattern carries information the wiring can use.

Spontaneous activity is activity that arises without sensory input or motor output. Developing neurons in many parts of the nervous system produce it, often as bursts lasting tens to hundreds of milliseconds, and the developing retina produces it as waves. The waves arrive at a time when the brain’s first draft of its connections is rough.

That draft is laid out by molecules. In the lesson on topographic maps you saw gradients of Eph receptors and ephrins steer retinal axons to roughly the right place, so that neighbouring points in the retina project to neighbouring points in the target. Rough is the word. Early in development each axon branches over a broad area, and in animals with two forward-facing eyes the axons from the two eyes overlap in the lateral geniculate nucleus (LGN), the relay in the thalamus that passes visual signals to the cortex. In the adult they end in separate, eye-specific layers. Getting from overlap to layers, and from broad branches to focal ones, is called refinement.

In 1991 Markus Meister, Rachel Wong, Denis Baylor and Carla Shatz laid isolated retinas from newborn ferrets and fetal cats on an array of electrodes and recorded up to 100 ganglion cells at once. The cells fired in nearly synchronous bursts, and each burst was a wave several hundred micrometres wide crossing the retina. Two features matter for wiring. Cells near each other fire close together in time, and cells far apart do not. Later, in 2012, James Ackman, Timothy Burbridge and Michael Crair used calcium imaging in live neonatal mice and saw waves of retinal activity propagate through the visual system before the eyes opened, so the pattern is not an artefact of a dish.

The three stages of retinal waves

The waves are not driven by one mechanism throughout development. In the mouse, where the timing is best mapped, the dominant driver changes in three stages. The ages below are approximate, and ferret, cat and primate retinas follow the same order on different timetables.

Stage I, before birth (from roughly embryonic day 16). Waves depend on gap junctions, channels that connect the cytoplasm of neighbouring cells directly, and also on nicotinic receptors; embryonic waves are less well studied than later ones.
Stage II, from around birth to about postnatal day 10. Starburst amacrine cells, interneurons of the inner retina, release acetylcholine onto nicotinic receptors on one another and on ganglion cells. In 1996 Marla Feller and colleagues showed in the developing mammalian retina that waves require this synaptic activation of nicotinic receptors, and that the only cholinergic cells present were these amacrine cells. Many of the receptors on ganglion cells contain the beta2 subunit.
Stage III, from about day 10 until the eyes open (around day 12 to 14 in mice). Bipolar cells, which will later carry the photoreceptor signal, release glutamate and drive the waves.

The stages overlap, and some experiments find the boundaries blur. What stays constant is the output: correlated bursts among neighbouring ganglion cells, with no fixed timing between the two eyes.

The idea: activity is a signal every cell carries with it

Start with the plain question: “Why is activity needed?” Molecular gradients can say roughly where an axon belongs, but two ganglion cells 50 micrometres apart carry almost the same molecular labels. What distinguishes them is when they fire. Activity is a signal available at the cell of origin, generated by the cell’s own position in the retina, and it can give a precision that labels alone do not.

In 1949 the psychologist Donald Hebb proposed a rule for how connections might change. In paraphrase: when an axon of cell A is near enough to excite cell B and repeatedly takes part in firing it, some growth or metabolic change makes A more effective at firing B. The summary “Cells that fire together, wire together” is associated with Carla Shatz; it is a paraphrase of Hebb, not his words.

Apply the rule to a neuron in the LGN. Its inputs from neighbouring cells of one eye fire in the same wave, so together they drive it and are strengthened. An input from the other eye fires on its own schedule, rarely helps drive the cell, and is weakened. Repeat this for every neuron and the two eyes sort into separate territories, while neighbours within each eye end up next to each other. The waves therefore carry two kinds of information: which eye, and roughly where in that eye.

A target cell needs a detector for “together”. A good candidate is the NMDA receptor, a glutamate-gated channel that opens only when glutamate is bound and the membrane is already depolarized, so it reports coincident input (see the lesson on ion channels as receptors). It admits calcium, which can set off lasting changes in the synapse (see the lesson on calcium signaling).

Testing the idea: block, tilt and add an eye

Block everything. In 1988 Shatz and Michael Stryker implanted osmotic pumps in fetal cats and infused tetrodotoxin (TTX), which blocks voltage-gated sodium channels and so stops action potentials, into the brain for two weeks during the period when segregation normally happens. Tracers injected into the eyes showed that the two projections had not separated. A student once asked: “Does TTX cut off only one eye or both?” Both, and more: infused into the brain, the drug would be expected to reach axons from both eyes and the LGN cells themselves. So the experiment shows that spikes are needed, but cannot tell you whether the eyes compete by their activity, or whether the spikes that matter are retinal.

Design problem

You can raise or lower wave activity in one eye only, or block waves in both eyes, in young ferrets. Predict the eye-specific territories in the LGN in each case. What would each result tell you about how the inputs compete?

One way to do it

Start with one eye. Inject a drug that disrupts cholinergic waves, such as the nicotinic agonist epibatidine, into the left eye and leave the right one alone. If inputs compete by activity, the active right eye should win. That is what Anna Penn, Patricio Riquelme, Marla Feller and Carla Shatz reported in ferrets in 1998: the projection from the active retina expanded into territory that normally belongs to the other eye, and the treated eye’s projection shrank.

Now raise activity instead. Daniel Stellwagen and Shatz made waves more frequent with drugs that raise cAMP. When one eye was made more active, its layer was larger even though the other eye was normal. When both eyes were made more active, the layers were normally sized. Territory follows the difference between the eyes, not the total.

Blocking both eyes removes the difference and the correlation together, so segregation should fail. Global silencing in the fetal cat, and the disrupted waves of beta2 knockout mice, point that way. The one-eye experiments are the ones that show competition, because only they leave one input active to compete with.

Change the pattern, keep the spikes. Todd McLaughlin, Chandra Torborg, Marla Feller and Dennis O’Leary used mice lacking the beta2 nicotinic subunit. In their recordings these mice had no waves in the first postnatal week, yet their ganglion cells fired at high rates without correlation. Their title states the result: “Retinotopic map refinement requires spontaneous retinal waves during a brief critical period of development”. By day 8, axons of neighbouring ganglion cells in normal mice had converged on small termination zones in the superior colliculus, the midbrain visual target. In the mutants the zones stayed broad. Glutamatergic waves and then vision arrived later, and the zones still failed to remodel. Two hedges belong here. Later laboratories recorded correlated but altered waves in beta2 knockout retinas under some conditions, so the mutants lack normal waves rather than all waves. And a knockout removes the subunit everywhere, not only in the retina. Later work that restored beta2 in retinal ganglion cells alone has kept the retina at the centre of the explanation.

Add an eye. A frog’s optic tectum, its main visual target, normally receives one eye only, so it has no reason to make eye-specific stripes. In 1978 Martha Constantine-Paton and Martha Law implanted an extra eye primordium into frog embryos, and the normal and extra eyes sent axons into a single tectal lobe. The two projections formed alternating bands. Competition between inputs created stripes where none exist in nature. In 1987 Hollis Cline, Elizabeth Debski and Constantine-Paton laid slow-release polymer containing APV, an NMDA receptor antagonist, on the tecta of three-eyed tadpoles. The active form of APV desegregated the stripes, while the retinal axons still responded; the inactive form left them normal; NMDA itself sharpened their borders; and after APV was removed the stripes returned within two weeks. Keeping the stripes needed NMDA receptors on the receiving side, the detector the Hebbian account calls for.

How we measure it

Multielectrode array recording

A piece of retina is laid on a grid of electrodes, each picking up the action potentials of nearby ganglion cells. Recording many cells at once shows whether activity is correlated and whether it travels across the tissue, which pairs of electrodes cannot show.

Chronic drug delivery in the developing animal

A drug is supplied for days to weeks from an osmotic pump, repeated injections into one eye, or a slow-release polymer laid on the brain. Where the drug acts, and whether it reaches one eye or both, decides which question the experiment can answer.

Anterograde tracing of each eye's projection

A tracer injected into one eye is carried down the ganglion cell axons and labels their endings in the brain. Labelling the two eyes differently shows whether their territories in the target overlap or have separated.

Knockout mice

A knockout mouse lacks one gene in every cell. Removing the beta2 nicotinic subunit changes the retina's spontaneous activity, so the map can be compared with normal, though the gene is also lost outside the retina.