Lecture 14

Question 1

What is meant by topographic mapping

Answer 1

When neighbouring neurons send axons to neighbouring sites in their target to maintain the topology/order

Question 2

Describe how the visual system topology is maintain in the retinotectal system

Answer 2

When an imaged is produced on the back of the retina this appears flipped in both axes relative to its true position in world. However the axonal projections cross over on the way to the tectum so that the image is flipped back to its correct topology. In addition topology is also conserved via nasal axons projecting to the posterior tectum/superior colliculi and temporal projecting to the anterior tectum/superior colliculi to conserve spatial representation of an image

Question 3

Describe the role of the gradients in the tectum in the topographic mapping of retinal axons

Answer 3

Cells from the posterior tectum make ephrin A2/A5 (non-permissive factors) that repel temporal retinal axons. Hence ephrins A2/A5 are expressed high posteriorly and low anteriorly forming a gradient. There is also a differential sensitivity of the nasal and temporal axons to ephrins expressed in this gradient with temporal axons being sensitive to the ephrins whereas nasal axons are not. Therefore nasal axons can extend through to posterior regions of the tectum where ephrins are high whereas temporal axons are unable to extend through this region of the tectum. In addition the expression of the ephrin receptors by the retinal ganglion axons is also graded with eph A3 high temporally to low nasally

Question 4

What is seen in the knockout of ephrin A2/A5 in the retinotectal system and what is the significance of this

Answer 4

Knockout of both ephrin A2 and A5 leads to temporal axons projecting posteriorly and the disruption of the topographic map. Specifically the double knockout shows disruptions in mapping whereby axons are actually projecting everywhere. However you’d expect that all the axons would actually project through the entire tectum and synapse posteriorly. This would be due to the loss of inhibitory ephrins that would normally prevent this.

Question 5

Explain the unusual results seen in the mammalian ephrin A2/A5 double knockout and why these occur

Answer 5

In mammals all axons actually initially grow throughout the entire length tectum and these axons then branch. It is in fact the branching of these neurons that is sensitive to the ephrin gradient. The effect of the ephrins is to bias the pre-existing competition between the retinal axons for synapses that is present in the tectum. This competition involves electrical activity with weaker synapses and axons getting eliminated. Ephrins are being used to make the temporal axon synapses weaker so that the nasal axons win the competition hence posterior synapses of temporal axons get pruned back. Thus knockout of ephrins leads to loss of disadvantage by the temporal axons which can now compete with the nasal axons throughout the tectum. This explains why visualisation of these axons with lipophilic dyes reveals axon synapses randomly distributed throughout the tectum

Question 6

Give examples of experimental evidence the implicate the role of electrical stimulation in the modulation of guidance cue responses

Answer 6

Electrical stimulation can both enhance the attraction to cues in the case of netrin or reverse the effects of repulsive cues such as MAG. For example 5μg ml-1 netrin from a pipette leads to a significant attraction of axons at 16-20 hours. However 1μg ml-1 of netrin from a pipette results in very little/no axon attraction. Surprisingly 1μg ml-1 netrin combined with electrical stimulation of the neuron results in much greater attraction of the axons over the 16-20 hour time period. Hence electrical stimulation can enhance the attraction of attractive cues. Conversely electrical stimulation can reverse the repulsion by MAG in vitro. In the presence of the secreted rMAG there is a repulsion of axons. However if rMAG is paired with electrical stimulation there is a significant attraction of the axons.

Question 7

What are the mediators of electrical stimulation in modulating the response to guidance cues

Answer 7

Electrical stimulation leads to an increase in [cAMP] that is Ca2+-dependent

Question 8

How can the effects of electrical stimulation on the modulation of neuronal responses to guidance cues be blocked

Answer 8

Can block the effects of electrical stimulation with PKA and AC inhibitors

Question 9

Refinement of connections in the retinotectal system depends on activity and competition between axons T or F

Answer 9

T

Question 10

How is it proposed that axons that fire together wire together

Answer 10

This is thought to be in part mediated through the localised release of neurotrophic factors like BDNF which acts to stabilise synapses. As such weaker synapses release less BDNF and are subsequently eliminated

Question 11

Tectal mapping occurs before birth in mammals so where does the electrical activity come from if there is no light

Answer 11

The retina becomes spontaneously active as axons reach the tectum. This is indicated by the injection of calcium indicators that fluoresce upon Ca2+ binding. Ca2+ waves in the neurons correspond with the firing of these neurons with two neurons that are firing together creating Ca2+ waves at the same time. This thus strengthens their synapses with the correct target in the tectum by releasing BDNF etc.

Question 12

How can refinement of projections in the tectum be blocked experimentally

Answer 12

Through the use of Na+ channel blockers such as tetrodotoxin which inhibits action potential generation

Question 13

Give an example of a genetic approach that can be used to disrupt neural activity and topographic map refinement in the tectum

Answer 13

If you were to knockout either the whole receptor or a subunit of the nAChR. For example mice lacking the β2 subunit of nAChR the main RGC receptor in early postnatal life have uncorrelated RGC activity (retinal waves are disrupted) and their topographic maps do not refine

Question 14

Give another example of where ephrin gradients are used to maintain topographic mapping

Answer 14

In the somatosensory cortex gradients of ephrins high medially and low laterally. Incoming thalamic axons also have a graded response to these ephrins. This ultimately results in the sensory information from various organs being spatially represented in the cortex.

Question 15

Describe how ephrin gradients are set up in early development

Answer 15

This is due to an interaction between the graded expression of both EMX2 and FGF8. The EMX2 gene is expressed in a high caudal to low rostral gradient during cortical development whereas FGF8 is expressed at high levels rostrally. The interactions between these gradients determines the expression of ephrins

Question 16

How is the mapping of the olfactory system different to spatial senses

Answer 16

There are over 1000 different olfactory receptors (GPCRs) but each olfactory neuron expresses only one of these receptors. The neurons expressing each receptor are scattered throughout the olfactory epithelium. However the axons from the neurons expressing the same olfactory receptor will converge on the same glomeruli. Thus receptor expression is dispersed in the nasal epithelium but axons become organised in the olfactory bulb. Unusually beyond the olfactory bulb mitral cells project to the piriform cortex but this too shows no spatial orientation. Hence it seems olfactory information if first very diffused before becoming organised into the glomeruli but then becomes diffuse again as this information is relayed to the piriform cortex.

Question 17

Describe the experiments that lead to the determination of mapping from the olfactory epithelium to the olfactory bulb

Answer 17

Knock in reporter animals were generated expressing a gene which contained an olfactory neuron specific gene such as P2 or M12 followed by an IRES upstream of a tau-lacZ fusion protein. This leads to the expression of the endogenous olfactory gene and a tau-β-galactosidase fusion protein specifically in the olfactory neurons. Tau is a MAP which would act to direction the β-galactosidase specifically to microtubules located in the axonal shaft hence allowing for the easy staining of the axon specifically. This showed that axons expressing the same olfactory receptor although dispersed throughout the olfactory epithelium actually projected to the same glomeruli.

Question 18

Glomeruli responding to the same/related odorants are diffuse throughout the nasal epithelium T or F

Answer 18

F – they often cluster together

Question 19

Describe how the effect of receptor expression on olfactory neuron guidance was determined

Answer 19

Receptor swap experiments demonstrate that where axons go is determined by which receptor is expressed. Swapping the protein coding and promoter regions of olfactory receptors resulted in a change in navigation of those axons specifically dependant on the receptor protein encoded. Hence the olfactory receptor expressed dictates where the olfactory axon will synapse.

Question 20

Early guidance is activity-independent T or F

Answer 20

T

Question 21

Alike to the visual system mapping in the olfactory system is occurring before exposure to the stimulus (i.e. the ligand for these receptors/odorant) explain how this can be the case

Answer 21

In the absence of ligand (odour) each olfactory receptor has a characteristic basal activity. Thus the levels of AC stimulation and subsequent basal cAMP levels are different. This determines via CREB the level of transcription of similar guidance cues (e.g. Robo/Slit Neuropilin/Sema). Therefore each neuron has a unique olfactory receptor and a unique repertoire of guidance molecules and receptors associated with it. This results in receptor/cue protein levels characteristically associated with expression of a particular OR which in turn determines mapping in olfactory bulb (OB). Neurons expressing the same olfactory receptor have similar basal cAMP signalling which determines where the axons go

Question 22

What is the effect of the differential expression of guidance cues (and their receptors) by specific groups of neurons that express the same olfactory receptor

Answer 22

These early incoming olfactory axons set up a gradient of guidance cues which guides a later set of olfactory axons

Question 23

Once olfactory axons are in roughly the correct place following guidance by the specific olfactory receptor they express how is a more discrete map created in which the exact synapse is determined

Answer 23

Once axons entering OB have been pre-sorted due to type I cue/receptor interactions cue expression switches. For example Robo/slits are expressed early whereas Neuropilin/Semas are expressed later. This new cue/receptor expression by the early entering axons then guides the later entering axons. Discrete sorting into glomeruli is also then activity-dependant whereby the activity drives higher cAMP levels which turns on the expression of these type II cues. These cues include homophilic adhesion molecules (Kirrels and contactins) as well as mutual repellents (ephs and ephrins)

Question 24

Outline olfactory neuron mapping beyond the olfactory bulb and what is significant about this regarding its mapping

Answer 24

Mitral cells project to the piriform cortex however this is now with no obvious spatial orientation. Individual odorants now activate neurons dispersed throughout piriform cortex with individual neurons in the piriform cortex being able to respond to multiple structurally dissimilar odorants. Hence olfactory mapping has gone from dispersed in the olfactory epithelium to spatially organised in the glomeruli back to dispersed again in the piriform cortex.

Question 25

In mammals the majority of odours only drive behaviour after learning T or F

Answer 25

T - hence the significance of odours is learnt by association

Question 26

Explain how it was investigated whether the piriform cortex is the site of olfactory learning

Answer 26

This was investigated by introducing a channelrhodopsin (ChR2) into subset of piriform cortex neurons. This would allow the experimental firing of piriform cortex neurons independently of mitral cell input. Stimulation of the ChR2 positive subset of neurons with light paired with either an aversive or appetitive (appetite inducing) stimulus in naïve (unconditioned) animals could then be used to condition the animals. After conditioning it could then be tested whether the light stimulus alone could elicit the appropriate behavioural response. This would indicate if the piriform cortex could be used for olfactory learning.

Question 27

Describe two ways used to introduce channelrhodopsins into the piriform cortex

Answer 27

The simplest way was to express the channelrhodopsin under control of the synapsin promoter which would direct the ChR2 expression in all neurons of the piriform cortex. This could be done by injection of the AAV retrovirus containing the DNA encoding the ChR2 under the control of the synapsin promoter. This infection would result in a 50% infection of the total number of cells at injection site of the virus in the piriform cortex. Activation of the neurons could then be measured using c-Fos. c-Fos is and early response gene as a result of ChR2 activation and is expressed early after activation. The second method would be to generate a transgenic animal expressing cre-recombinase under the control of the EMX1 promoter. EMX1 is a gene that is a marker of excitatory neurons hence directing the expression of cre only in excitatory neurons. This is followed by the injection into the piriform cortex of a virus containing genetic information encoding a floxed ChR2 gene that is reversed. In addition the loxP sites flanking the ChR2 gene are opposite in direction. This means that upon injection of the virus cre-recombinase will actually cut and recombine the Chr2 gene in a way in which it is flipped round and is now part of the reading frame and can be transcribed. This will occur only in the excitatory neurons of the piriform cortex as there are the only ones expressing cre recombinase. This results in the infection of 50% of only excitatory neurons

Question 28

Describe the results of the optogenetic experiments used to determine if the piriform cortex could be the site of olfactory learning

Answer 28

Photostimulation (PS) of ChR2-expressing neurons in the piriform cortex was the conditioned stimulus (CS). This was paired with a foot shock or the unconditioned stimulus (US). This shock was only elicited one side of the chamber to condition the animals to move to the other side. After 10 repeats the light alone was found to sufficient to cause the mice to move to the other side of the chamber. It was found that this photostimulation equivalent to the effects of odorant stimulation with the foot shock. In addition the results could also be replicated with appetitive stimuli for example pairing photostimulation with taking water resulted in the same effects. This associative learning was also subsequently found to be plastic. Changes in the unconditioned stimulus over time from aversive to appetitive and back again could change the conditioned response. This strongly suggested that the random connections from olfactory bulb to the piriform cortex are used to associate odours with particular experiences.

Question 29

All responses to odorants are learned T or F

Answer 29

F - a small subset of odours elicit innate responses of which the piriform cortex is not involved in

Question 30

Does the fact that the neurons in the piriform cortex can be used to associate odours with behaviour prove that the piriform cortex is the site of olfactory learning

Answer 30

No all this shows is that these neurons are capable of being utilised for this. You would need to investigate the effects of inhibiting activity in this region on the ability of the animals to elicit olfactory learning.