GluA4 facilitates cerebellar expansion coding and enables associative memory formation
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Evaluation Summary:
This work explores the cellular and behavioural effects of a genetically induced reduction of the expression of a glutamate (excitatory) receptor (GluA4), focusing on the cerebellum , a structure involved in the acquisition of arbitrary, complex motor reflexes. The authors show that synaptic transmission at the input layer to the cerebellar cortex is reduced, despite some compensation by other mechanisms, which are characterised. Locomotion is little affected while acquisition of a "conditioned eyeblink" is abolished. The authors try to link the cellular and behavioural phenomena via modelling of the cerebellar computation, although this is not definitive. The work is of high quality, of interest to cerebellar physicists and neurocomputational modellers in particular.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
AMPA receptors (AMPARs) mediate excitatory neurotransmission in the central nervous system (CNS) and their subunit composition determines synaptic efficacy. Whereas AMPAR subunits GluA1–GluA3 have been linked to particular forms of synaptic plasticity and learning, the functional role of GluA4 remains elusive. Here, we demonstrate a crucial function of GluA4 for synaptic excitation and associative memory formation in the cerebellum. Notably, GluA4-knockout mice had ~80% reduced mossy fiber to granule cell synaptic transmission. The fidelity of granule cell spike output was markedly decreased despite attenuated tonic inhibition and increased NMDA receptor-mediated transmission. Computational network modeling incorporating these changes revealed that deletion of GluA4 impairs granule cell expansion coding, which is important for pattern separation and associative learning. On a behavioral level, while locomotor coordination was generally spared, GluA4-knockout mice failed to form associative memories during delay eyeblink conditioning. These results demonstrate an essential role for GluA4-containing AMPARs in cerebellar information processing and associative learning.
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Reviewer #2 (Public Review):
In this manuscript, the authors characterise a GluA4-knockout mouse with respect to changes of cerebellar cortical circuit properties and behaviours.
They demonstrate a clear reduction in the component of mossy fibre--granule cell synaptic transmission mediated by AMPA receptors, as expected. They also show two parallel changes in granule cells that could be considered partially compensatory: tonic inhibition of granule cells is reduced and the NMDAR-mediated component of the mossy fibre input is upregulated. The overall effect of the mutation is nevertheless to reduce the efficacy of the mossy fibre input; spike emission is therefore reduced in frequency, delayed, and has less precise timing.
Two other key synapses in the mossy fibre pathway are shown to be apparently unaffected in the knockout mouse, …
Reviewer #2 (Public Review):
In this manuscript, the authors characterise a GluA4-knockout mouse with respect to changes of cerebellar cortical circuit properties and behaviours.
They demonstrate a clear reduction in the component of mossy fibre--granule cell synaptic transmission mediated by AMPA receptors, as expected. They also show two parallel changes in granule cells that could be considered partially compensatory: tonic inhibition of granule cells is reduced and the NMDAR-mediated component of the mossy fibre input is upregulated. The overall effect of the mutation is nevertheless to reduce the efficacy of the mossy fibre input; spike emission is therefore reduced in frequency, delayed, and has less precise timing.
Two other key synapses in the mossy fibre pathway are shown to be apparently unaffected in the knockout mouse, namely mossy fibre to Golgi cell transmission and also granule cell to Purkinje cell transmission.
The authors then model representation in the granule cell layer and downstream learning by the Purkinje cell, focusing on a reduction of the effective coding space available in the expansion performed by the granule cell layer and the downstream reduction of learning speed in the Purkinje cell.
In a final, behavioural, section, the authors show that locomotion is little affected but that eyelid conditioning is essentially abolished, with two different conditioned stimuli.
Overall, the experiments, analysis and presentation are of excellent quality.
However, the conceptual framework and broader interpretation of the work is quite ambitious and I believe that it requires more nuanced presentation.
A first and reasonably straightforward issue is the fact that the authors are, as they are well aware, working with a systemic knockout. Logically, therefore, the behavioural effects on eyeblink conditioning could reflect interference with any part of the input-output loop. Within the cerebellar circuit, the authors address this reasonably comprehensively, by confirming that mossy fibre to Golgi cell and granule cell to Purkinje cell transmission are unaffected. Nevertheless, one quickly wonders whether the activity of interneurones, climbing fibres or cerebellar nuclei might somehow be altered. The authors address possible extracellular effects of the knockout by showing that eyeblink conditioning is essentially abolished with two different modalities of conditioned stimulus. Again, it remains logically possible that both inputs or the common output could be altered.
Experimentally verifiying all possible stages of the behavioural input-output loop is not feasible, while the ideal experiment of a granule-cell-specific knockout would amount to redoing the whole project, which is obviously out of scope. Nevertheless, I believe the issue does require slightly more open and detailed discussion; maybe the developmental down-regulation of GluA4 in relevant tissues could be substantiated better with reference, for instance, to expression atlases of the Allen Brain Institute. Ultimately, if the locus of action is not completely certain, that should be reflected in the conclusions.
Finally, I'm a little uncomfortable with the ambitious conclusion that learning and behaviour have been constrained by the reduced coding expansion by the granule cell layer. Although the changes observed are indeed almost certain to reduce coding expansion as defined, I feel that the failure of learning could also be understood in more prosaic terms. In particular, the inputs to the Purkinje cell may simply be too weak, too delayed or too unreliable to be an effective plasticity substrate for rapidly developing a conditioned response before the air puff. To a large extent the lower-level modifications will correlate with the higher-level coding expansion, so the concepts are more or less synonymous. Yet, it feels different to conclude that patterns can't be separated because they produced no granule cell activity (to consider a logical extreme) and to conclude that their separation is too difficult because of output similarity and saturation of learning.
Furthermore, there are ways to view coding expansion that wouldn't necessarily align with the authors' conclusion. Specifically, the combinatorial pattern separation analysed in the original Marr paper would, I believe, increase as the ratio of mossy fibre input strength to granule cell threshold decreases. In other words, for given overlapping mossy fibre inputs, the overlap between granule cells outputs could decrease as the input/threshold ratio decreases.
Addressing these issues experimentally is certainly unfeasible. However, it might be possible to explore correlations/overlaps between input and output patterns in the modelling. The discussion could be made a little less assertive on these issues, and the question of input delay should be addressed.
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Reviewer #1 (Public Review):
This study focuses on the consequences of deleting the GluA4 subunit of AMPA receptors for cerebellar synaptic transmission and cerebellar-dependent behaviors. The manuscript is well organized and the information is clearly presented. The first aim of the study is to investigate the effect of the deletion at the level of synaptic function. This is well achieved by a combination of patch-clamp recordings from cerebellar slices and modeling. It is found that deletion of the GluA4 subunit results in a strong decrease in synaptic currents from mossy fibers (MF) to granule cells (GC) as well as in two «compensatory» changes pertaining to NMDA Rs and tonic inhibition. As a consequence, MF-GC transfer is strongly reduced at high frequencies but less affected al low frequencies. The second part of the work …
Reviewer #1 (Public Review):
This study focuses on the consequences of deleting the GluA4 subunit of AMPA receptors for cerebellar synaptic transmission and cerebellar-dependent behaviors. The manuscript is well organized and the information is clearly presented. The first aim of the study is to investigate the effect of the deletion at the level of synaptic function. This is well achieved by a combination of patch-clamp recordings from cerebellar slices and modeling. It is found that deletion of the GluA4 subunit results in a strong decrease in synaptic currents from mossy fibers (MF) to granule cells (GC) as well as in two «compensatory» changes pertaining to NMDA Rs and tonic inhibition. As a consequence, MF-GC transfer is strongly reduced at high frequencies but less affected al low frequencies. The second part of the work investigates the effect of the GluA4 deletion on cerebellar-dependent behaviors. GluA4 knock-out mice are found to have no deficits in locomotion but exhibit a total absence of associative learning in an eye-blink conditioning paradigm. Both, at the slice level and at the behavioral level the strength of this work resides on the quality of the data and the rigorous analysis. A shortcoming of the work stems from the «compensatory» changes which complicate interpretation. However modeling strategies are implemented incorporating those changes and they are able to well predict the observed alterations in GC firing pattern, thus limiting the negative impact.
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Evaluation Summary:
This work explores the cellular and behavioural effects of a genetically induced reduction of the expression of a glutamate (excitatory) receptor (GluA4), focusing on the cerebellum , a structure involved in the acquisition of arbitrary, complex motor reflexes. The authors show that synaptic transmission at the input layer to the cerebellar cortex is reduced, despite some compensation by other mechanisms, which are characterised. Locomotion is little affected while acquisition of a "conditioned eyeblink" is abolished. The authors try to link the cellular and behavioural phenomena via modelling of the cerebellar computation, although this is not definitive. The work is of high quality, of interest to cerebellar physicists and neurocomputational modellers in particular.
(This preprint has been reviewed by eLife. We …
Evaluation Summary:
This work explores the cellular and behavioural effects of a genetically induced reduction of the expression of a glutamate (excitatory) receptor (GluA4), focusing on the cerebellum , a structure involved in the acquisition of arbitrary, complex motor reflexes. The authors show that synaptic transmission at the input layer to the cerebellar cortex is reduced, despite some compensation by other mechanisms, which are characterised. Locomotion is little affected while acquisition of a "conditioned eyeblink" is abolished. The authors try to link the cellular and behavioural phenomena via modelling of the cerebellar computation, although this is not definitive. The work is of high quality, of interest to cerebellar physicists and neurocomputational modellers in particular.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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