Cerebellar climbing fibers impact experience-dependent plasticity in the mouse primary somatosensory cortex

Reviewer #1 (Public review):

Summary:

Silbaugh, Koster, and Hansel investigated how the cerebellar climbing fiber (CF) signals influence neuronal activity and plasticity in mouse primary somatosensory (S1) cortex. They found that optogenetic activation of CFs in the cerebellum modulates responses of cortical neurons to whisker stimulation in a cell-type-specific manner and suppresses potentiation of layer 2/3 pyramidal neurons induced by repeated whisker stimulation. This suppression of plasticity by CF activation is mediated through modulation of VIP- and SST-positive interneurons. Using transsynaptic tracing and chemogenetic approaches, the authors identified a pathway from the cerebellum through the zona incerta and the thalamic posterior medial (POm) nucleus to the S1 cortex, which underlies this functional modulation.

Strengths:

This study employed a combination of modern neuroscientific techniques, including two-photon imaging, opto- and chemo-genetic approaches, and transsynaptic tracing. The experiments were thoroughly conducted, and the results were clearly and systematically described. The interplay between the cerebellum and other brain regions - and its functional implications - is one of the major topics in this field. This study provides solid evidence for an instructive role of the cerebellum in experience-dependent plasticity in the S1 cortex.

Weaknesses:

There may be some methodological limitations, and the physiological relevance of the CF-induced plasticity modulation in the S1 cortex remains unclear. In particular, it has not been elucidated how CF activity influences the firing patterns of downstream neurons along the pathway to the S1 cortex during stimulation.

(1) Optogenetic stimulation may have activated a large population of CFs synchronously, potentially leading to strong suppression followed by massive activation in numerous cerebellar nuclear (CN) neurons. Given that there is no quantitative estimation of the stimulated area or number of activated CFs, observed effects are difficult to interpret directly. The authors should at least provide the basic stimulation parameters (coordinates of stim location, power density, spot size, estimated number of Purkinje cells included, etc.).

(2) There are CF collaterals directly innervating CN (PMID:10982464). Therefore, antidromic spikes induced by optogenetic stimulation may directly activate CN neurons. On the other hand, a previous study reported that CN neurons exhibit only weak responses to CF collateral inputs (PMID: 27047344). The authors should discuss these possibilities and the potential influence of CF collaterals on the interpretation of the results.

(3) The rationale behind the plasticity induction protocol for RWS+CF (50 ms light pulses at 1 Hz during 5 min of RWS, with a 45 ms delay relative to the onset of whisker stimulation) is unclear.

a) The authors state that 1 Hz was chosen to match the spontaneous CF firing rate (line 107); however, they also introduced a delay to mimic the CF response to whisker stimulation (line 108). This is confusing, and requires further clarification, specifically, whether the protocol was designed to reproduce spontaneous or sensory-evoked CF activity.

b) Was the timing of delivering light pulses constant or random? Given the stochastic nature of CF firing, randomly timed light pulses with an average rate of 1Hz would be more physiologically relevant. At the very least, the authors should provide a clear explanation of how the stimulation timing was implemented.

(4) CF activation modulates inhibitory interneurons in the S1 cortex (Figure 2): responses of interneurons in S1 to whisker stimulation were enhanced upon CF coactivation (Figure 2C), and these neurons were predominantly SST- and PV-positive interneurons (Figure 2H, I). In contrast, VIP-positive neurons were suppressed only in the late time window of 650-850 ms (Figure 2G). If the authors' hypothesis-that the activity of VIP neurons regulates SST- and PV-neuron activity during RWS+CF-is correct, then the activity of SST- and PV-neurons should also be increased during this late time window. The authors should clarify whether such temporal dynamics were observed or could be inferred from their data.

(5) Transsynaptic tracing from CN nicely identified zona incerta (ZI) neurons and their axon terminals in both POm and S1 (Figure 6 and Figure S7).

a) Which part of the CN (medial, interposed, or lateral) is involved in this pathway is unclear.

b) Were the electrophysiological properties of these ZI neurons consistent with those of PV neurons?

c) There appears to be a considerable number of axons of these ZI neurons projecting to the S1 cortex (Figure S7C). Would it be possible to estimate the relative density of axons projecting to the POm versus those projecting to S1? In addition, the authors should discuss the potential functional role of this direct pathway from the ZI to the S1 cortex.

Reviewer #2 (Public review):

Summary:

The authors examined long-distance influence of climbing fiber (CF) signaling in the somatosensory cortex by manipulating whiskers through stimulation. Also, they examined CF signaling using two-photon imaging and mapped projections from the cerebellum to the somatosensory cortex using transsynaptic tracing. As a final manipulation, they used chemogenetics to perturb parvalbumin-positive neurons in the zona incerta and recorded from climbing fibers.

Strengths:

There are several strengths to this paper. The recordings were carefully performed, and AAVs used were selective and specific for the cell types and pathways being analyzed. In addition, the authors used multiple approaches that support climbing fiber pathways to distal regions of the brain. This work will impact the field and describes nice methods to target difficult-to-reach brain regions, such as the inferior olive.

Weaknesses:

There are some details in the methods that could be explained further. The discussion was very short and could connect the findings in a broader way.

Reviewer #3 (Public review):

Summary:

The authors developed an interesting novel paradigm to probe the effects of cerebellar climbing fiber activation on short-term adaptation of somatosensory neocortical activity during repetitive whisker stimulation. Normally, RWS potentiated whisker responses in pyramidal cells and weakly suppressed them in interneurons, lasting for at least 1h. Crusii Optogenetic climbing fiber activation during RWS reduced or inverted these adaptive changes. This effect was generally mimicked or blocked with chemogenetic SST or VIP activation/suppression as predicted based on their "sign" in the circuit.

Strengths:

The central finding about CF modulation of S1 response adaptation is interesting, important, and convincing, and provides a jumping-off point for the field to start to think carefully about cerebellar modulation of neocortical plasticity.

Weaknesses:

The SST and VIP results appeared slightly weaker statistically, but I do not personally think this detracts from the importance of the initial finding (if there are multiple underlying mechanisms, modulating one may reproduce only a fraction of the effect size). I found the suggestion that zona incerta may be responsible for the cerebellar effects on S1 to be a more speculative result (it is not so easy with existing technology to effectively modulate this type of polysynaptic pathway), but this may be an interesting topic for the authors to follow up on in more detail in the future.