A system of feed-forward cerebellar circuits that extend and diversify sensory signaling

Reviewer #1 (Public Review):

The manuscript by Hariani et al. presents experiments designed to improve our understanding of the connectivity and computational role of Unipolar Brush Cells (UBCs) within the cerebellar cortex, primarily lobes IX and X. The authors develop and cross several genetic lines of mice that express distinct fluorophores in subsets of UBCs, combined with immunocytochemistry that also distinguishes subtypes of UBCs, and they use confocal microscopy and electrophysiology to characterize the electrical and synaptic properties of subsets of so-labelled cells, and their synaptic connectivity within the cerebellar cortex. The authors then generate a computer model to test the possible computational functions of such interconnected UBCs.

Using these approaches, the authors report that:

1. GRP-driven TDtomato is expressed exclusively in a subset (20%) of ON-UBCs, defined electrophysiologically (excited by mossy fiber afferent stimulation via activation of UBC AMPA and mGluR1 receptors) and immunocytochemically by their expression of mGluR1.

2. UBCs ID'd/tagged by mCitrine expression in Brainbow mouse line P079 are expressed in a similar minority subset of OFF-UBCs defined electrophysiologically (inhibited by mossy fiber afferent stimulation via activation of UBC mGluR2 receptors) and immunocytochemically by their expression of Calretinin. However, such mCitrine expression was also detected in some mGluR1 positive UBCs, which may not have shown up electrophysiologically because of the weaker fluorophore expression without antibody amplification.

3. Confocal analysis of crossed lines of mice (GRP X P079) stained with antibodies to mGluR1 and calretinin documented the existence of all possible permutations of interconnectivity between cells (ON-ON, ON-OFF, OFF-OFF, OFF-ON), but their overall abundance was low, and neither their absolute nor relative abundance was quantified.

4. A computational model (NEURON ) indicated that the presence of an intermediary UBC (in a polysynaptic circuit from MF to UBC to UBC) could prolong bursts (MF-ON-ON), prolong pauses (MF-ON-OFF), cause a delayed burst (MF-OFF-OFF), cause a delayed pause (MF-OFF-ON) relative to solely MF to UBC synapses which would simply exhibit long bursts (MF-ON) or long pauses (MF-OFF).

The authors thus conclude that the pattern of interconnected UBCs provides an extended and more nuanced pattern of firing within the cerebellar cortex that could mediate longer-lasting sensorimotor responses.

The cerebellum's long-known role in motor skills and reflexes, and associated disorders, combined with our nascent understanding of its role in cognitive, emotional, and appetitive processing, makes understanding its circuitry and processing functions of broad interest to the neuroscience and biomedical community. The focus on UBCs, which are largely restricted to vestibular lobules of the cerebellum reduces the breadth of likely interest somewhat. The overall design of specific experiments is rigorous and the use of fluorophore expressing mouse lines is creative. The data that is presented and the writing are clear. However, the overall experimental design has issues that reduce overall interpretation (please see specific issues for details), which combined with a lack of thorough analysis of the experimental outcomes severely undermines the value of the NEURON model results and the advance in our understanding of cerebellar processing in situ (again, please see specific issues for details).

Specific issues:

1. All data gathered with inhibition blocked. All of the UBC response data (Fig. 1) was gathered in the presence of GABAAR and Glycine R blockers. While such an approach is appropriate generally for isolating glutamatergic synaptic currents, and specifically for examining and characterizing monosynaptic responses to single stimuli, it becomes problematic in the context of assaying synaptic and action potential response durations for long-lasting responses, and in particular for trains of stimuli, when feed-forward and feed-back inhibition modulates responses to afferent stimulation. That is, even for single MF stimuli, given the >500ms duration of UBC synaptic currents, there is plenty of time for feedback inhibition from Golgi cells (or feedforward, from MF to Golgi cell excitation) to interrupt AP firing driven by the direct glutamatergic synaptic excitation. This issue is compounded further for all of the experiments examining trains of MF stimuli. Beyond the impact of feedback inhibition on the AP firing of any given UBC, it would also obviously reduce/alter/interrupt that UBC's synaptic drive of downstream UBCs. This issue fundamentally undermines our ability to interpret the simulation data of Vm and AP firing of both the modeled intermediate and downstream UBC, in terms of applying it to possible cerebellar cortical processing in situ.

2. No consideration for the involvement of polysynaptic UBCs driving UBC responses to MF stimulation in electrophysiology experiments. Given the established existence (in this manuscript and Dino et al. 2000 Neurosci, Dino et al. 2000 ProgBrainRes, Nunzi and Mugnaini 2000 JCompNeurol, Nunzi et al. 2001 JCompNeurol) of polysynaptic connections from MFs to UBCs to UBCs, the MF evoked UBC responses established in this manuscript, especially responses to trains of stimuli could be mediated by direct MF inputs, or to polysynaptic UBC inputs, or possibly both (to my awareness not established either way). Thus the response durations could already include extension of duration by polysynaptic inputs, and so would overestimate the duration of monosynaptic inputs, and thus polysynaptic amplification/modulation, observed in the NEURON model.

3. Lack of quantification of subtypes of UBC interconnectivity. Given that it is already established that UBCs synapse onto other UBCs (see refs above), the main potential advance of this manuscript in terms of connectivity is the establishment and quantification of ON-ON, ON-OFF, OFF-ON, and OFF-OFF subtypes of UBC interconnections. But, the authors only establish that each type exists, showing specific examples, but no quantification of the absolute or relative density was provided, and the authors' unquantified wording explicitly or implicitly states that they are not common. This lack of quantification and likely small number makes it difficult to know how important or what impact such synapses have on cerebellar processing, in the model and in situ.

4. Lack of critical parameters in NEURON model.
   A) The model uses # of molecules of glutamate released as the presumed quantal content, and this factor is constant. However, no consideration of changes in # of vesicles released from single versus trains of APs from MFs or UBCs is included. At most simple synapses, two sequential APs alters release probability, either up or down, and release probability changes dynamically with trains of APs. It is therefore reasonable to imagine UBC axon release probability is at least as complicated, and given the large surface area of contact between two UBCs, the number of vesicles released for any given AP is also likely more complex.
   B) the model does not include desensitization of AMPA receptors, which in the case of UBCs can paradoxically reduce response magnitude as vesicle release and consequent glutamate concentration in the cleft increases (Linney et al. 1997 JNeurophysiol, Lu et al. 2017 Neuron, Balmer et al. 2021 eLIFE), as would occur with trains of stimuli at MF to ON-UBCs.

5. Lack of quantification of various electrophysiological responses. UBCs are defined (ON or OFF) based on inward or outward synaptic response, but no information is provided about the range of the key parameter of duration across cells, which seems most critical to the current considerations. There is a similar lack of quantification across cells of AP duration in response to stimulation or current injections, or during baseline. The latter lack is particularly problematic because, in agreement with previous publications, the raw data in Fig. 1 shows ON UBCs as quiescent until MF stimulation and OFF UBCs firing spontaneously until MF stimulation, but, for example, at least one ON UBC in the NEURON model is firing spontaneously until synaptically activated by an OFF UBC (Fig. 11A), and an OFF UBC is silent until stimulated by a presynaptic OFF UBC (Fig. 11C). This may be expected/explainable theoretically, but then such cells should be observed in the raw data.

Reviewer #2 (Public Review):

In this paper, the authors presented a compelling rationale for investigating the role of UBCs in prolonging and diversifying signals. Based on the two types of UBCs known as ON and OFF UBC subtypes, they have highlighted the existing gaps in understanding UBCs connectivity and the need to investigate whether UBCs target UBCs of the same subtype, different subtypes, or both. The importance of this knowledge is for understanding how sensory signals are extended and diversified in the granule cell layer.

The authors designed very interesting approaches to study UBCs connectivity by utilizing transgenic mice expressing GFP and RFP in UBCs, Brainbow approach, immunohistochemical and electrophysiological analysis, and computational models to understand how the feed-forward circuits of interconnected UBCs transform their inputs.

This study provided evidence for the existence of distinct ON and OFF UBC subtypes based on their electrophysiological properties, anatomical characteristics, and expression patterns of mGluR1 and calretinin in the cerebellum. The findings support the classification of GRP UBCs as ON UBCs and P079 UBCs as OFF UBCs and suggest the presence of synaptic connections between the ON and OFF UBC subtypes. In addition, they found that GRP and P079 UBCs form parallel and convergent pathways and have different membrane capacitance and excitability. Furthermore, they showed that UBCs of the same subtype provide input to one another and modify the input to granule cells, which could provide a circuit mechanism to diversify and extend the pattern of spiking produced by mossy fiber input. Accordingly, they suggested that these transformations could provide a circuit mechanism for maintaining a sensory representation of movement for seconds.

Overall, the article is well written in a sound detailed format, very interesting with excellent discovery and suggested model, however, I have some comments/suggestions that may help to improve this manuscript:

• The discovery of UBCs innervating each other and their own subtypes, suggesting the presence of feed-forward networks in the cerebellum, is an incredibly fascinating and exciting finding followed by an intriguing model by authors. However, it is worth considering an alternative model as well. I acknowledge that visualizing such interactions using current tools and methods can be challenging ("The approaches used here were not able to determine the existence of networks of more than 2 UBCs connected one after the other. If present, 3 or more UBCs in series could extend and transform the input in even more dramatic ways. The temporal diversity that UBC circuits generate may underlie the flexibility of the cerebellum to coordinate movements over a broad range of behaviors."). Therefore, if this is the case in which more than 2 UBCs connected one after the other, then an alternative model PERHAPS resembles the basal nuclei, with its direct and indirect circuits, can be considered (maybe a type of circular model). The basal nuclei circuits are also regulated by modulators such as D1 dopamine receptors in the direct pathway, causing depolarization, and D2 dopamine receptors in the indirect pathway, resulting in hyperpolarization upon dopamine activation. This approach could involve using computational models to gain insight into potential alternatives within this pathway (may be a future direction).

• GRP UBCs are more densely distributed in lobes VI-IX, while P079 UBCs are more densely distributed in the dorsal leaflet of lobe X in sagittal sections. While the cerebellum is well known for its characteristic stripy pattern, are UBC distributions the same in coronal/transverse section?

• The extension of the axons from both subtypes of UBCs show they are long enough to pass several UBCs and even projections are directed toward the white matter (e.g. Fig 9A), suggesting targeting the UBCs or granule cells in other lobules. Is it suggesting UBCs connectivity between different lobules (perhaps longitudinal connectivity)? Is there any observation or information in coronal/transverse section to visualize mediolateral connectivity?

• The limitation in identifying networks involving more than two sequentially connected UBCs was briefly noted. I suggest including a paragraph describing limitations and discussing the implications of the findings would enhance the overall impact of the research and broaden our understanding of cerebellar function.

• It is a pity that there is no clear conclusion to the discussion of this very interesting study. I suggest providing the key points as a conclusion.

• Please make the correction in Figure 2A by relabeling it as IXa, IXb, and IXc to correct the typographical error.

• I recommend rotating Figure 7A to align its orientation with the other figures for consistency.

Author Response

Thank you for taking the time to manage the reviews of this manuscript. Many helpful suggestions were presented by the two reviewers that will certainly strengthen the revised version of the manuscript. We would like to take the opportunity to provide a provisional response to address concerns and factual errors in the eLife assessment and public reviews. Please see below.

Response to eLife assessment:

The assessment does not appear to reflect reviews entirely accurately. While reviewer 1 was unsatisfied by the “lack of thorough analysis of the experimental outcomes”, the criticism of a lack of sufficient support of our claims was not present in the reviews. Thus, the sentence, “The evidence supporting the claims are interesting although incomplete in some areas”, seems to us excessively negative. Furthermore, while we agree that this work inspires new studies to determine how UBC circuits function in the intact brain and how they promote behaviors, and that “substantial work remains to be conducted” to explore these new avenues, the way the sentence is constructed, and placed directly after “incomplete in some areas”, makes it read as a negative related to the current manuscript, whereas opening doors to new lines of research is certainly positive for the field.

Response to Reviewer 1:

• One of the main criticisms appears to be a lack of quantification of our electrophysiological data and clear explanation of how the model reproduces the behavior of the cells reported here and in previous work. We are thankful for the identification of these omissions. Our extensive work in UBC electrophysiology instructed the development of these models and they reproduce the essential features of ON and OFF UBC spiking responses and mGluR2 and AMPAR conductances accurately, although we agree that we did not present sufficient evidence for this in the manuscript.

• Another major criticism was a lack of consideration of feedback and feedforward inhibition. The goal of Figure 1 was to determine the cell types of labeled UBCs in transgenic mouse lines, which is determined entirely by their synaptic responses to glutamate (Borges-Merjane & Trussell, 2015). Thus, blocking inhibition was essential to produce clear results. Feedback and feedforward inhibition from Golgi cells, which is certainly important in the intact circuit, is not possible to produce in a physiologically realistic way in acute brain slices, because electrical stimulation produces synchronous excitation and inhibition (by directly exciting Golgi cells, rather than their synaptic inputs). The main inhibition that UBCs receive is through mGluR2, which lasts for 100-1000s of milliseconds, and the main excitation that UBCs receive is through mGluR1 and AMPA, which also both last 100-1000s of milliseconds. Thus, these large conductances are unlikely to be significantly shaped by 1-10 ms IPSCs from feedforward and feedback inhibition. For these reasons, it was not our intention to explore GABAergic/glycinergic feedforward and feedback inhibition in the present study.

Factual errors in public reviews:

Reviewer 1, specific point 4:

A) The reviewer accurately points out that the model did not incorporate a change in the amount of glutamate released across release events during trains of presynaptic spikes. We did not find this to be necessary to reproduce the AMPA and mGluR2 currents accurately, because the majority of the response occurs after the last presynaptic stimulus. Short term plasticity during the stimulus train would be expected to change the total amount of glutamate released, but not the time course of the slow current response. We previously showed that the predominant synaptic plasticity that occurs at this synapse during the train is short-term depression that is due in large part to postsynaptic desensitization of AMPA receptors, rather than a change in presynaptic release.

B) The reviewer states that the model does not include desensitization of AMPA receptors. Although there is not a variable that defines desensitization explicitly, the detailed kinetic AMPA receptor model used here accounts for desensitization, which, in fact, mediates slow ON UBC current and is the focus of our previous work. This AMPA receptor model (developed in Balmer et al., 2021 using UBC data from Lu et al., 2017) is a 13-state model, including 4 open states with 1-4 glutamates bound, 4 closed states with 1-4 glutamates bound, 4 desensitized states with 1-4 glutamates bound, and 5 closed states with 0-4 glutamates bound. The transition rates between different states in the model were fit to AMPA receptor currents recorded from dissociated UBCs and they approximate well the ON UBC currents evoked by synaptic stimulation (Balmer et al., 2021).