Abstract
The cerebellum consists of parallel parasagittal modules that contribute to diverse behaviors, spanning motor to cognitive. Recent work employing cell-type specific tracing has identified circumscribed output channels of the cerebellar nuclei that could confer tight functional specificity. These studies have largely focused on excitatory projections of the cerebellar nuclei, however, leaving open the question of whether inhibitory neurons also constitute multiple output modules. We mapped output and input patterns to intersectionally restricted cell types of the interposed and adjacent interstitial nuclei. In contrast to the widespread assumption of primarily excitatory outputs and restricted inferior olive-targeting inhibitory output, we found that inhibitory neurons from this region ramified widely within the brainstem, targeting both motor- and sensory-related nuclei, distinct from excitatory output targets. Despite differences in output targeting, monosynaptic rabies tracing revealed largely shared afferents to both cell classes. We discuss the potential novel functional roles for inhibitory outputs in the context of cerebellar theory.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This is a very brief summary of the major changes to the revised manuscript and does not replace our submitted response to reviewers. In the revised manuscript, we have added two independent methods to validate the transgenic Cre driver mouse lines used in the study: in situ hybridization and immunohistochemistry. Of critical importance, we learned that the Gad1-Cre line promiscuously expressed Cre in multiple cell types that project from the interposed nucleus. This promiscuity was observed both in immunohistochemistry, where we found terminal varicosities that expressed Vglut2 or Gad65/67, as well as in somata that expressed Vglut2 and not Vgat, or vice versa, inconsistent with specific label of inhibitory neurons. We could find no report of this promiscuity in the literature, despite the use of this line by other groups. Therefore, as a service to the field we include the description of this validation step in this report, while also entirely removing tracing datasets derived from the Gad1-Cre line from the manuscript. By contrast, we found the Vgat-Cre line to be well controlled with corroborating IHC and ISH data specific to inhibitory cell types. Importantly for the main focus of the manuscript, and as reported in the original submission, we observed that these cells innervated multiple targets outside of IO. The discovery that the Gad1-Cre line was not specific, necessitated replacing Gad1-Cre with Vgat-Cre mice for monosynaptic rabies tracing. We describe details of this discovery, decision, and consequences of this change more thoroughly in the main text of the revised manuscript. All terminal varicosities of Ntsr1-Cre derived projections were Vglut2 immunoreactive, mitigating concerns that these outputs were mixed. However, despite the specificity of immunolabel in terminals, in situ hybridization data in the cerebellar nuclei of Ntsr1-Cre mice revealed a small population of Vgat neurons, putative interneurons, that colocalized with Cre-driven YFP. Therefore, we replaced the Ntsr1-Cre dataset in the monosynaptic rabies experiments with Vglut2-Cre mice. Beyond these major changes, a variety of other revisions have been made addressing reviewer concerns.
Abbreviations
- APN
- Anterior Pretectal Nucleus
- CbCtx
- Cerebellar Cortex
- CbN
- Cerebellar Nuclei
- CM
- Centromedial nucleus of the thalamus
- CN
- Cochlear Nucleus
- CU
- Cuneate Nucleus
- CUN
- Cuneiform Nucleus
- DTN
- Dorsal Tegmental Nucleus
- ECU
- External Cuneate Nucleus
- GoC
- Golgi Cells
- GRN
- Gigantocellular Reticular Nucleus
- IC
- Inferior Colliculus
- III
- Oculomotor Nucleus
- IN
- Interposed Nucleus
- IntA
- Anterior Interposed Nucleus
- IO
- Inferior Olive
- IRN
- Intermediate reticular nucleus
- LAV
- Lateral vestibular Nucleus
- LC
- Locus Coeruleus
- LDT
- Lateral dorsal tegmental nucleus
- LN
- Lateral Cerebellar Nucleus
- LRN
- Lateral Reticular Nucleus
- MARN
- Magnocellular reticular nucleus
- MD
- Mediodorsal nucleus of the thalamus
- MDRNd
- Medullary reticular nucleus dorsal
- MDRNv
- Medullary reticular nucleus ventral
- MLI
- Molecular Layer Interneurons
- MN
- Medial Cerebellar Nucleus
- MRN
- Midbrain reticular nucleus
- MV
- Medial vestibular nucleus
- NLL
- nucleus of the lateral lemniscus
- NTS
- Nucleus of the solitary tract
- PAG
- Periaqueductal grey
- PARN (PCRt)
- Parvicellular reticular nucleus
- PAS
- Parasolitary nucleus
- PB
- Parabrachial nuclei
- PC
- Purkinje Cells
- PCG
- Pontine Central Gray
- PCN
- Paracentral nucleus of the thalamus
- PDTg
- Posterodrosal tegmental nucleus
- PF
- Parafascicular nucleus of the thalamus
- PG (PN, BPN)
- Pontine gray
- PGRN
- Paragigantocellular reticular nucleus
- PHY
- Perihypoglossal nuclei
- PMR
- Paramedian reticular nucleus
- PO
- Posterior complex of the thalamus
- PPN
- Pedunculopontine nucleus
- PPY
- Parapyramidal nucleus
- PRN
- Pontine reticular nucleus
- PRP
- Prepositus nucleus
- PRT
- Pretectal region
- PSV
- Principal sensory nucleus of the trigeminal
- RAmb
- Midbrain raphe nucleus
- RM
- Nucleus raphe magnus
- RN
- Red nucleus
- RPS
- Relative Projection Strength
- SAG
- Nucleus sagulum
- SC
- Superior colliculus
- SLC
- Subceruleus nucleus
- SLD
- Sublaterodorsal nucleus
- SNr
- Substantia nigra, reticulata
- SPIV
- Spinal vestibular nucleus
- SPVc
- Spinal nucleus of the trigeminal, caudal
- SPVi
- Spinal nucleus of the trigeminal, interpolar
- SPVo
- Spinal nucleus of the trigeminal, oral
- SUT
- Supratrigeminal nucleus
- SUV
- Superior vestibular nucleus
- TRN (NRTP)
- Tegmental reticular nucleus of the pons
- V
- Motor nucleus of the trigeminal
- VAL
- Ventral anterior-lateral complex of the thalamus
- VEST
- Vestibular nuclei
- VII
- Facial motor nucleus
- VM
- Ventromedial nucleus of the thalamus
- VPL
- Ventral posterolateral nucleus of the thalamus
- VPM
- Ventral posteromedial nucleus of the thalamus
- VTA
- Ventral tegmental area
- X
- Nucleus X
- XII
- Hypoglossal nucleus
- Y
- Nucleus Y
- ZI
- Zona incerta