A Novel FN-MdV Pathway and Its Role in Cerebellar Multimodular Control of Sensorimotor Behavior


 The cerebellum is crucial for various associative sensorimotor behaviors. Delay eyeblink conditioning (DEC) depends on the simplex lobule-interposed nucleus (IN) pathway, yet it is unclear how other cerebellar modules cooperate during this task. Here, we demonstrate the contribution of the vermis-fastigial nucleus (FN) pathway in controlling DEC. We found that task-related modulations in vermal Purkinje cells and FN neurons predict conditioned responses (CRs). Coactivation of the FN and the IN allows for the generation of proper motor commands for CRs, but only FN output fine-tunes unconditioned responses. The vermis-FN pathway launches its signal via the contralateral ventral medullary reticular nucleus, which converges with the command from the simplex-IN pathway onto facial motor neurons. We propose that the IN pathway specifically drives CRs whereas the FN pathway modulates the amplitudes of eyelid closure during DEC. Thus, associative sensorimotor task optimization requires synergistic modulation of different olivocerebellar modules that provide unique contributions.


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Sensorimotor associative behaviors allow vertebrates to convert perceptions from the 33 environment into specific motor executions. Pavlovian delay eyeblink conditioning 34 (DEC) is an ideal model for studying the neuronal and circuit mechanisms for 35 associative tasks in which the motor response is precisely timed with respect to 36 sensory input 1-3 . In this paradigm, animals are presented with a neutral conditioned 37 stimulus (CS) followed, at a fixed interval, by an unconditioned stimulus (US) that 38 reliably causes an unconditioned eyeblink reflex (UR). Prior to conditioning, the CS amplitudes of CRs 17 (Fig. 1f-i). A group of FN neurons raised their facilitation peaks 116 with an increase in CR peak amplitudes across trials (P < 0.05, linear regression, n = 117 10 units; Fig. 1f, g). In other words, the modulation amplitudes of these FN neurons 118 were faithfully correlated with CR peak amplitudes. Interestingly, we found a similar 119 portion of facilitation neurons in which their CS-related modulation correlated 120 negatively with CR amplitude (P < 0.05, linear regression, n = 5 units; Fig. 1h, i), 121 suggesting diverse coding mechanisms for conditioned eyelid closure in FN neurons. 122 To analyze the temporal relationship between FN activity and CR performance, 123 we generated a three-dimensional correlation matrix for all modulating FN neurons 124 (see Methods and our previous work 17 ). In short, we computed the significance of trial-125 by-trial correlations between FN neuronal activity and eyelid position at various epochs 126 throughout the task. Significant correlations between FN facilitation and CR 127 performance were found above the diagonal line of the matrix within the CS-US 128 interval, revealing that the across-trial correlations were strongest when FN facilitation 129 preceded eyelid closure (Fig. 1j). The peak correlation was found when FN neuron 130 facilitation occurred 40 ms prior to the CR (Fig. 1j). In line with this, both the onset and 131 peak timings of FN facilitation were significantly earlier than CR onset (P < 0.001, 132 paired t-test; Fig. 1k) and peak time (P < 0.01, paired t-test; Fig. 1l). In contrast, FN 133 neuron suppression had a minimal trial-by-trial correlation with CR performance 134 ( Supplementary Fig. 3a, b). Even so, the onset and trough timings of neuronal 135 suppression were also significantly earlier than CR onset (P < 0.01, paired t-test;  ChrimsonR-tdTomato into the FN of VGluT2-ires-Cre or Gad2-ires-Cre mice (Fig. 2a). 148 ChrimsonR-expressing neurons showed robust short-latency facilitation (7.1 ± 4.2 ms    Fig. 3a). Well-isolated PCs were identifiable with their stereotypical simple spike and 185 complex spike waveforms (Fig. 3b). A majority of the vermal PCs modulated their 186 activity during the CS-US interval (Fig. 3c, d). Specifically, one-third of the PCs 187 decreased their simple spike firing rates during the CS-US interval (SS suppression, 188 firing rate decreased 18.9 ± 2.9%, n = 23 units; Fig. 3c, d), similar to the PC activity  Fig. 6c).

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Given the significant trial-by-trial correlation between FN firing rate and CR  in response to the CS and US. In total, 29 vermal PCs significantly increased their 218 complex spike firing rate following CS (CpxCS, Wilcoxon rank-sum test, P < 0.05; Fig.   219 4a). Among these PCs, 23 neurons presented short-latency complex spikes in 220 response to the US (CpxUS, 26.9 ± 2.6 ms after US, Supplementary Fig. 8a), 41.9 ± 221 2.8 ms before the UR peak (mean ± s.e.m., Supplementary Fig. 8c). The other 6   (Fig. 5a). Precise muscimol injection targeting the FN, ipsilaterally to the 251 10 trained eye, largely abolished CRs in conditioned mice (Fig. 5b, c). Interestingly, 252 inhibiting FN activity also suppressed eyelid closure in response to the US by reducing 253 the UR peak amplitudes over 40% (Fig. 5b, c). Both CR and UR performance 254 recovered fully after washing out the muscimol (Fig. 5b, c). These results suggest the 255 functional necessity of FN neuron activity for CR and UR performance during DEC.

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To pinpoint whether FN modulation, specifically during the CS-US interval, is 257 essential for CRs and URs, we transiently suppressed FN activity during the CS-US driving, CR and UR performance. 287 We next tested whether the vermis-FN module was also required for the 288 acquisition of CR by using chemogenetic (long-term) and optogenetic (timing-specific) 289 suppression of FN outputs throughout DEC training. Inhibitory DREADDs were virally 290 expressed in the FN unilateral to the eye that received DEC training, and tdTomato 291 was delivered to control mice (Fig. 6a). The activity of DREADD-expressing FN 292 neurons in awake mice was significantly decreased after i.p. clozapine-N-oxide (CNO) 293 administration (Fig. 6b, c). Therefore, we injected CNO daily in both groups, 15-20 min     The Vermis-FN-MdV module is essential for eyelid closure during DEC 400 We found that excitatory FN neurons and vermal PCs had task-related modulation in

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Mice were allowed to recover for at least two weeks after surgery and prior to behavior 606 studies. For in-vivo electrophysiological experiments, a small craniotomy (F = 2.0 mm) 607 was made on the skull over the recording sites. We built a chamber around the 608 craniotomy with Charisma and sealed it with Picodent twinsil after recording.

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For intracranial viral/CTB injections, anesthetized animals were fixed on a 610 mouse stereotaxic surgical plate. The skull was exposed, and the head was positioned 611 so that the bregma and lambda were leveled. See the coordinates for different brain 612 regions in Table S1. We gently lowered the glass capillary (tip opening F = 8 μm), and 613 AAV viral vector/CTB was slowly injected in the targeted regions. Glass capillaries 614 were left on the injection sites for > 5 mins before slowly retracted from the brain. To   In-vivo electrophysiology 634 We used single-channel or multichannel electrophysiological acquisition system in this 635 study. We recorded vermal Purkinje cells at a depth of 1.5-2.0 mm and FN neurons at 636 a depth of 2.0-2.7 mm, as measured from the cerebellar surface. For single-channel 637 recordings, a glass capillary (tip opening F = 2 μm) filled with 2M NaCl solution was 638 slowly penetrated in the cerebellum till a well-isolated neuronal signal was observed.    Table 1) and their stereotypical firing patterns in response to eyelid movements.

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To specifically suppress the FN-MdV pathway in well-trained animals (Fig. 9),   to test the learning outcomes. In order to prove effectiveness of the DREADD-CNO 715 system, we recorded FN neuron activity changes over time after the CNO injection in 716 awake mice (Fig. 6b, c).  were band-pass filtered at 300-3000 Hz to subtract noise and field potential signals. 772 We extracted spike events with amplitudes that crossed the threshold at three SDs of To quantify the fluorescence signals from our tracing experiments, we first registered 814 slices into the Allen Mouse Common Coordinate Framework (CCF) 83 in order to 815 standardize the brain slices across mice and to annotate nuclei. Detailed registration 816 method has been described in our previous work 38 . Briefly, we manually selected the 817 coronal plane from the CCF template (10 µm per voxel) that best corresponded to our 818 section. Next, at least 30 control points were placed at the corresponding locations of 819 section and CCF template. Sections were warped to the CCF template by using an