Purkinje cell intrinsic activity shapes cerebellar development and function

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
The emergence of functional cerebellar circuits is heavily influenced by activity-dependent processes. However, the contribution of intrinsic Purkinje cell activity to cerebellar development remains less understood. Here, we demonstrate that before synaptic networks mature, Purkinje cell intrinsic activity is essential for regulating dendritic growth, establishing connections with cerebellar nuclei, and ensuring proper cerebellar function. Disrupting this activity during the postnatal period impairs motor function, with earlier perturbations causing more severe deficits. Importantly, only early developmental disruptions lead to pronounced defects in cellular morphology, highlighting key temporal windows for dendritic growth and maturation. Transcriptomic analyses reveal that early intrinsic activity drives the expression of activity-dependent genes, including Prkcg and Car8, which are essential for dendritic development. Our findings emphasize the importance of temporally regulated intrinsic activity in Purkinje cells in guiding cerebellar circuit development, providing a potential unifying mechanism underlying cerebellum-associated disorders.

Introduction
Neural development relies on a complex interplay between genetic and early activity-dependent processes that guide morphogenesis and establish functional neural circuits1, 2. Early electrical activity regulates a wide range of developmental programs in various regions of the nervous system, including the retina3, 4, spinal cord5, 6, cochlea7, and neocortex8, 9. Genetic programs are especially relevant during cerebellar ontogenesis, where more than half of the brain’s neurons10 converge to form an intricate network of connections.

The cerebellum undergoes a prolonged maturation period, making it particularly susceptible to developmental errors11. Indeed, evidence suggests that a common feature of several cerebellar-associated disorders is the impairment of Purkinje cell intrinsic activity12, which is the spontaneous activity generated by these principal neurons in the absence of synaptic inputs13, 14. However, the requirement for early Purkinje cell activity in their maturation and its potential contribution to cerebellar dysfunction remains largely unexplored. Purkinje cells are at the core of cerebellar circuitry, and their development is intertwined with that of the surrounding cerebellar elements15. The genetic and cellular programs governing the genesis, migration, diversity, and differentiation of cerebellar cells have been explored and cataloged through recent single-cell transcriptomic endeavors16, 17. Although less studied, activity-dependent mechanisms also play a crucial role in the early stages of cerebellar development. For example, the organization of Purkinje cell parasagittal banding requires Purkinje cell output signals18, while the competition and subsequent elimination of climbing fiber afferents depend on Purkinje cell activity19. Furthermore, electrical stimulation of cerebellar slices induces the expression of Arc in Purkinje cells, contributing to the elimination of climbing fiber synapses20. Recent in vitro studies have revealed that even when synaptic transmission is blocked, Purkinje cells exhibit intrinsic activity as early as postnatal day 3, with this activity gradually increasing until it stabilizes at mature levels by the end of the second postnatal week21. Nevertheless, the extent to which early intrinsic activity influences the molecular programs underlying Purkinje cell maturation, circuit assembly, and overall cerebellar function remains unclear.

To investigate the role of Purkinje cell intrinsic activity in cerebellar development, we genetically reduced their intrinsic activity at distinct postnatal stages in mice. Our findings reveal that early activity is fundamental for the development of Purkinje cell dendritic arbors, axonal targeting of cerebellar nuclei neurons, and the acquisition of cerebellar functions. Intrinsic activity is particularly important during the first two postnatal weeks for Purkinje cell maturation and the establishment of balance and coordination functions. Molecularly, early intrinsic activity modulates the expression of several activity-dependent genes, including Prkcg and Car8. Functional shRNA knockdown of Prkcg and Car8 genes demonstrates that they are key regulators of Purkinje cell development. Overall, our results unveiled a selective requirement for early Purkinje cell activity in shaping cerebellar development and function.

Results
Overexpression of Kir2.1 reduces Purkinje cell intrinsic activity
To reduce the intrinsic activity of Purkinje cells during postnatal development, we generated a mouse model that allows for temporal control of activity. The tamoxifen-inducible Pcp2creER mouse line22 was crossed with a conditional line expressing the inward rectifying potassium channel 2.1 (Kir2.1) fused to the mCherry reporter gene23, generating Pcp2creER;Kir2.1 mutant mice (Supplementary Fig. 1a, c). As an inducible control, Pcp2creER;Ai1424 mice conditionally expressing the tdTomato reporter were also generated (Supplementary Fig. 1a, b). To examine Purkinje cell activity in a cell-autonomous manner, we administered a single low dose of tamoxifen at postnatal day (P) 1 to achieve sparse labeling of Purkinje cells expressing either Kir2.1-mCherry or tdTomato in mutant and control pups, respectively (Fig. 1a and Supplementary Fig. 1a, d). At P21, when cerebellar development is complete, we performed ex vivo recordings from sagittal cerebellar slices to evaluate the levels of intrinsic activity in Purkinje cells expressing tdTomato (Pcp2creER;Ai14), Kir2.1-mCherry (Pcp2creER;Kir2.1), or neither (Pcp2creER;Kir2.1;Ctl, unlabeled controls). Recordings were conducted in the presence of synaptic blockers for NMDA, AMPA, GABA and glycine receptors. In control recordings, 77% of Purkinje cells exhibited spontaneous firing, confirming intrinsic activity. In contrast, Kir2.1 overexpression abolished spontaneous firing in 90% of the recorded Pcp2creER;Kir2.1 Purkinje cells by hyperpolarizing the resting membrane potential (Fig. 1b, c). Treatment with 300 µM barium, a potassium channel blocker, restored the membrane potential to control levels in Pcp2creER;Kir2.1 Purkinje cells (Supplementary Fig. 2a). Kir2.1 overexpression also caused a significant shift in the current-voltage (I-V) curve of mutant Purkinje cells compared to controls, notably more hyperpolarized current at -140 mV. This I-V curve could also be restored to control levels by the presence of barium (Supplementary Fig. 2b-d). Despite the loss of intrinsic activity, mutant Pcp2creER;Kir2.1 Purkinje cells retained the ability to fire action potentials in response to depolarizing current injection (Fig. 1d, e). However, their lower input resistance (Supplementary Fig. 2e) made them less excitable, requiring larger current injections (≥ 600 pA) to evoke the firing of action potentials (Fig. 1e and Supplementary Fig. 2f).

Next, we examined Purkinje cell activity in vivo using Pcp2cre/+;Kir2.1 mice, in which Kir2.1 is overexpressed in all Purkinje cells, and control littermates Pcp2+/+;Kir2.1 at P28 ± 3 days (Fig. 1f and Supplementary Fig. 1e-g). Purkinje cells were identified by the presence of simple and complex spikes, and single unit recordings were confirmed by the presence of the characteristic pause in simple spikes following each complex spike (Fig. 1g). The simple spike firing rate was significantly reduced in Pcp2cre/+;Kir2.1 Purkinje cells (65 ± 4 Hz) compared with controls (13 ± 3 Hz) (Fig. 1g, h), and mutant cells displayed increased irregularity in simple spike firing (Fig. 1i). Conversely, the complex spike rate was significantly higher in Pcp2cre/+;Kir2.1 Purkinje cells than in controls (Fig. 1j), while their firing regularity remained unchanged (Fig. 1k). In line with previous findings that simple spike firing rate primarily reflects the intrinsic activity of Purkinje cells25, these results demonstrated that Kir2.1-overexpression also minimizes Purkinje cell firing activity in vivo. Taken together, these findings indicate that Purkinje cells overexpressing Kir2.1 exhibit reduced intrinsic activity but retain the capacity to generate action potentials when stimulated.

Loss of intrinsic activity impairs Purkinje cell morphological maturation
To investigate whether disrupting intrinsic activity impacts the morphological development of Purkinje cells, we used inducible Pcp2creER;Ai14 and Pcp2creER;Kir2.1 mice to sparsely label Purkinje cells from P1 to P21 (Fig. 2a). We analyzed the dendritic arbors of Purkinje cells at P21 across different cerebellar regions. Sholl analyses revealed that Purkinje cells in Pcp2creER;Kir2.1 mice exhibited reduced dendritic complexity compared with Pcp2creER;Ai14 control cells (Fig. 2.b-d). Moreover, other morphological parameters, including area, the length of the longest dendrite, and extension in the molecular layer (ML), were significantly reduced in Kir2.1-expressing Purkinje cells relative to controls (Fig. 2b, e-g). To corroborate these findings, we performed gain-of-function experiments by electroporating plasmids encoding GFP (control) and Kir2.1-T2A-tdTomato (Kir2.1) into Purkinje cell progenitors at embryonic day (E) 12.5 (Supplementary Fig. 3a). At P21, Kir2.1 Purkinje cells exhibited markedly reduced dendritic complexity compared with controls (Supplementary Fig. 3b-d), along with decreased area, longest dendritic length and extension in the molecular layer (Supplementary Fig. 3b, e-g).

To assess whether deficits in intrinsic activity affected the presynaptic targeting of Purkinje cells as a population, we used Pcp2cre/+;Kir2.1 mice and control littermates (Fig. 2h). We quantified the number of calbindin-expressing (Calb+) and vesicular GABA transporter (VGAT+) boutons contacting NeuN+ cerebellar nuclei neurons. Loss of intrinsic activity resulted in a significant reduction in the number of Calb+ VGAT+ contacts that Purkinje cells made onto lateral cerebellar nuclei NeuN+ neurons compared with controls (Fig. 2i, j). To determine how these structural changes translated into cerebellar output, we performed extracellular recordings from cerebellar nuclei neurons in vivo in Pcp2cre/+;Kir2.1 mice and controls. Cerebellar nuclei were localized post hoc using Evans blue. Neurons in the cerebellar nuclei of Pcp2cre/+;Kir2.1 mice displayed significantly higher firing rates (Fig. 2k, l) and increased irregularity in firing compared with controls (Fig. 2m). Overall, these results indicate that loss of Purkinje cell intrinsic activity during the early postnatal period disrupts dendritic growth and impairs presynaptic targeting.

Disrupted intrinsic activity in Purkinje cells impairs motor performance and learning
Mutations that affect Purkinje cell development are known to cause motor impairments26-29. To investigate how disrupting intrinsic activity from birth to adulthood affects motor behavior, we tested adult (P60-P90) Pcp2cre/+;Kir2.1 mice and control littermates, Pcp2+/+;Kir2.1. In the balance beam test, Pcp2cre/+;Kir2.1 mice required more time to cross a 12 mm flat beam and made more missteps than controls (Fig. 3a, b and Supplementary Movies 1, 2). On the accelerating rotarod, Pcp2cre/+;Kir2.1 mice had a shorter latency to fall on the rotating rod at both 40 and 80 rpm compared to controls (Fig. 3c and Supplementary Movies 3). Using the LocoMouse test30, 31, we found that Pcp2cre/+;Kir2.1 mice exhibited abnormal body axis swings and increased body-axis angles (Fig. 3d, e). While stride and stance distances were similar between groups, mutants took significantly longer to complete these movements (Supplementary Fig. 4a and Supplementary Movies 4, 5). Interlimb coordination was also disrupted in the mutants, with diagonal limb pairs failing to move synchronously across different walking speeds (Fig. 4c). However, in the open field test, while the distance traveled was similar between groups, mutants moved faster than controls (Supplementary Fig. 4b). Overall, these data revealed that disruption of intrinsic Purkinje cell activity from early development to adulthood results in an ataxic phenotype characterized by impaired gait coordination and balance.

Given the importance of Purkinje cell activity for motor learning, we examined the impact of reduced intrinsic activity on cerebellum-dependent learning behaviors, such as vestibular-ocular reflex (VOR) adaptation32, 33 and eyeblink conditioning (EBC)34, 35. We observed no differences in VOR and visual VOR gain (amplitude) and phase (timing) between genotypes (Supplementary Fig. 4e, f). However, Pcp2cre/+;Kir2.1 mutants exhibited lower gain and higher phase in the optokinetic reflex (OKR) compared to controls (Supplementary Fig. 4d). In a phase-reversal VOR adaptation protocol, control animals successfully reversed their VOR direction by increasing phase over a 5-day experiment. In contrast, Pcp2cre/+;Kir2.1 mutants failed to adapt their phase or gain, indicating impaired motor learning (Fig. 3f, h and Supplementary Fig. 4g). Similarly, in the EBC paradigm, where mice learn to associate a visual conditioned stimulus (CS – LED light) with an eyeblink-inducing unconditioned stimulus (US – air puff delivered to the mouse cornea), mutant mice exhibited significantly reduced conditioned response (CR – preventive eyelid closure) percentage and amplitude compared to controls (Fig. 3g, i and Supplementary Fig. 4h, i). Importantly, both genotypes showed similar unconditioned response peak times, confirming that their ability to close the eyelid was intact (Supplementary Fig. 4j).

To further investigate the neural correlates of these behavioral deficits, we performed in vivo recordings of Purkinje cell and cerebellar nuclei neuron activity in adult mice (Supplementary Fig. 5a). Consistent with findings in juvenile animals, Pcp2cre/+;Kir2.1 Purkinje cells exhibited significantly lower simple spike firing rates and larger firing irregularity than controls (Supplementary Fig. 5b-d), whereas complex spike rate and regularity were unaffected (Supplementary Fig. 5b, e, f). Unlike the juvenile mice, cerebellar nuclei neurons in adult mutant mice showed reduced firing rates with unchanged firing regularity (Supplementary Fig. 5g-i). A pathological feature shared by different subtypes of ataxia is the alteration of excitatory synaptic inputs onto the Purkinje cells, particularly, the loss of climbing fibers (CFs)36-39 and impairment of parallel fibers (PFs)40, 41. Using VGLUT2 as a marker for CF terminals, we found a significant reduction in the percentage of CF extension within the molecular layer and VGLUT2 puncta density in mutants compared to controls (Supplementary Fig. 6a-d). Furthermore, the number of VGLUT2 puncta apposed to the Purkinje cell soma was increased in mutants, suggesting impaired CF translocation (Supplementary Fig. 6b, e). Examination of PF terminals using VGLUT1 revealed a marked decrease in puncta density and enlarged bouton size in mutants relative to controls (Supplementary Fig. 6f-h). Collectively, these results demonstrate that suppressing Purkinje cell intrinsic activity from early development to adulthood results in impaired motor coordination and cerebellum-dependent learning, accompanied by disrupted cerebellar firing patterns and synaptic organization.

The requirement of intrinsic activity for Purkinje cell morphological development decreases with age
After establishing the importance of Purkinje cell intrinsic activity for dendritic growth and presynaptic targeting, we asked whether this requirement persisted across different developmental stages. To address this, we administered low doses of tamoxifen at P1, P7, or P14 in Pcp2creER;Ai14 and Pcp2creER;Kir2.1 mice to achieve sparse labeling in order to analyse Purkinje cell dendritic arbor morphology one week later, at P7, P14, and P21, respectively (Fig. 4a, e, i and Supplementary Fig. 7). Following Kir2.1 overexpression from P1 to P7, mutant Purkinje cells displayed a greater number of primary neurites emerging from the soma compared with controls (Fig. 4b, c, d). While control P7 Purkinje cells exhibited a characteristic single flattened dendrite with a vertical orientation, Kir2.1-overexpressing Purkinje cells retained an immature, multipolar morphology with neurites extending in multiple directions, resembling an earlier developmental stage42. Despite these differences in dendritic organization, the area and the length of the longest neurite did not differ between genotypes (Fig. 4d). Overexpression of Kir2.1 during the second postnatal week (P7-P14) caused a marked reduction in dendritic complexity, area, dendrite length, and extension in the molecular layer in mutant Purkinje cells compared with controls at P14 (Fig. 4f-h). When intrinsic activity was suppressed during the third postnatal week (P14-P21), mutant Purkinje cells at P21 still exhibited significant reductions in dendritic complexity, area, and dendrite length relative to controls (Fig. 4j-l). However, the magnitude of these deficits - 10% in dendritic complexity, 21% in area, and 13% in dendrite length - was less pronounced than that observed with activity disruption throughout the entire developmental period (P1- P21: 38%, 49%, and 26%, respectively; Fig. 2c-f). Notably, at this later stage, the percentage of dendritic extension in the molecular layer was similar between mutant and control Purkinje cells (Fig. 4l).

To confirm the effectiveness of Kir2.1-mediated suppression of intrinsic activity throughout these stages, we performed ex vivo recordings in control and Kir2.1-overexpressing Purkinje cells. In control recordings, 77%, 92%, and 81% of Purkinje cells exhibited spontaneous firing at P7, P14, and P21, respectively. In contrast, only 6%, 0% and 8% of Pcp2creER;Kir2.1 Purkinje cells were intrinsically active at the corresponding ages (Supplementary Fig. 8a, e, i). Kir2.1 overexpression also significantly altered intrinsic electrophysiological properties, including a hyperpolarized resting membrane potential (Supplementary Fig. 8b, f, j), increased hyperpolarizing currents at −140 mV (Supplementary Fig. 8c, g, k), and reduced input resistance (Supplementary Fig. 8d, h, l) across all developmental stages examined. To examine how intrinsic activity influences Purkinje cell connectivity with cerebellar nuclei neurons at different developmental stages, we used Pcp2cre/+;Kir2.1 mice from P1-P7 to achieve Kir2.1-overexpression in all Purkinje cells from birth. For the intermediary stages, P7-P14 and P14-P21, we employed the inducible Pcp2creER;Kir2.1 mice and administered high doses of tamoxifen to maximize Kir2.1 expression in all Purkinje cells, alongside the respective control groups (Supplementary Fig. 9a, b, e, f, i, j). Kir2.1 overexpression from P1 to P7 significantly reduced the number of Calb+ VGAT+ contacts between Purkinje cells and NeuN+ neurons in the lateral cerebellar nuclei compared with controls (Supplementary Fig. 9c, d). Suppression of intrinsic activity from P7 to P14 also led to a significant decrease in these inhibitory contacts (Supplementary Fig. 9g, h). In contrast, reducing activity from P14 to P21 had no effect on the number of Calb+ VGAT+ inhibitory contacts between Purkinje cells and NeuN+ neurons in the mutant compared with the control (Supplementary Fig. 9k, l). These findings indicate that Purkinje cell intrinsic activity is essential for proper dendritic morphology and presynaptic targeting during development, with the most pronounced deficits occurring when activity is disrupted during the first two postnatal weeks.

The onset of Purkinje cell intrinsic activity loss determines the severity of motor impairment
Next, we examined whether the timing of intrinsic activity disruption in Purkinje cells during development influences the severity of motor impairments. High doses of tamoxifen were administered for three consecutive days at P7, P14, or P21 in inducible Pcp2creER;Kir2.1 mice and respective controls to induce Kir2.1-overexpression in all Purkinje cells (Fig. 5a and Supplementary Fig. 10c-h). Motor performance in adulthood was assessed using the balance beam test. Regardless of the timing of intrinsic activity suppression, Pcp2creER;Kir2.1 mice were significantly slower and made more missteps when crossing the flat 12 mm beam compared to controls, indicating impaired balance and coordination (Fig. 5b, c). However, the severity of these deficits depended on when intrinsic activity was disrupted. Mice with disrupted Purkinje cell activity from the second or third postnatal week performed significantly better than those in which Purkinje cell activity was suppressed from birth (Fig. 5b-f, and Supplementary Fig. 10a, b). These findings suggest that while intrinsic Purkinje cell activity is crucial for optimizing motor movements, motor control is particularly sensitive to disruptions in Purkinje cell activity during early postnatal weeks.

Because delaying the onset of Kir2.1-overexpression by just one week was sufficient to improve balance beam performance, we hypothesized that the early deficits observed in the most severe phenotype, where Kir2.1 was overexpressed from birth (Supplementary Fig. 11a), might also impair the maturation of other cerebellar cell types whose development depends on proper Purkinje cell maturation, namely molecular layer interneurons (MLIs)43, 44 and granule cells (GCs)45, 46. To test this, we examined MLI density in adult Pcp2cre/+;Kir2.1 mice and their respective controls (Supplementary Fig. 11a, b). While parvalbumin (PV) is a known marker for both Purkinje cells and MLIs47, it does not label all MLIs within the molecular layer48. Hence, we quantified the number of PV-positive (PV+) cells co-labeled with NeuroTrace (NeuT+), a fluorescent Nissl stain, within the molecular layer. The number of PV+ NeuT+ cells, but not NeuT+ cells, was reduced in Pcp2cre/+;Kir2.1 mice compared with controls (Supplementary Fig. 11b-d). The molecular layer was also thinner than in controls (Supplementary Fig. 11b, e). We next assessed MLI density in mice in which Purkinje cell activity was disrupted at P7, thereby sparing the first week of development from changes in activity (Supplementary Fig. 11f). The density of PV+ NeuT+ and NeuT+ MLIs, as well as the molecular layer thickness, were unchanged between groups (Supplementary Fig. 11g-j). Lastly, we analysed the granule cell layer thickness (GCL) as a proxy for GC numbers, and measured the size of vermal cerebellar lobules in Pcp2cre/+;Kir2.1 mice and P7-inducible Pcp2creER;Kir2.1 mice and their respective controls (Supplementary Fig. 11k). There was no difference in the GCL thickness of the cerebellar cortex between Pcp2cre/+;Kir2.1 or P7-inducible Pcp2creER;Kir2.1 mice and respective controls (Supplementary Fig. 11l, n). However, lobules I-II, IV-V, and IX were smaller in Pcp2cre/+;Kir2.1 mice (Supplementary Fig. 11m), while lobules IV-V and VI were reduced in the P7-inducible Pcp2creER;Kir2.1 mice compared to controls (Supplementary Fig. 11o). Together, these results indicate that early disruption of Purkinje cell intrinsic activity not only leads to more severe motor deficits but also interferes with the proper maturation of cerebellar circuitry and structure.

Early loss of Purkinje cell intrinsic activity activates gene networks underlying cerebellar disease
Our findings revealed that loss of Purkinje cell activity during the first week of development was sufficient to alter neuron development (Fig. 4a-d). To uncover the molecular mechanisms by which intrinsic activity influences Purkinje cell development, we isolated Purkinje cells from P7 Pcp2cre/+;Kir2.1 and control mice and compared their transcriptomics profiles using bulk RNA sequencing (RNAseq) (Supplementary Fig. 12a-c). Differential expression analysis identified 1602 differentially expressed genes (DEGs) (Fig. 6a). Gene ontology (GO) enrichment analysis of DEGs revealed a significant overrepresentation of biological processes related to synaptic signaling (e.g., Grid2, Snca), calcium ion transport (e.g., Trpc3, Itpr1), and regulation of nervous system development (e.g., Hes5, Sema6d) (Fig. 6b), reflecting the morphological and synaptic alterations observed in our model (Fig. 6b). Enriched cellular component terms included the postsynaptic density membrane, dendritic spines, presynaptic membrane and axon terminus, further supporting transcriptional dysregulation at the synaptic level (Supplementary Fig. 12d). At the molecular function level, most DEGs were associated with transmembrane transport activity and calcium channel activity, indicating altered calcium signaling mechanisms (Supplementary Fig. 12e). These data evidence that the transcriptional changes following Purkinje cell intrinsic activity suppression, converge on pathways essential for neuronal excitability, synaptic integrity, and cerebellar circuit development. To identify functional networks affected by the loss of Purkinje cell intrinsic activity, we performed KEGG pathway enrichment analysis on the DEGs. This analysis revealed significant enrichment for pathways associated with synaptic formation and function, including axon guidance, GABAergic, dopaminergic, and glutamatergic synapses. In addition, several intracellular signaling mechanisms were enriched, such as calcium, phosphatidylinositol, and retrograde endocannabinoid signaling, as well as long-term depression, underlying the broad impact of intrinsic activity on intracellular communication and synaptic plasticity. Notably, KEGG enrichment also identified disease-related pathways, including those associated with neurodegeneration, spinocerebellar ataxia, and Huntington’s disease, suggesting that early disruption of Purkinje cell activity engages molecular programs commonly implicated in cerebellar and neurodegenerative disorders (Fig. 6c). Consistent with these transcriptional alterations, loss of Purkinje cell intrinsic activity led to severe motor impairments in our mouse model. To explore disease-relevant transcriptional changes, we examined the KEGG Disease Database, which identified 216 genes linked to movement disorders with a cerebellar component, 25 of which overlapped with our DEG list (Fig. 6d and Supplementary Fig. 12f, g). These overlapping genes were key regulators of neuronal development, excitability, synaptic and mitochondrial function49, most of which were downregulated in our dataset (Fig. 6e).

Prkcg and Car8 regulate early Purkinje cell dendritic development
To investigate whether genes implicated in cerebellar disease also contribute to Purkinje cell development, we selected two candidates from our RNA-seq analysis for functional studies: Prkcg (PKC, protein kinase C gamma)50 and Car8 (CAR8, carbonic anhydrase VIII)51 (Fig. 6e). The selection criteria included: 1) fold-change in expression, 2) statistical significance, and 3) known involvement in cerebellar disease52, 53, 54 (Supplementary Fig. 12g). We performed Purkinje cell-specific in vivo gene knockdown using effective short-hairpin RNAs (shRNAs) targeting Prkcg (shPrkcg) and Car8 (shCar8), with shLacZ as a control (Supplementary Fig. 13a, c). Each shRNA was delivered via adeno-associated virus (AAVs) carrying an mCherry reporter gene (Supplementary Fig. 13e). AAVs were injected into the lateral ventricles of neonatal Pcp2cre/+ mice, and tissue was collected at P7 for analysis (Fig. 7a). In situ hybridization confirmed successful downregulation of Prkcg or Car8 in mCherry-positive Purkinje cells (Supplementary Fig. 13b, d). Morphological analyses revealed that Prkcg knockdown significantly increased dendritic complexity, area, and dendritic length, while reducing the number of neurites emerging from the soma compared to controls (Fig. 7b-g). These results indicate that PKC acts as a negative regulator of dendritic growth. In contrast, Car8 downregulation did not affect dendritic complexity or cell area (Fig. 7b-e). However, it reduced dendritic length and increased the number of neurites extending from the soma compared to controls (Fig. 7b, f, g), suggesting a delay in dendritic maturation similar to that observed in Kir2.1-overexpressing Purkinje cells. These findings implicate CAR8 in the transition from an immature multipolar morphology to a more mature, vertically oriented flattened dendrite.

Methods
Mouse strains
The following transgenic mouse lines were used in this study and maintained on a C57BL/6 background (Charles River Laboratories): Pcp2creER (Tg(Pcp2-creERT2)17.8.ICS)22, Ai14 (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J)24, Kir2.1 (Gt(ROSA)26Sortm2(CAG-KCNJ2/mCherry)Fmr)23 and Pcp2cre/+(B6.Cg-Tg(Pcp2-cre)3555Jdhu/J)118. Ai14 (Jackson Laboratories #007908) and Kir2.1 (kindly provided by Guillermina López-Bendito) mouse lines were maintained as homozygous and crossed with cre-driver lines, resulting in progeny that were always heterozygous for Ai14 and Kir2.1. Ai14 and Kir2.1 mice were crossed with inducible Pcp2creER mice to drive tdTomato or Kir2.1-mCherry expression in Purkinje cells, respectively, following tamoxifen administration. Tamoxifen (Sigma-Aldrich, Cat. #T5648, 20 mg/ml in corn oil) was administered subcutaneously or intraperitoneally to induce Cre recombination. The Ai14 and Kir2.1 lines were also crossed with Pcp2cre/+ mice to conditionally express tdTomato or Kir2.1 in all Purkinje cells, respectively. Both male and female mice were used in all experiments. For in utero electroporation, time-mated FVB/NHsd (Envigo) female mice were used and housed individually. The day of the vaginal plug detection was designated as embryonic day (E) 0.5. All animals were maintained under standard temperature-controlled laboratory conditions on a 12 h:12 h light/dark cycle, with water and food available ad libitum. All procedures were approved by the Dutch Ethical Committee for Animal Experiments and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Erasmus Medical Center, and with the European and Dutch National Legislation.

Tamoxifen protocols
Tamoxifen dosing and timing were adjusted to achieve either sparse or complete Purkinje cell labeling at different developmental stages, driving the expression of tdTomato or Kir2.1-mCherry, in Pcp2creER;Ai14 and Pcp2creER;Kir2.1, respectively. Protocols were designed with consideration of the developmental onset and pattern of Pcp2 expression. Sparse labeling, which allows visualization of individual cell morphology, was achieved by administering tamoxifen at 100 mg/kg body weight at postnatal day (P) 1.5 mg/kg at P7, and 1 mg/kg at P14. For recombination in all Purkinje cells, in short-term studies, 100 mg/kg tamoxifen was administered at P7 and 50 mg/kg at P14 to label Purkinje cells over a defined one-week window. For long-term recombination extending into adulthood, Pcp2creER;Kir2.1 mice and their control littermates received tamoxifen at 100 mg/kg for three consecutive days at P7, P14, or P21. To achieve recombination in all Purkinje cells from birth while avoiding multiple injections in neonatal pups, constitutive Pcp2cre/+;Kir2.1 mice and their control littermates were used, ensuring expression of tdTomato or Kir2.1-mCherry in all Purkinje cells from the earliest developmental stages. An overview of all experimental designs is provided in Supplementary Fig. 14.

Ex vivo Purkinje cell recordings and analysis
Ex vivo slice recordings of Purkinje cells were performed on cerebellar slices obtained from a total of 52 mice at three developmental ages: P7, P14, and P21 (± 1 day). Cerebellar tissue was collected from Pcp2creER;Ai14 or Pcp2creER;Kir2.1 mice that had been injected with tamoxifen at P1, P7, or P14 to induce sparse expression of tdTomato or Kir2.1-mCherry, respectively, in Purkinje cells. Brains were rapidly removed and placed in an ice-cold slicing solution continuously equilibrated with 95% O2 and 5% CO2, containing (in mM): 240 Sucrose, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 1 CaCl2, 26 NaHCO3, and 10 D-glucose. Sagittal vermal cerebellar slices (250 µm thick) were obtained in ice-cold slicing solution using a vibratome (VT1000S, Leica Biosystems, Wetzlar, Germany) equipped with a ceramic blade (Campden Instruments Ltd, Manchester, United Kingdom). Slices were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) at 34ºC for one hour before being transferred to a recording chamber. The chamber was maintained at 34ºC using a feedback temperature controller (Scientifica, Uckfield, United Kingdom) and continuously perfused with oxygenated aCSF containing (in mM): 124 NaCl, 5 KCl, 1.25 Na2HPO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 20 D-glucose21.

For all the recordings, the aCSF was supplemented with synaptic receptor blockers to isolate the intrinsic activity of Purkinje cells. The following antagonists were included: the NMDA receptor antagonist D-AP5 (50 µM), the selective and competitive AMPA receptor antagonist NBQX (10 µM), and the non-competitive GABA receptor antagonist and glycine receptor inhibitor picrotoxin (100 µM; all from Hello Bio Ltd, Bristol, United Kingdom). For some recordings, the aCSF was additionally supplemented with 300 µM barium (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Purkinje cells were selected based on their fluorescent labeling: tdTomato expression in Pcp2creER;Ai14, absence of mCherry in Pcp2creER;Kir2.1,Ctl, and mCherry expression in Pcp2creER;Kir2.1. Neurons were visualized using a SliceScope Pro 3000 microscope, with a CCD camera, a trinocular eyepiece (Scientifica, Uckfield, UK), and an ocular (Teledyne Qimaging, Surrey, Canada).

Whole-cell recordings were obtained using borosilicate pipettes (Harvard apparatus, Holliston, MA, USA) with a resistance of 4-6 MΩ, filled with an internal solution containing (in mM): 9 KCl, 3.48 MgCl2, 4 NaCl, 120 K+-Gluconate, 10 HEPES, 28.5 Sucrose, 4 Na2ATP, and 0.4 Na2GTP, adjusted to pH 7.25-7.35 and osmolarity 290-300 mOsmol/Kg (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Whole-cell recordings were performed on Purkinje cells held at -65 mV. Before establishing the whole-cell recording configuration, capacitance was optimally compensated. Input resistance was determined using the seal test protocol integrated into PatchMaster software (HEKA Elektronik). Immediately after achieving the giga-ohm seal (>1 GΩ), a test pulse was applied through PatchMaster (a 10 mV hyperpolarizing voltage step for 10–20 ms) to assess passive membrane properties without activating voltage-gated currents. Membrane resistance (Rm) was calculated by subtracting the series resistance (Rs) from the total input resistance (Rin) (Rm = Rin - Rs). Recordings with a series resistance greater than 25 MΩ were excluded. After achieving whole-cell configuration, zero current was injected in current-clamp mode to assess intrinsic action potential firing and resting membrane potential, defined as the mode of the voltage trace. Intrinsic excitability was tested by holding the cells at -65 mV using a holding current, which was then labeled as ‘0 pA’. From this holding current, 500 ms current steps increasing by 100 pA for every step were applied, and the number of action potentials in the resulting voltage trace was counted. The current-voltage (I-V) curves were generated in voltage-clamp mode by applying 500 ms voltage steps from -140 mV to 0 mV in 10 mV increments and measuring the mode of the resulting current. Recordings were acquired with Patchmaster (HEKA Electronics, Lambrecht, Germany) using an ECP-10 amplifier (HEKA Electronics, Lambrecht, Germany) and digitized at 20 kHz. Data files were imported into and analyzed with a custom-built MATLAB code (Mathworks, Natick, MA, USA).

In vivo extracellular recordings and analysis
In vivo extracellular recordings of Purkinje cells and cerebellar nuclei neurons were performed in juvenile (P28 ± 3 days; n = 10) and adult (P60-P90) (n = 9) Pcp2cre/+;Kir2.1 mice and their control littermates. Mice were anesthetized with isoflurane in oxygen (4 % for induction and 1.5–2 % for maintenance). The scalp was shaved and incised along the rostrocaudal midline, and the medial neck muscles overlaying the occipital bone were removed. Next, a craniotomy was made in the occipital bone using a high-speed diamond-tipped drill (Kulzer, #H71104003). A custom-made recording chamber (Charisma Flow Row composite, #66095845) was positioned around the craniotomy and temporarily sealed with bonewax (Ethicon, W30) to protect the exposed brain. Then, a custom-built metal pedestal with a square magnet was fixed to the frontal and parietal bones to allow for head fixation during recordings. Mice were allowed to recover for three days post-surgery. On the day of recording, mice were head-fixed in a mouse holder, and the bonewax was removed to expose the cerebellar surface for extracellular recordings25.

Extracellular activity was recorded using borosilicate glass pipettes (World Precision Instruments, BF200-116-10) pulled to have a long, thin taper and a resistance of 2-4 MΩ, filled with 2 M NaCl solution. One silver chloride electrode wire was inserted into the pipette to record electrical activity, and a second silver chloride electrode wire was placed in the saline-filled recording chamber as a reference electrode. Pipettes were advanced into the cerebellar cortex or nuclei using a micromanipulator (SM-5, Luigs & Neumann, Ratingen, Germany). Signals were pre-amplified (custom-made preamplifier, 1000x DC), filtered (CyberAmp 320, Axon Instruments, Molecular Devices, Sunnyvale, CA, USA), digitized (Power1401, CED, Cambridge, UK), and stored for offline analysis. To histologically verify the recording site of cerebellar nuclei neurons, at the end of some recording sessions, 0.5 % Evans Blue solution was injected at the recording location with a micropipette (Hirschmann, Z611239-250EA). Recordings from Purkinje cells and cerebellar nuclei neurons were analyzed using SpikeTrain software (Neurasmus BV, Rotterdam, The Netherlands), running in MATLAB 2014a (MathWorks, Natick, MA, USA). SpikeTrain employs wave-clustering algorithms to discriminate simple and complex spikes. Purkinje cells were identified by the presence of both simple and complex spikes and confirmed as single units by the characteristic pause in simple spike firing following each complex spike. For each neuron and spike type, the firing rate and the coefficient of variation (CV) 2 were calculated. The CV2 measures local, spike-to-spike variability in firing pattern and was calculated as 2 × |ISI n+1 - ISI n|)/(ISI n+1 + ISI n)119 where ISI = inter-spike intervals.

Immunohistochemistry
Mice were deeply anesthetized with sodium pentobarbital by intraperitoneal injection and transcardially perfused with sodium chloride solution, followed by 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4). Dissected brains were post-fixed for 2 h at 4°C in 4 % PFA and cryoprotected overnight at 4°C in 30 % sucrose/0.1 M PB. The next day, brains were embedded in 14 % gelatin/ 30 % sucrose/0.1 M PB solution, fixed for 2 h at room temperature, and incubated overnight at 4°C in 30 % sucrose/0.1 M PB. Sagittal or coronal brain sections (50 µm) were cut using a freezing microtome.

Free-floating sections were rinsed in 0.1 M PB and subjected to antigen retrieval by incubation for 2 h in 10 mM sodium citrate solution at 80°C. Sections were then rinsed and incubated for 2 h at room temperature in a blocking solution containing 0.5 % Triton X-100 and 10 % normal horse serum in 0.1 M PB to prevent nonspecific binding. Subsequently, sections were incubated overnight at 4°C with primary antibodies diluted in antibody solution (0.5 % Triton X-100 and 2 % normal horse serum in 0.1 M PB).

The following primary antibodies were used: rabbit anti-RFP (1:1000, Rockland, #600-401-379), chicken anti-RFP (1:1000, Rockland, #600-901-379), goat anti-GFP (1:1000, Rockland, #600-101-215), mouse anti-calbindin D-28K (1:10000, Swant, #CB300), guinea pig-VGAT (1:500, Synaptic Systems, #131004), guinea pig-VGLUT1 (1:2000, Merck, AB5905), guinea pig-VGLUT2 (1:2000, Merck, #AB2251-I), mouse-parvalbumin (1:5000, ThermoFisher Scientific, MA5-47410), and rabbit-NeuN (1:1000, Millipore, #ABN78). Sections were rinsed in 0.1 M PB and incubated for 2 h at room temperature with secondary antibodies: Cy3-AffiniPure Donkey anti-Rabbit (1:1000, #711-165-152), Cy3-AffiniPure Donkey anti-Chicken (1:1000, #703-165-155), Alexa Fluor 488-AffiniPure Donkey anti-Goat (1:1000, #705-545-147), Alexa Fluor 488-AffiniPure Donkey anti-Mouse (1:1000, #715-545-150), Cy3-AffiniPure Donkey anti-Mouse (1:1000, #715-165-150), Alexa Fluor 488-AffiniPure Donkey anti-Guinea Pig (1:1000, #706-545-148), Alexa Fluor 647-AffiniPure Donkey Anti-Mouse (1:1000, #715-605-151), and Cy5-AffiniPure Donkey anti-Rabbit (1:1000, #711-175-152) all from Jackson ImmunoResearch.

Finally, sections were counterstained with the fluorescent nuclear dye 4′,6-diamidino-2-phenylindole (DAPI, Thermo Scientific, #D3571) and mounted with Mowiol (Polysciences, Inc., #17951). Sections used for molecular layer interneuron quantification were counterstained with NeuroTrace 435/455 Blue Fluorescent Nissl Stain (1:300, ThermoFisher Scientific, N21479).

Image acquisition and analysis
Images were acquired at a resolution of 1024 x 1024 pixels and 8-bit depth using either an LSM 700 or LSM 900 confocal laser scanning microscope, and an Axio Imager.M2 epifluorescence microscope (Carl Zeiss Microscopy, LLC, USA). For each experiment, all imaging parameters, including laser power, detection filter settings, pinhole size, and photomultiplier gain, were kept constant across samples.

To image individual Purkinje cells, sagittal sections were acquired using a 20x/0.8 NA or 40x/1.3 NA oil-immersion objective with a z-step of 1 µm. Given the morphological variability of Purkinje cells across cerebellar regions21, cells were imaged throughout the cerebellar cortex and plotted individually. Dendrite complexity was quantified from maximum intensity projections of z-stack images using the Sholl analysis macro in FIJI (ImageJ) software120. To quantify Purkinje cell area excluding the axon, the maximum projection of each image was thresholded in FIJI, and the area was then measured. The longest dendrite length was obtained as a result of Sholl analysis. The molecular layer (ML) ratio was calculated as the percentage of Purkinje cell height (the distance from the top of the Purkinje cell soma to the apical edge of the neuron) divided by the molecular layer height. Neurite number was measured as the number of processes originating from the soma of a Purkinje cell.

To image calbindin-positive (+) and VGAT+ presynaptic puncta, coronal cerebellar sections were acquired using a 63X/1.4NA oil-immersion objective with a z-interval of 1 µm. For quantification of presynaptic densities, the perimeter of NeuN+ cerebellar nuclei neuron soma was measured using FIJI. Presynaptic puncta calbindin+ and VGAT+ were automatically detected using the Find Maxima function to count the number of puncta colocalizing with the soma perimeter. The number of calbindin+ signal colocalizing with VGAT+ puncta was normalized to the corresponding soma perimeter of each analyzed cell. Given the diversity in soma size among cerebellar nuclei neurons121, 122, data were plotted individually.

Tile-scan images of Purkinje cells and VGLUT2 puncta were acquired with a 40X/1.3NA oil-immersion objective with a z-interval of 0.8 µm. Maximum intensity projections of sagittal sections were used for quantification. The molecular layer thickness was measured as the distance from the top of a Purkinje cell soma to the apical edge of the molecular layer. Climbing fiber (CF) height was measured as the distance from the top of a Purkinje cell soma to the most distal VGLUT2 puncta. Molecular layer thickness and CF height were determined from three measurements per image. CF extension was quantified as the percentage of the CF height relative to molecular layer thickness. VGLUT2 density was quantified by counting VGLUT2 puncta within a specific region of interest (ROI) using the ‘Analyze Particles’ function in FIJI, and normalizing the count to the ROI area. To quantify VGLUT2 perisomatic puncta, the perimeter of individual Purkinje cell soma was measured, and VGLUT2 puncta colocalizing with the soma perimeter were counted, excluding those inside the soma. Measurements were performed from three Purkinje cells per image, and the number of VGLUT2 puncta was normalized to the soma perimeter of each analyzed cell.

Images of VGLUT1 puncta were acquired using a 63X/1.4NA oil-immersion objective, with a scan zoom of 2 and a z-interval of 0.5 µm. Maximum intensity projections of sagittal sections near the apical edge of the molecular layer were used for quantification. VGLUT1 puncta were identified using the Trainable Weka Segmentation (v4.0.0) plugin123. Both the number and size of VGLUT1 puncta were quantified, and VGLUT1 density was calculated as the number of puncta per ROI area.

Molecular layer interneurons (MLIs) stained with NeuroTrace and parvalbumin (PV) were imaged using a 20X/0.8NA objective with a z-interval of 1 µm. Maximum intensity projections of sagittal sections were analyzed using a macro implemented in FIJI. The number of NeuroTrace+, PV+, and colocalized NeuroTrace+ PV+ cells was counted. MLI density was calculated as the number of neuronal cells per ROI area.

Images of sagittal cerebellar sections were acquired using an Axio Imager.M2 epifluorescence microscope with a 10x/0.45 NA objective. Granule cell layer (GCL) thickness was measured at two distinct positions within each lobule: at the base and in the middle region. At each position, two independent measurements were taken, defined as the distance from the bottom of the Purkinje cell soma to the white matter. The mean value of these measurements was used for subsequent analysis. The area of each cerebellar lobule was determined by manually delineating lobule boundaries and measuring the enclosed area using FIJI.

In utero electroporation
Timed-pregnant FVB/NHsd females at E12.5 were deeply anesthetized with isoflurane (5% induction, 2% maintenance) in oxygen at a flow rate of 1 l/min, and body temperature was maintained at 37°C. Buprenorphine (0.1 mg/kg) was administered subcutaneously for analgesia. The abdominal cavity was then opened, and the uterus was carefully exposed to access the embryos. A beveled glass micropipette was used to inject a combination of 3 μg/μl of pCAG-GFP (Addgene; #11150; a gift from C. Cepko124) and 1 μg/μl of pCAG-Kir2.1-T2A-tdTomato (Addgene; #60598; a gift from M. Scanziani125) with 0.05% of FastGreen into the fourth ventricle of each embryo. Electroporation was performed by delivering five pulses of 35 V for 50 ms at 900 ms intervals using a 2 mm tweezertrode (CUY650P2, Nepa Gene) connected to an electroporator (BTX, ECM830). The electrodes were positioned to target Purkinje cell progenitors in the cerebellar ventricular zone. After electroporation, the uterus was placed back into the abdominal cavity, and the abdominal wall and skin were sutured. The female was monitored until complete recovery.

Balance beam test
All behavioral tests were performed on adult mice (P60-P90).
Mice were tested on a 1-meter-long bean with a flat surface, 12 mm wide, elevated 50 cm above the surface between two platforms. A cage was placed at the end of the beam as a finish point. The test consisted of two training days followed by a testing day, with three trials per day. Trials in which a mouse fell five consecutive times were considered completed. Motor coordination was determined by averaging the time taken to cross the beam and the number of missteps per run126.

Accelerating rotarod test
Mice were tested on an accelerating rotarod over five consecutive days, with four non-consecutive trials per day. On days 1 to 4, the rotating rod was accelerated to 40 rpm, and on day 5, the maximum speed was increased to 80 rpm. Mice were given an hour to rest between trials. A trial was considered complete when the mouse reached a maximum latency of 300 s, fell off the rod, or rotated three consecutive times. Motor coordination was quantified by averaging the latency to fall across trials on the accelerating rod (Ugo Basile Biological Research Apparatus).

LocoMouse test
The LocoMouse test was performed to assess whole-body coordination during overground locomotion30. Mice crossed a glass corridor (66.5 cm long, 4.5 cm wide, and 20 cm high) between two dark boxes. A mirror (66 cm × 16 cm) was placed below the corridor at a 45˚ angle to allow simultaneous recording of side and bottom views. Side and bottom views of walking behavior were recorded using a high-speed camera (Basler, 1440 x 250 pixels, 400 fps). The test consisted of two consecutive habituation days followed by three consecutive experimental days. During habituation, mice walked freely between the two dark boxes for 15 min. On each experimental day, 15 trials per animal were collected; each trial consisted of single corridor crossing, and the animals were not required to walk continuously throughout a trial. A DeepLabCut network was trained using the side camera view of the LocoMouse to track the paws, nose, and tail base positions of each individual mouse127. Data were processed using custom-written Python (v3.7) code available at GitHub (GitHub, San Francisco, CA, USA, https://github.com/BaduraLab/DLC_analysis)31.

Open field test
The open field test was performed in a white-opaque polypropylene square arena (50 x 50 x 40 cm) under uniform lighting. Each animal was placed in the center of the arena and allowed to explore freely for 10 min. A digital video system recorded the overall activity inside the box (Carl Zeiss Tessar 2.0/3.7 2MP V-UBM46 Autofocus Camera). Data were analyzed using OptiMouse, an open-source MATLAB program128, which automatically calculated the total distance traveled and speed to evaluate spontaneous locomotor activity.

Compensatory eye movements
Mice were surgically prepared for head-restrained recordings by placing a custom-built metal pedestal with a square magnet on the frontal and parietal bones under general anesthesia with isoflurane/O2. Three days after recovery, mice were head-fixed in a holder at the center of a turntable (60 cm diameter), surrounded by a cylindrical screen (63 cm diameter) with a random-dotted pattern (drum). Eye movements were recorded with a CCD camera fixed to the turntable, and eye-tracking software was used (ETL-200, ISCAN Systems, Burlington, NA, USA). Eyes were illuminated with two table-fixed infrared emitters and a third emitter attached to the camera, which produced a tracked corneal reflection used as the reference point. The recording camera was calibrated by moving it left-right 20° peak-to-peak while the eye remained stationary129. Gain and phase values of eye movements were calculated using custom-made MATLAB scripts, available at GitHub (GitHub, San Francisco, CA, USA, https://github.com/MSchonewille/iMove;130). Mice were familiarized with the experimental setup three days before the experiments by providing visual and/or vestibular stimulations for 30 min. Compensatory eye movements were evoked using a sinusoidal drum rotation in light (optokinetic reflex-OKR), turntable rotation in dark (vestibular ocular reflex-VOR), or turntable rotation in light (visual VOR-VVOR) with 5° amplitude at 0.1–1 Hz. To assess motor learning, mice were subjected to a mismatch between visual and vestibular input to adapt the VOR. The ability to perform VOR phase reversal was tested over five consecutive days, with six 5 min training sessions per day and VOR recordings before, between, and after each training session. Mice were kept in the dark between recording sessions to avoid unlearning of the adapted responses. On the first training day, the visual (drum) and vestibular (turntable) stimuli rotated in phase at 0.6 Hz, each with an amplitude of 5°, resulting in a decrease in gain. In the following days, the drum amplitude was increased to 7.5° (day 2) and 10° (days 3, 4, and 5), while the turntable amplitude remained at 5°. This led to a reversal of the VOR direction, an inversion of the compensatory eye movement driven by vestibular input, moving the eye in the same direction as the head rotation rather than the normal compensatory opposite direction. Motor performance in response to these stimulations was measured by calculating the response gain (ratio of eye movement amplitude per stimulus amplitude) and phase (difference between eye and stimulus in degrees)21.

Eyeblink conditioning
A custom-built metal pedestal with a square magnet on top was placed on the frontal and parietal bones of a mouse under general anesthesia with 2% isoflurane/O2. Three days after recovery, mice were head-fixed on a foam-cylindrical treadmill, on which they could walk freely inside custom-built sound- and light-attenuating boxes. Eyelid movements were monitored under infrared illumination using a high-speed (333 fps) monochrome Basler video camera (Basler ace 750-30 gm). Stimuli and measurement devices were controlled by National Instruments hardware and custom-written LabVIEW software. The conditioned stimulus (CS) was a blue LED light (duration 280 ms, diameter 5 mm) placed 10 cm in front of the mouse. The unconditioned stimulus (US) consisted of a 30 ms mild corneal air puff, controlled by a VHS P/P solenoid valve with a back pressure of 30 psi (Lohm rate, 4750 Lohms; Internal volume, 30 µl, The Lee Company®, Westbrook, USA) and delivered via a 27.5 mm gauge needle perpendicularly positioned at approximately 5 mm from the center of the left cornea. Prior to the experiments, mice were habituated to the set-up for one day (two 30 min sessions spanning 6 h) with no stimuli delivered. To assess motor learning, mice were subjected to eyeblink conditioning training for 5 consecutive days, with two sessions per day separated by 6 h of rest. Before the first training session, a baseline session was conducted to confirm that the CS did not elicit any reflexive eyelid closure. This baseline session consisted of 15 CS-only trials and 3 US-only trials. Immediately after this, the training sessions began. During each session, every animal received a total of 200 paired CS-US trials, 20 US-only trials, and 20 CS-only trials. These trials were presented across 20 blocks, with each block consisting of 1 US-only trial, 10 paired CS-US trials, and 1 CS-only trial. The interval between CS onset and US onset was 250 ms. All experiments were performed at approximately the same time of the day by the same experimenter. Individual eyeblink traces were analyzed with a custom-written MATLAB script available at GitHub (GitHub, San Francisco, CA, USA, https://github.com/francescafiocchi91/Eyeblink_Conditioning36) (R2018a, Mathworks). Eyeblink traces (2000 ms) were imported from a MySQL database into MATLAB and aligned at zero for the 500 ms pre-CS baselines. Trials with significant activity in the 500 ms pre-CS period (> 7 times the interquartile range) were considered invalid and disregarded for further analysis. The eyelid signal was min-max normalized so that a fully open eye corresponded to a value of 0 and a fully closed eye to a value of 1131. In valid normalized CS-only trials, eyelid responses were considered as conditioned responses (CR) if the maximum amplitude was larger than 0.10 between 100-500 ms after CS onset and showed a positive slope in the 150 ms before the time point of the expected US delivery (US is omitted in CS-only trials)132, 133.

Purkinje cell collection for RNA sequencing
To isolate Purkinje cells, the cerebellar tissue was extracted from either P7 Pcp2+/+;Kir2.1 or P7 Pcp2cre/+;Kir2.1 mice. Sagittal slices of vermal cerebellar tissue were obtained and maintained as described in the ex vivo Purkinje cell recordings section. For cell collection, individual slices were transferred to a recording chamber and maintained at 34C under continuous perfusion with equilibrated aCSF. In P7 Pcp2cre/+;Kir2.1 slices, Purkinje cells were selected based on Kir2.1-mCherry expression. Neurons were extracted into a glass pipette using negative pressure, and 50 cells were sampled across the cerebellar cortex. Eight biological samples per genotype were collected for RNA sequencing. Cells were harvested in a mild hypotonic lysis buffer containing 0.2% Triton X and 2 U/µl RNase inhibitor, kept on ice during collection, and either processed immediately or stored at -80°C until RNA extraction.

cDNA library preparation and RNA sequencing
Library preparation and RNA sequencing experiments were performed at the Erasmus Center for Biomics (Erasmus MC, Rotterdam, The Netherlands). For each biological replicate, cDNA libraries were generated using the Smart-seq2 method134. Briefly, cells were lysed, and poly(A)+ RNA was reverse-transcribed using an oligo(dT) primer. Template switching during reverse transcription was achieved using a locked nucleic acid-containing template-switching oligonucleotide. The reverse-transcribed cDNA was preamplified with primers for 18 cycles, followed by cleanup. Tagmentation was performed on 1 ng of the pre-amplified cDNA using the Illumina Nextera DNA Flex kit. The tagmented library was extended with Illumina adaptor sequences by PCR for 12 cycles and purified. The resulting sequencing library was measured on a Bioanalyzer and equimolarly loaded onto a flow cell for sequencing according to the Illumina TruSeq v3 protocol on the Illumina HiSeq 2500 platform, with a single read of 50 base pairs and dual 9-base-pair indices.

Bioinformatic analysis of RNA sequencing
Illumina adapter sequences and poly-A stretches were trimmed from the reads prior to mapping to the mouse reference sequence GRCm38 (mm10) using HISAT2 (version 2.1.0). From the alignments, the reads per gene were determined using htseq-count (version 0.11.2). Gene and transcript annotations were retrieved from Ensembl (build 101) using consensus coding sequences for each gene. Count data were analyzed using RStudio (version 0.99.484) following the edgeR-limma package workflow135. Briefly, Ensembl IDs were annotated to Entrez gene IDs, gene symbols, gene names, and chromosome locations using the Mus.musculus package. Raw counts were transformed into counts per million (CPM) and log2-CPM, and genes with a CPM below 0.2 in at least three samples were filtered out. Normalization of gene expression distributions was performed using the Trimmed Mean of M-values method.

Differentially expressed genes (DEGs) between Purkinje cells from Pcp2+/+;Kir2.1 and Pcp2cre/+; Kir2.1 mice were visualized using a hierarchical clustering heatmap with normalized row z-scores in R. To identify biologically significant genes, data were organized in a volcano plot with a fold change cutoff of 0.5 and a –log10 (false discovery rate; FDR) cutoff of 1.3. Gene ontology (GO) analyses were performed using the Gene Ontology tool (http://pantherdb.org/), and KEGG enrichment analyses were performed using the clusterProfiler package (version 4.14.6)136. DEGs were selected for GO and KEGG enrichment analysis. The complete gene set identified from the biological samples was used as the reference list, and GO terms with corrected P ≤ 0.05 were considered significantly enriched. GO term visualization was generated using the ggplot2 package (version 3.5.1), and network visualizations using the igraph package (version 2.1.4)137. A list of 216 human movement disorder-associated genes was extracted from the KEGG Disease database (https://www.genome.jp/kegg/disease/), which compiles known genetic and environmental contributors to human diseases. This list was compared to the 1602 DEGs identified from RNA sequencing, revealing 25 overlapping genes. The shared genes were ranked by -log10 (FDR) and log fold change, Prkcg and Car8 emerging as the top hits.

Single-molecule fluorescence in situ hybridization
Mice were perfused as described above, and brains were postfixed for 2 h at 4°C before being cryoprotected in 30% sucrose/0.1 M PB overnight at 4°C. Sagittal sections 30 µm thick were obtained using a freezing microtome, mounted on RNAse-free SuperFrost Plus slides (Epredia), and probed against the candidate genes according to the manufacturer's protocol. Single-molecule fluorescence in situ hybridization was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio, #323100) with probes targeting Car8 (Mm-Car8-C2, #514171) and Prkcg (Mm-Prkcg-C2, #417911). Sections were co-immunostained with rabbit anti-RFP (1:1000, Rockland, #600-401-379) and Cy3-AffiniPure Donkey anti-Rabbit (1:1000, Jackson ImmunoResearch, #711-165-152).

Cell culture and transfection
HEK293T cells were cultured in Dulbecco’s Modified Eagle’s medium supplemented with high glucose, Glutamax, and pyruvate (DMEM; ThermoFisher Scientific, #31966-047). In addition, 10% fetal bovine serum, 1% of penicillin (10,000 U/ml), and of streptomycin (10,000 µg/ml). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. HEK293T cells were plated in 6-well plates at a density of 0.5 x 106 cells/well and transfected the following day with plasmids expressing the full-length genes of interest and respective shRNA-expressing plasmids at a 1:3 ratio using Lipofectamine 2000 (ThermoFisher Scientific, #11668019). The plasmids used to express Prkcg and Car8 were pcDNA3.1+/C-(K)DYK-Prkcg (GenScript, OMu17867) and pcDNA3.1+/C-(K)DYK-Car8 (GenScript, OMu00430), respectively. shRNA sequences targeting Prkcg and Car8 were designed by The RNAi Consortium (TRC) and obtained from Horizon Discovery. The antisense sequence and respective clone IDs were as follows: Prkcg shRNA1 5’-TTG ATG GCA TAG AGT TCG TCG-3’ (TRCN0000022684), Prkcg shRNA2 5’-TAA TAT GGA TCT CAT CCG ACG-3’ (TRCN0000022685), Prkcg shRNA3 5’-ATA GGC AAT GAT CTC AGG TGC-3’ (TRCN0000022686), Prkcg shRNA4 5’-TTC ACA TAA GTA AAG CCC TGG-3’ (TRCN0000022687), Prkcg shRNA5 5’-AAA CGA GCG GTG AAC TTG TGG-3’ (TRCN0000022688), Car8 shRNA1 5’-AAT ATC CAG GTA ACT CCT TCG-3’ (TRCN0000114512), Car8 shRNA2 5’-TTT AGG TTG ATA GGT GAC TGG-3’ (TRCN0000114513), Car8 shRNA3 5’-TAG CAT CAG GAA ACA CTA AGC-3’ (TRCN0000114514) and Car8 shRNA4 5’-ATC TGG GAT ATA GTT AAG GGG-3’ (TRCN0000114515). A non-targeting control shRNA against lacZ 5’-AAA TCG CTG ATT TGT GTA GTC-3’ was used as a negative control.

Western blotting
HEK293T cells were collected 24 h after transfection in lysis buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured, and equal amounts of proteins were separated by SDS-PAGE in Criterion TGX Stain-Free Gels (Bio-Rad) and transferred onto nitrocellulose membranes using the Trans-Blot Turbo Blotting System (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline (TBS)-Tween (20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1%, Tween 20) for 1 h at room temperature and incubated overnight at 4C with the following primary antibodies: rabbit anti-DYKDDDDK-tag (1:2000, GenScript, #A00170) and mouse anti-actin (1:1000, Millipore, #MAB1501). After washing, membranes were incubated for 1 h at room temperature with the following secondary antibodies: goat anti-Rabbit Immunoglobulins/HRP (1:10000, Agilent Dako, #P0448) or goat anti-Mouse Immunoglobulins/HRP (1:10000, Agilent Dako, #P0447). Protein bands were detected by the luminol-based enhanced chemiluminescence method (SuperSignal West Femto Maximum Sensitivity Substrate or SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific). Membranes were stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) when necessary. Densitometry analyses of protein bands were normalized to actin levels using the Image Studio Lite software (LI-COR Biosciences).

Generation of DNA constructs
DNA vectors were generated using standard molecular biology procedures. To produce viral vectors expressing shRNA for the target genes, pairs of oligonucleotides were designed, obtained (Integrated DNA Technologies) and hybridized for shlacZ (strand 1: 5’-cta ggA AAT CGC TGA TTT GTG TAG TCT GAT ATG TGC AGA CTA CAC AAA TCA GCG ATT TTT TTTg-3’and strand 2: 5’-aat tca aaaa AAA TCG CTG ATT TGT GTA GTC TGC ACA TAT CAG ACT ACA CAA ATC AGC GAT TTc-3’), shPrkcg (strand 1 5’-cta ggT AAT ATG GAT CTC ATC CGA CGC TCG AGC G TC GGA TGA GAT CCA TAT TAT TTTTg-3’and strand 2 5’-aat tca aaaa TAA TAT GGA TCT CAT CCG ACG CTC GAG CGT CGG ATG AGA TCC ATA TTAc-3’) and shCar8 (strand 1 5’-cta ggT TTA GGT TGA TAG GTG ACT GGC TCG AGC CAG TCA CCT ATC AAC CTA AAT TTTTg-3’and strand 2 5’-aat tca aaaa TTT AGG TTG ATA GGT GAC TGG CTC GAG CCA GTC ACC TAT CAA CCT AAAc-3’). Each double-stranded oligonucleotide was subsequently cloned into the pDIO-DSE-mCherry-PSE-MCS vector (Addgene; #129669; a gift from B. Rico138) using EcoRI and AvrII restriction sites.

Generation of viral particles
The adenovirus-associated virus (AAV) preparation AAV-FLEX-GFP was commercially acquired (Addgene; #28304-PHPeB; a gift from E. Boyden). HEK293T cells cultured in DMEM (with 10% fetal calf serum and penicillin/streptomycin) were co-transfected with AAV2/9 serotype helper plasmid, pAdΔF6 helper plasmid, and the pAAV-shRNA construct using polyethylenimine (PEI; 25 kDa, linear). Seventy-two hours after transfection, cells were harvested, lysed, and centrifuged to remove cellular debris. AAV particles were purified from the supernatant using an iodixanol density gradient and subsequently concentrated in 5% sucrose/PBS using an Amicon Ultra-15 centrifugal filter. Viral titers were determined by quantitative PCR targeting the mCherry reporter sequence present in the viral genome (vg). The mCherry primers used were: 5′-tcc cac aac gag gac tac ac-3′ and 5′-ctt gta cag ctc gtc cat gc-3’. The final viral titers ranged from 5 x 1012 to 2 x 1013 vg/ml139.

Intraventricular viral injection in neonates
For intraventricular injections, P0 Pcp2cre/+ were cryo-anesthetized with ice for 1 min before injection. Pups were placed in a custom-made platform, and a solution of AAVs diluted 1:100 in sterile NaCl containing 0.05% FastGreen was injected bilaterally into the lateral ventricles using a Nanoject III (Drummond Scientific Company). The injection site was located at approximately two-fifths of the distance between the lambda and each eye, and 1 μl of viral solution was injected into each lateral ventricle. After injection, pups were placed on a 37 °C warming pad to recover from anesthesia, and subsequently returned to the mother140.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA, USA; www.graphpad.com), MATLAB (MathWorks, Natick, MA, USA), and R software. Data normality was assessed using the Shapiro–Wilk test, and equality of variances was tested using the F-test. Statistical comparison between two normally distributed groups was performed using a two-tailed unpaired Student’s t-test, or a two-tailed Mann-Whitney U test for non-parametric data. For comparisons among more than two groups, one-way or two-way ANOVA was used for normally distributed data, while the Kruskal-Wallis test or a mixed-effects model was applied for non-parametric data. Statistical significance was defined as p < 0.05.

Data availability
Sequencing data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under the accession number GSE294208 and are publicly available. Source data are provided with this paper.

Code availability
This study did not generate original codes. For LocoMouse, Compensatory Eye Movements and Eyeblink Conditioning analyses the codes we previously reported and deposited on GitHub: https://github.com/BaduraLab/DLC_analysis31, https://github.com/MSchonewille/iMove130 and https://github.com/francescafiocchi91/Eyeblink_Conditioning36, respectively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors aimed to study how a Purkinje cell-specific reduction of intrinsic activity, achieved through the\noverexpression of the inward rectifier Kir2.1 channel, affects Purkinje cell (PC) development and cerebellar-dependent\nbehaviors in adult mice. Using a conditional Pcp2creERT2 strain of mice, they demonstrated that a single dose of tamoxifen\nadministered at P1 led to PC hyperpolarization and morphological alterations in PC dendrites. In non-conditional\nPcp2cre;Kir2.1 mice, they identified synaptic alterations at PC-DCN contacts and showed that adult mice exhibited\nimpairments in specific cerebellar learning tasks, such as VOR adaptation and eyeblink conditioning. The authors also\nexamined how Kir2.1 expression at specific time points during postnatal development influenced dendritic morphology after\n7 days and affected motor balance in adulthood. Finally, they compared gene expression profiles between non-conditional\nPcp2cre;Kir2.1 mice and WT mice at P7, identifying two differentially expressed genes, Prkcg and car8. By targeting them\nusing ShRNA at birth they showed morphological alterations at P7\nOverall, this is a potentially interesting study that aggregates a collection of well-performed and analyzed experiments. While\nthis is not the first study to investigate the effects of Purkinje cell (PC) hyperpolarization (e.g., see PMID: 19805323), this\nmanuscript provides convincing evidence that PC hyperpolarization during development, particularly within the first two\npostnatal weeks, leads to morphological, functional, and behavioral impairments. However, several flaws significantly limit\nthe impact of this manuscript in its current form.\nMajor Concerns:\n1-The study lacks controls to confirm that: (1) The different strains of Pcp2 mice express Kir2.1 in similar subsets of PCs. (2)\nDifferent tamoxifen injection protocols (single dose, low dose, high dose, or injections at different ages) result in interpretable\nand consistent levels of Kir2.1 expression.\n2- The study is essentially descriptive with no attempt to assess mechanisms involved in the dysfunctions observed. Even\nthe RNA sequencing analysis falls short in identifying putative mechanisms.\nSpecific points:\n- Expression of Kir2.1 across mice strains: line 73 suggests that not all Purkinje cells are targeted in Pcp2creERT2;Kir2.1\nmice. At a minimum, a thorough evaluation of transgene expression in both strains of mice is necessary to understand its\npotential influence on cerebellar development and behavior.\n- Functional assessment of PC activity: it is critical to evaluate Purkinje cell activity in vivo, as well as the output activity of\nthe cerebellum in the deep cerebellar nuclei (DCN).\n- Integration of parallel fiber and climbing fiber inputs: understanding how parallel fiber and climbing fiber inputs are\nintegrated by Purkinje cells is essential to interpret the behavioral outcomes. For example, see PMID: 19805323.\nAdditionally, assessing climbing fiber multi-innervation (e.g., through acute slice experiments) would be informative. At the\nvery least, VGluT2 staining to evaluate climbing fiber inputs is missing.\n- PC-DCN immunostaining: immunostaining in non-conditional mice or mice injected with high doses of tamoxifen provides\nlimited information, as co-localization staining lacks functional relevance. Functional data, such as electrophysiological\nrecordings from brain slices, would be far more informative. Furthermore, the switch to high-dose tamoxifen when studying\nPC-DCN connections during development is abrupt and unexplained. Was the single-dose protocol used in Figure 1\nineffective?\n- Non-conditional expression extrapolations: the activity of PCs in Pcp2cre;Kir2.1 (non-conditional) mice was not tested, yet\nconclusions are extrapolated from the conditional model injected with tamoxifen at P1. Similarly, VOR and eyeblink\nconditioning experiments were only performed in the non-conditional model. This discrepancy should be addressed.\n- A diagram summarizing the experimental design, including the different strains of mice, tamoxifen doses, and the resulting\neffects, is necessary to improve clarity and guide readers through the manuscript.", "reviewer_2": "This is an elegant and well-written manuscript that tries to demonstrate the role that Purkinje cell activity plays in the\ndevelopment of the cerebellum. Using an inducible transgenic gene approach, the authors explore the temporal properties of\nintrinsic activity of Purkinje cells in the proper formation of cerebellar circuits.\nThe figures are clear and the experiments are for the most part quite thorough. The authors try to reduce Purkinje cell activity\nboth early during development and later using inducible expression of Kir2.1 at different ages. Thus, the authors try to\nestablish that its role is most important during early development, with the strongest effects on Purkinje cell dendritic length\nand behavior being observed when activity is impaired at the earliest postnatal ages.\nOverall I think that this manuscript reasonably demonstrates the importance of Purkinje cell activity in Purkinje cell dendritic\ngrowth and in cerebellar function, although more evidence about exactly what is happening with firing would help the\nmanuscript (see below). If these changes are incorporated, I believe that it will be a classic paper that will be well cited both\nin the cerebellar field and in the field of circuit development, meaning its impact will be large and thus is suitable for\npublication in Nature Comms.\nWhile I think that the manuscript is very interesting, I am not yet fully convince by the data that expressing Kir2.1 is doing so\nvia Purkinje cell activity and not some other means (such as making the cells sick). Points 1 and 2 would clarify this and\nprovide additional strength to their hypothesis.\n1. The firing data presented in Extended Data Figure 1d-e shows that the Purkinje cells expressing Kir2.1 at 21 days old can\nstill spike, although it could be highlighted better by showing current injection steps for additional representative traces to\nbetter demonstrate the right-ward shift, which is unfortunately not particular evidence with the 3 traces shown for each\nexample. It would be beneficial to see traces with fewer action potentials so a better comparison of individual action\npotentials could be observed as well. I am surprised that there is no activity at 0 pA for the two control conditions given that\nPurkinje cells typically fire spontaneously at P21, and would like the authors to comment on this. Also, the magnitude of the\nvoltage deflection to the negative current injection is much smaller in the Kir2.1 expressing cells, likely because the cells are\nsmaller. I think this could be supported by reporting the whole cell capacitance and input resistance (Rin) for their\nrecordings. I think this data should be incorporated into Fig. 1 as without that, the impression arising from Figure 1 is that\nfiring is completely abolished, which is likely incorrect (and also brings up the question of whether the cells can fire action\npotentials at all).\n2. How Kir2.1 affects Purkinje cell activity at young ages is demonstrated in Extended Figure 4. However, the authors do not\nfocus on activity, but rather report membrane potential and the amplitude of a current evoked at a negative holding potential.\nThis seems counter-intuitive to me, given that we know that Purkinje cells fire action potentials at these young ages. Firing\ndata would be much more convincing and informative in my opinion, since firing is what they are trying to manipulate. I\nsuggest that this is done as described above in point 2. The authors argue that the effects of Kir2.1 are mediated through the\nalteration in firing activity, so I think that needs to be shown directly. Without direct evidence of firing differences, it could be\nthat Kir2.1 is having an effect by making the cells sicker or decreasing their Rin and making them leakier, rather than directly\nthrough firing.\n3. The manuscript focuses heavily on Purkinje cell structure, and relate this to behavior. Yet Purkinje cells are known to\ninfluence the development of other parts of the cerebellar circuit. It would be useful if the authors could determine if other\nparts of the circuit are changed by this manipulation of Purkinje cell activity. While I do not think it is required to characterize\neverything, at least the size of the lobules and the thickness of the granule cell layer (as a read-out of granule cell number),\nwould be informative. The MLI number would also be interesting, since there are dendritic changes in Purkinje cells. This\nwould help understand the extent of the changes in the circuit and whether behavioral changes are likely to arise entirely\nfrom Purkinje-cell related changes or may also involve other parts of the circuit. I think this is most important for the P1 and\nP7 injections since this is when granule cells and MLIs are still migrating.\n4. Finally, the authors state that they label only a subset of Purkinje cells with these methods. It would be beneficial to\nunderstand approximately how many Purkinje cells are labeled and in which lobules they tend to see labeling, if indeed\nthere is some regionalization of expression of their Kir2.1 transgene."}, "author_response": "Response to reviewers for Nature Communications manuscript NCOMMS-24-63742-T by\nOsório et al.\nWe sincerely thank the reviewers for their insightful and constructive comments. Their feedback\nhas been invaluable in improving the clarity, rigor, and overall quality of our manuscript.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, the authors aimed to study how a Purkinje cell-specific reduction of intrinsic\nactivity, achieved through the overexpression of the inward rectifier Kir2.1 channel, affects\nPurkinje cell (PC) development and cerebellar-dependent behaviors in adult mice. Using a\nconditional Pcp2creERT2 strain of mice, they demonstrated that a single dose of tamoxifen\nadministered at P1 led to PC hyperpolarization and morphological alterations in PC dendrites. In\nnon-conditional Pcp2cre;Kir2.1 mice, they identified synaptic alterations at PC-DCN contacts and\nshowed that adult mice exhibited impairments in specific cerebellar learning tasks, such as VOR\nadaptation and eyeblink conditioning. The authors also examined how Kir2.1 expression at\nspecific time points during postnatal development influenced dendritic morphology after 7 days\nand affected motor balance in adulthood. Finally, they compared gene expression profiles\nbetween non-conditional Pcp2cre;Kir2.1 mice and WT mice at P7, identifying two differentially\nexpressed genes, Prkcg and car8. By targeting them using ShRNA at birth they showed\nmorphological alterations at P7.\nOverall, this is a potentially interesting study that aggregates a collection of well-performed and\nanalyzed experiments. While this is not the first study to investigate the effects of Purkinje cell\n(PC) hyperpolarization (e.g., see PMID: 19805323), this manuscript provides convincing evidence\nthat PC hyperpolarization during development, particularly within the first two postnatal weeks,\nleads to morphological, functional, and behavioral impairments. However, several flaws\nsignificantly limit the impact of this manuscript in its current form.\nResponse: We thank the reviewer for their overall appreciation of our work and their constructive\nfeedback.\n1-The study lacks controls to confirm that: (1) The different strains of Pcp2 mice express Kir2.1 in\nsimilar subsets of PCs. (2) Different tamoxifen injection protocols (single dose, low dose, high\ndose, or injections at different ages) result in interpretable and consistent levels of Kir2.1\nexpression.\nResponse: We thank the reviewer for the recommendation to include additional controls\naddressing Purkinje cell labeling across mouse strains and tamoxifen protocols at different ages.\nTamoxifen dosing and timing were adjusted to achieve either sparse labeling or labeling in all\nPurkinje cells at different developmental ages, enabling controlled expression of tdTomato or\nKir2.1-mCherry in Pcp2creER;Ai14 and Pcp2creER;Kir2.1, respectively. To ensure recombination of\nall Purkinje cells from birth while avoiding multiple injections in neonatal pups, we also used\nconstitutive Pcp2cre/+;Kir2.1 mice and their control littermates. This approach ensured robust\nexpression of tdTomato or Kir2.1–mCherry in all Purkinje cells from birth onward. An overview of\nall experimental designs is now provided in Supplementary Fig. 14. We have included the\nrequested controls in the revised manuscript, specifically:\nSupplementary Fig. 1a-d Sparse labeling of Purkinje cells at P21 in Pcp2creER;Ai14 and\nPcp2creER;Kir2.1;\nSupplementary Fig. 7 Sparse labeling of Purkinje cells at P7, P14, or P21 in Pcp2creER;Ai14 and\nPcp2creER;Kir2.1;\nSupplementary Fig. 1e-g Labeling of all Purkinje cells at P21 in Pcp2+/+;Kir2.1 and\nPcp2cre/+;Kir2.1;\nSupplementary Fig. 9 Labeling of all Purkinje cells at P7 in Pcp2+/+;Kir2.1 and Pcp2cre/+;Kir2.1\nand at P14 or P21 in Pcp2+/+;Kir2.1 and Pcp2creER;Kir2.1;\nSupplementary Fig. 10 Labeling of all Purkinje cells in the adult in Pcp2+/+;Kir2.1 and\nPcp2cre/+;Kir2.1, and Pcp2+/+;Kir2.1 and Pcp2creER;Kir2.1.\nThese additions clarify that labeling patterns are consistent across mouse strains and tamoxifen\ninduction protocols. The corresponding text has been incorporated into the Methods section\n(pages 20-21, lines 475-488) and Supplementary Information of the revised manuscript.\n2\n2- The study is essentially descriptive with no attempt to assess mechanisms involved in the\ndysfunctions observed. Even the RNA sequencing analysis falls short in identifying putative\nmechanisms.\nResponse: We appreciate the reviewer's valuable comment. Although our initial Discussion\naddressed potential mechanisms underlying the observed dysfunctions, we appreciate the\nopportunity to expand and clarify this important aspect of our study. To address the reviewer's\nsuggestion, we have extended our RNA sequencing analysis to more comprehensively identify\nmolecular pathways and gene networks contributing to the observed phenotypes. These analyses\nare now presented in new Figure 6 (page 53-54, lines 1286-1298) and Supplementary Fig. 12.\nWe have clarified the specific purpose of each analysis and more explicitly linked the\ntranscriptional changes to the morphological, functional, and behavioral phenotypes observed.\nThis includes highlighting pathways related to neuronal maturation, synaptic signaling, ion\nchannel regulation, and cerebellar circuit development that may contribute to the dysfunctions\nobserved upon Kir2.1 expression. Please find these new changes in the revised manuscript in\nthe Methods (pages 31-32, lines 767-790), Results (pages 12-13, lines 252-283), and\nDiscussion (pages 15-19, lines 336-340, 349-360, 417-422, 447-455) sections. We hope the\nreviewer finds that these additions strengthen the mechanistic insights and overall impact of our\nstudy.\nSpecific points:\n- Expression of Kir2.1 across mice strains: line 73 suggests that not all Purkinje cells are targeted\nin Pcp2creERT2;Kir2.1 mice. At a minimum, a thorough evaluation of transgene expression in\nboth strains of mice is necessary to understand its potential influence on cerebellar development\nand behavior.\nResponse: We thank the reviewer for highlighting the need to further clarify Kir2.1 expression\nacross mouse strains. The goal of the experiments shown in new Figure 2a-g and Figure 4 was\nto investigate whether disrupting intrinsic activity impacted Purkinje cell morphological\ndevelopment. To enable high-resolution dendritic reconstruction in a densely packed brain\nstructure, we deliberately employed a sparse labeling strategy using the Pcp2creER line with a\nsingle tamoxifen injection (100mg/kg) at postnatal day 1. This approach generated sparse\n3\ntdTomato or Kir2.1-mCherry expression in Purkinje cells, preventing overlap with neighboring\ncells and thereby allowing reliable quantification of individual dendritic arbors. It is important to\nnote that in this model, Kir2.1 expression in Purkinje cells following tamoxifen injection is an all-\nor-none event, as Cre-mediated recombination removes the STOP cassette, allowing Kir2.1\nexpression (now shown in the new Supplementary Fig. 1). Thus, once recombined, Kir2.1\nexpression is consistent within each labeled Purkinje cell. Furthermore, both Ai14 and Kir2.1\nmouse lines were maintained as homozygous and crossed with Cre-driver lines, ensuring that all\nprogeny were heterozygous for Ai14 and Kir2.1 and therefore maintained consistent transgene\nexpression levels. To further validate the robustness of our findings, we obtained comparable\nresults using a different approach for sparse labeling, i.e. using in utero electroporation, as shown\nin new Supplementary Fig. 3. While the promoter driving Kir2.1 is the same between approaches\n- the CAG promoter in the in utero electroporation experiments and a CAG-lox-STOP-lox-Kir2.1-\nmCherry cassette inserted into the Rosa26 locus in the mouse model - we cannot directly\ncompare Kir2.1 expression levels across these systems. Nevertheless, the convergent results\nstrongly suggest that small differences in Kir2.1 expression levels do not materially affect the\nobserved phenotype. Representative images of sparse labeling are now included in the revised\nmanuscript in Supplementary Fig. 1a-d and Supplementary Fig. 7.\nFor all other experiments, in vivo extracellular recordings, synaptic terminal quantification,\nbehavioral tests, and RNA sequencing, we employed a genetic strategy to achieve labeling of all\nPurkinje cells. This was accomplished either by modifying tamoxifen dosing and injection\nfrequency in the inducible Pcp2creER;Ai14 and Pcp2creER;Kir2.1 mice, or by using the non-inducible\nPcp2cre/+;Kir2.1 mouse model. Corresponding images of these protocols have been added to\nSupplementary Fig. 1e-g, Supplementary Fig. 9, and Supplementary Fig. 10.\nFor experiments requiring recombination from birth, we used non-inducible Pcp2cre/+;Kir2.1 mice\nand their littermate controls, while avoiding multiple injections in neonatal pups. This ensured\nKir2.1-mCherry expression in all Purkinje cells from the earliest developmental stages. Previous\nstudies detected Pcp2 mRNA at P11 and GFP expression at E17 in a Pcp2-GFP transgenic mice2.\nWe found that in Pcp2cre/+(B6.Cg-Tg(Pcp2-cre)3555Jdhu/J)3 we could detect tdTomato or Kir2.1-\nmCherry at P1 in Pcp2cre/+;Ai14 and Pcp2cre/+;Kir2.1 mice, respectively (Reviewers Fig. 1 below).\nAt P1, it is possible to distinguish parasagittal bands of tdTomato (Reviewers Fig. 1c) and Kir2.1-\nmCherry (Reviewers Fig. 1c) expression characteristic of the Pcp2 gene at this age. By P7,\nPcp2cre/+;Kir2.1 mice show labeling in all Purkinje cells, but with a higher intensity in the nodular\nregions of the cerebellum due to the Pcp2 gene expression pattern (this gene is first expressed\n4\nin parasagittal bands overlapping Zebrin-positive cells, and then the expression extends to all\nPurkinje cells) (new Supplementary Fig. 9a, b).\nReviewers Fig. 1 Specific labeling of Purkinje cells at P1 in Pcp2cre/+;Ai14 and Pcp2cre/+;Kir2.1\na Experimental design. Brain from conditional Pcp2cre/+;Ai14 was collected at P1. b Schematic illustrating\nthe genetic strategy used to express tdTomato specifically in Purkinje cells, showing that upon Cre-\nmediated recombination, it is an all-or-none event. c Coronal cerebellar sections at P1 from Pcp2cre/+;Ai14\nmice show labeling of tdTomato (magenta) and DAPI (grey). d Experimental design. Brain from conditional\nPcp2cre/+; Kir2.1 mice was collected at P1. e Schematic illustrating the genetic strategy used to express\nKir2.1-mCherry specifically in Purkinje cells. f Coronal cerebellar sections at P1 from Pcp2cre/+;Kir2.1 mice\nshow labeling of Kir2.1-mCherry (magenta) and DAPI (grey). Pcp2, Purkinje cell protein 2 promoter; cre,\nrecombinase; Rosa26, ubiquitous promoter/enhancer; STOP, sequence interrupting gene expression;\ntdTomato, red fluorescent protein; Kir2.1, inward-rectifier potassium channel; mCherry, red fluorescent\nprotein. Scale bar, 500 µm (c, f).\nAchieving labeling of all Purkinje cells in the inducible Pcp2creER;Kir2.1 mice at comparable stages\nrequired daily tamoxifen injections, due to the developmental expression pattern of the Pcp2\ngene, which significantly increased neonatal stress and mortality. An overview of all experimental\ndesigns is now provided in Supplementary Fig. 14. These additions clarify that labeling patterns are consistent across mouse strains and tamoxifen induction protocols, ensuring that the\nobserved phenotypes are not attributable to differences in recombination efficiency. The\n5\ncorresponding text and images have been incorporated into the Methods section (pages 20-21,\nlines 475-488) and Supplementary Information in the revised manuscript.\n- Functional assessment of PC activity: it is critical to evaluate Purkinje cell activity in vivo, as well\nas the output activity of the cerebellum in the deep cerebellar nuclei (DCN).\nResponse: In response to the reviewer's suggestions, we performed a new set of in vivo\nextracellular recordings to assess Purkinje cell activity and cerebellar output in the cerebellar\nnuclei. Specifically, we recorded Purkinje cells and cerebellar nuclei neurons at P28 ± 3 days and\nin adulthood in Pcp2cre/+;Kir2.1 and their control littermates, Pcp2+/+;Kir2.1. Purkinje cells were\nidentified based on the presence of both simple and complex spikes, as well as the characteristic\npause in simple spikes following each complex spike. In juvenile mice, the simple spike firing rate\nwas significantly reduced in Pcp2cre/+;Kir2.1 Purkinje cells compared with controls, and mutant\nPurkinje cells exhibited increased irregularity in simple spike firing. Conversely, the complex spike\nrate was significantly higher in Pcp2cre/+;Kir2.1 Purkinje cells than in controls, while its regularity\nremained unchanged. Simple spike firing primarily reflects the intrinsic activity of Purkinje cells\nas previously shown4 , thus, these findings demonstrate that intrinsic activity is reduced in vivo in\nKir2.1-overexpressing Purkinje cells. Importantly, despite reduced intrinsic activity, these neurons\nretained the ability to generate action potentials when stimulated (new Figure 1f-k). Recordings\nfrom the cerebellar nuclei neurons in Pcp2cre/+;Kir2.1 mice revealed significantly higher firing rates\nand increased firing irregularity relative to controls (new Figure 2k-m). To further investigate the\nneural correlates of the behavioral deficits we observed in the adult mutants, we also performed\nin vivo recordings in adult mice. Consistent with the juvenile data, adult Pcp2cre/+;Kir2.1 Purkinje\ncells exhibited significantly lower simple spike firing rates and greater firing irregularity than\ncontrols, while complex spike rate and regularity were unchanged. Surprisingly, cerebellar nuclei\nneurons in adult mutant mice showed reduced firing rates, whereas firing regularity was\nunaffected (new Supplementary Fig. 5). We have incorporated these new data into the revised\nMethods (pages 22-23, lines 534-562), Results (pages 5, 7-8, lines 90-101, 120-125, 156-162),\nand Discussion (pages 14, 17, lines 311-315, 391-401) sections.\n- Integration of parallel fiber and climbing fiber inputs: understanding how parallel fiber and\nclimbing fiber inputs are integrated by Purkinje cells is essential to interpret the behavioral\noutcomes. For example, see PMID: 19805323. Additionally, assessing climbing fiber multi-\n6\ninnervation (e.g., through acute slice experiments) would be informative. At the very least, VGluT2\nstaining to evaluate climbing fiber inputs is missing.\nResponse: We thank the reviewer for this important suggestion. While our initial study did not\ndirectly assess climbing fiber or parallel fiber innervation, we have now performed additional\nimmunohistochemical analyses using VGLUT2 and VGLUT1 as markers of climbing fiber and\nparallel fiber terminals, respectively. Alterations in excitatory inputs onto Purkinje cells are a\ncommon pathological feature across multiple ataxias, including loss or impaired climbing fibers5-8\nand deficits in parallel fiber synapses9, 10. Using VGLUT2 to label climbing fiber terminals, we\nobserved a significant reduction in the percentage of climbing fiber extension within the molecular\nlayer in Pcp2cre/+;Kir2.1 mutants compared with controls, as well as a decrease in VGLUT2 puncta\ndensity. In addition, the number of VGLUT2 puncta apposed to the Purkinje cell soma was\nincreased in mutants, indicating defective climbing fiber translocation and impaired synaptic\nmaturation. Analysis of parallel fiber terminals using VGLUT1 revealed a marked reduction in\npuncta density and an increase in bouton size in mutants relative to controls, consistent with\ndisrupted parallel fiber synaptic organization. Collectively, these results demonstrate that\nsuppressing Purkinje cell intrinsic activity from early development to adulthood disrupts the normal\nrefinement of both climbing fiber and parallel fiber excitatory inputs. This synaptic disorganization\nunderlies the impaired motor coordination, deficits in cerebellum-dependent learning, and altered\nin vivo firing patterns observed in mutant mice (new Supplementary Fig. 6). These findings\nsuggest that reduced intrinsic Purkinje cell activity interferes with the maturation of excitatory\nsynapses, ultimately affecting cerebellar circuit function and behavior. We have incorporated\nthese new data and their interpretation into the Methods (pages 25-26, lines 613-630), Results\n(page 8, lines 162-172), and Discussion (pages 17-18, lines 411-425) sections to highlight and\ninterpret their relevance for excitatory synaptic development and behavioral outcomes.\n- PC-DCN immunostaining: immunostaining in non-conditional mice or mice injected with high\ndoses of tamoxifen provides limited information, as co-localization staining lacks functional\nrelevance. Functional data, such as electrophysiological recordings from brain slices, would be\nfar more informative.\nResponse: We thank the reviewer for their comment and apologize for any confusion regarding\nthe purpose of the Purkinje cell–cerebellar nuclei neuron immunostaining analysis. The primary\n7\nobjective of this experiment was not to infer functional connectivity, but rather to assess whether\nPurkinje cell axons appropriately reach and target cerebellar nuclei neurons during development\nin our control and mutant mice. This experiment was intended to provide an anatomical framework\nfor understanding how reduced intrinsic Purkinje cell activity might alter the structural maturation\nof cerebellar circuitry. To address the reviewer's concern about functional relevance, we\nemphasize that cerebellar nuclei neuron activity was directly assessed through in vivo\nelectrophysiological recordings in both juvenile and adult mice (new Figure 2k-m and\nSupplementary Fig. 5g-i). These recordings provide complementary functional data that,\ntogether with the anatomical observations, help to build a mechanistic link between disrupted\nPurkinje cell intrinsic activity, altered cerebellar output, and the behavioral impairments observed.\nIn the revised manuscript, we have clarified the rationale for the Purkinje cell–cerebellar nuclei\nneuron immunostaining in the Results (pages 6-8, 10, lines 115-125, 160-162, 202-215) and\nexpanded the Discussion (pages 15, 17, lines 331-340, 391-401) to explain how the anatomical\nand functional findings together inform potential mechanisms underlying the deficits in motor\ncoordination and cerebellum-dependent learning.\nFurthermore, the switch to high-dose tamoxifen when studying PC-DCN connections during\ndevelopment is abrupt and unexplained. Was the single-dose protocol used in Figure 1\nineffective?\nResponse: We apologize for the lack of clarity regarding the tamoxifen protocols used in different\nexperiments. As noted earlier, the sparse-labeling strategy was employed specifically to visualize\nand quantify the dendritic arborization of individual Purkinje cells, where minimal overlap between\nneighboring cells is essential. This required a low-dose, single-tamoxifen induction to ensure\nsparse recombination. In contrast, the analysis of Purkinje cell–cerebellar nuclei neuron\nconnectivity and the behavioral experiments required all Purkinje cells to be labeled. For these\npurposes, we used a high-dose tamoxifen protocol to achieve recombination in all Purkinje cells.\nThus, the shift in tamoxifen dosage reflects different experimental goals, sparse labeling for\nsingle-cell morphological analyses versus labeling of all Purkinje cells for circuit-level anatomical\nand behavioral studies, rather than the single-dose protocol shown in Supplementary Fig. 1d.\nTo address the reviewer's concern, we have clarified these methodological distinctions in the\nrevised Methods section (pages 20-21, lines 475-488), and included representative control\nimages to illustrate the effectiveness of both sparse and complete labeling approaches in their\n8\nrespective contexts. An overview of all experimental designs is provided in Supplementary Fig.\n14.\n- Non-conditional expression extrapolations: the activity of PCs in Pcp2cre;Kir2.1 (non-\nconditional) mice was not tested, yet conclusions are extrapolated from the conditional model\ninjected with tamoxifen at P1.\nResponse: We appreciate the reviewer's concern regarding the extrapolation from the inducible\nto the non-inducible conditional models. To directly address this, we have now included in vivo\nPurkinje cell activity recordings from the non-inducible conditional model Pcp2Cre/+;Kir2.1 and\ncontrol littermates in the revised manuscript (new Figure 1f-k, and Supplementary Fig. 5a-f).\nImportantly, the Kir2.1-mCherry construct is identical in both models. In both cases, Kir2.1\nexpression is activated only after Cre-mediated excision of the floxed stop cassette. Thus, it is\nthe design of the floxed allele, not the type of Cre driver, that determines expression. Once\nrecombination occurs, whether constitutively or following tamoxifen induction, the excision is\npermanent, and Kir2.1 is expressed from the same promoter in both systems. Therefore, any\npotential differences would arise solely from differences in the timing of Cre activity, not from the\nKir2.1 system itself (new Supplementary Fig. 1). While inducible systems can exhibit variability\nin recombination efficiency, we carefully validated recombination in our lines. Our\nimmunohistochemical analyses (new Supplementary Fig. 9 and Supplementary Fig. 10)\ndemonstrate consistent labeling of all Purkinje cells in the inducible model. Additionally, prior to\ninitiating this study, we systematically tested our Cre lines to map the timing and pattern of\nrecombination during early development. To improve clarity, we now include Reviewers Fig. 1,\nshowing tdTomato and Kir2.1-mCherry expression patterns in Pcp2Cre/+ at P1.\nSimilarly, VOR and eyeblink conditioning experiments were only performed in the non-conditional\nmodel. This discrepancy should be addressed.\nResponse: We thank the reviewer for raising this point. The VOR and eyeblink conditioning\nexperiments were performed in the non-inducible conditional Pcp2cre/+;Kir2.1 model, and we\nrecognize the importance of explaining why these behavioral assays were not repeated in the\ninducible line. Because the Kir2.1 construct is identical in both the non-inducible and inducible\nconditional models, with Kir2.1 expression activated only after Cre-mediated excision of the same\n9\nfloxed stop cassette (new Supplementary Fig. 1), the two systems differ solely in the timing of\nrecombination. As described above, both models show highly consistent anatomical,\nelectrophysiological, and behavioral phenotypes, indicating that Kir2.1 expression has\ncomparable functional consequences regardless of whether recombination occurs constitutively\nor via tamoxifen induction. Our rationale for using the inducible conditional model was specifically\nto manipulate the timing of Kir2.1 expression, enabling us to examine how perturbing Purkinje cell\nintrinsic activity at intermediate developmental stages affects circuit maturation and later\nbehavioral outcomes. To assess these behavioral outcomes, we selected the balance beam test.\nBecause the non-conditional model already provided robust deficits in cerebellum-dependent\nlearning (VOR and eyeblink conditioning), and because the inducible model served a different\nmechanistic purpose (timing-specific disruption), we did not consider repeating the conditioning\nexperiments essential. We hope this additional explanation clarifies the rationale behind our\nexperimental design.\n- A diagram summarizing the experimental design, including the different strains of mice,\ntamoxifen doses, and the resulting effects, is necessary to improve clarity and guide readers\nthrough the manuscript.\nResponse: We thank the reviewer for this helpful suggestion. To enhance clarity and guide\nreaders through the different experimental strategies, we have now included a schematic diagram\nsummarizing the mouse strains used, the tamoxifen protocols, and the associated experimental\noutcomes (new Supplementary Fig. 14). This visual overview is intended to complement the\nmethodological descriptions and provide a clear framework for interpreting the data across\nmodels, developmental stages, and experimental approaches. We hope this addition addresses\nthe reviewer's concern and improves the accessibility of our manuscript.\nThis is an elegant and well-written manuscript that tries to demonstrate the role that Purkinje cell\nactivity plays in the development of the cerebellum. Using an inducible transgenic gene approach,\nthe authors explore the temporal properties of intrinsic activity of Purkinje cells in the proper\nformation of cerebellar circuits.\nThe figures are clear and the experiments are for the most part quite thorough. The authors try to\nreduce Purkinje cell activity both early during development and later using inducible expression\nof Kir2.1 at different ages. Thus, the authors try to establish that its role is most important during\nearly development, with the strongest effects on Purkinje cell dendritic length and behavior being\nobserved when activity is impaired at the earliest postnatal ages.\nOverall I think that this manuscript reasonably demonstrates the importance of Purkinje cell\nactivity in Purkinje cell dendritic growth and in cerebellar function, although more evidence about\nexactly what is happening with firing would help the manuscript (see below). If these changes are\nincorporated, I believe that it will be a classic paper that will be well cited both in the cerebellar\nfield and in the field of circuit development, meaning its impact will be large and thus is suitable\nfor publication in Nature Comms.\nWhile I think that the manuscript is very interesting, I am not yet fully convince by the data that\nexpressing Kir2.1 is doing so via Purkinje cell activity and not some other means (such as making\nthe cells sick). Points 1 and 2 would clarify this and provide additional strength to their hypothesis.\nResponse: We thank the reviewer for their positive and constructive evaluation of our manuscript.\nWe particularly appreciate the recognition of the potential impact of our findings for both the\ncerebellar and broader circuit development fields.\n11\n1. The firing data presented in Extended Data Figure 1d-e shows that the Purkinje cells expressing\nKir2.1 at 21 days old can still spike, although it could be highlighted better by showing current\ninjection steps for additional representative traces to better demonstrate the right-ward shift, which\nis unfortunately not particular evidence with the 3 traces shown for each example. It would be\nbeneficial to see traces with fewer action potentials so a better comparison of individual action\npotentials could be observed as well.\nResponse: We thank the reviewer for this helpful suggestion. To better illustrate the reduced\nexcitability of Kir2.1-expressing Purkinje cells at P21, we have expanded the set of representative\ntraces to include a broader range of current injection steps (new Supplementary Fig. 2f). These\nadditional examples more clearly reveal the rightward shift in the firing–current relationship and\nthe reduced spike output, complementing the data in Figure 1d-e. We have also included traces\nfrom lower-current injection steps that evoke fewer action potentials, enabling improved\nvisualization and comparison of individual spikes between control and Kir2.1-expressing Purkinje\ncells.\nI am surprised that there is no activity at 0 pA for the two control conditions given that Purkinje\ncells typically fire spontaneously at P21, and would like the authors to comment on this.\nResponse: We thank the reviewer for raising this important point. We apologize for the lack of\nclarity. The \"0 pA\" condition in our current-step protocol does not correspond to the cells' natural\nresting membrane potential. In this experiment, a holding current is used to clamp each Purkinje\ncell at –65 mV. This holding current is then labeled as “0 pA” prior to applying current-injection\nsteps. We have now clarified this point in the Methods (page 22, lines 513-528) to avoid\nconfusion.\nAlso, the magnitude of the voltage deflection to the negative current injection is much smaller in\nthe Kir2.1 expressing cells, likely because the cells are smaller. I think this could be supported by\nreporting the whole cell capacitance and input resistance (Rin) for their recordings. I think this\ndata should be incorporated into Fig. 1 as without that, the impression arising from Figure 1 is\n12\nthat firing is completely abolished, which is likely incorrect (and also brings up the question of\nwhether the cells can fire action potentials at all).\nResponse: We thank the reviewer for raising this important point. To clarify whether the effects\nof Kir2.1 expression reflect suppression of Purkinje cell activity rather than general cellular\ndysfunction, we have now quantified and included input resistance (Rin) from our recordings. As\nexpected, Kir2.1-overexpressing cells show a significantly reduced Rin, consistent with increased\npotassium conductance (new Supplementary Fig. 2e). Regarding whole-cell capacitance,\naccurate measurement in Purkinje cells is challenging due to their large and intricate architecture,\nwhich violates the assumption of membrane isopotentiality11. Additionally, intracellular calcium\nconcentrations can influence membrane capacitance12, 13, and Kir2.1-overexpressing Purkinje\ncells exhibit altered calcium signaling. Despite these limitations, we plotted capacitance values\nalongside rheobase in Reviewers Fig. 2 and found no significant differences in capacitance\nbetween control (Pcp2creER;Ai14, Pcp2creER;Kir2.1,Ctl) and mutant (Pcp2creER;Kir2.1) Purkinje cells\nat P21. Importantly, the Purkinje cells exhibited a higher rheobase, indicating that more current is\nrequired to elicit action potentials.\nReviewers Fig. 2 Overexpression of Kir2.1\nincreases the rheobase of Purkinje cells at P21.\na Quantification of the capacitance from Pcp2creER;Ai14\n(n = 11 cells/3 mice), Pcp2creER;Kir2.1,Ctl (n = 11\ncells/4 mice) and Pcp2creER;Kir2.1 (n = 11 cells/4 mice),\nKruskal-Wallis multiple comparisons test. b\nQuantification of Rheobase in Purkinje cells from\nPcp2creER;Ai14 (n = 11 cells/3 mice),\nPcp2creER;Kir2.1,Ctl (n = 11 cells/4 mice) and\nPcp2creER;Kir2.1 (n = 13 cells/4 mice). One-way\nANOVA Bonferroni's multiple comparisons test: ***P <\n0.001. Data related to Fig. 1.\nAfter achieving whole-cell configuration, zero current was injected in current-clamp mode to\nassess intrinsic action potential firing and resting membrane potential, defined as the mode of the\nvoltage trace. In control recordings, 77% of Purkinje cells exhibited spontaneous firing, confirming\nintrinsic activity. In contrast, Kir2.1 overexpression abolished spontaneous firing in 90% of the\nrecorded Pcp2creER;Kir2.1 Purkinje cells by hyperpolarizing the resting membrane potential (new\nPcp2creER;Kir2.1 Purkinje cell was able to fire action potentials if the current steps were large\nenough (new Figure 1e and Supplementary Fig. 2f). These data clarify that Purkinje cells\nexpressing Kir2.1 can still fire action potentials when depolarized, but spontaneous activity is\nlargely abolished. We have incorporated these new data and their interpretation into the Methods\n(page 22, lines 513-528) and Results (page 5, lines 75-89) sections.\nAdditionally, we performed a new set of in vivo extracellular recordings to assess Purkinje cell\nactivity and cerebellar output in the cerebellar nuclei. Specifically, we recorded Purkinje cells and\ncerebellar nuclei neurons at P28 ± 3 days and in adulthood in Pcp2cre/+;Kir2.1 and their control\nlittermates, Pcp2+/+;Kir2.1. In juvenile mice, the simple spike firing rate was significantly reduced\nin Pcp2cre/+;Kir2.1 Purkinje cells compared with controls, and mutant Purkinje cells exhibited\nincreased irregularity in simple spike firing. Conversely, the complex spike rate was significantly\nhigher in Pcp2cre/+;Kir2.1 Purkinje cells than in controls, while its regularity remained unchanged.\nAs previously shown, simple spike firing primarily reflects the intrinsic activity of Purkinje cells4,\nthus, these findings demonstrate that intrinsic activity is reduced in vivo in Kir2.1-overexpressing\nPurkinje cells. Importantly, despite reduced intrinsic activity, these neurons retained the ability to\ngenerate action potentials when stimulated (new Figure 1f-k). Recordings from the cerebellar\nnuclei neurons in Pcp2cre/+;Kir2.1 mice revealed significantly higher firing rates and increased\nfiring irregularity relative to controls (new Figure 2k-m). To investigate the neural correlates of the\nbehavioral deficits we observed in the adult mutants, we also performed in vivo recordings in adult\nmice. Consistent with the juvenile data, adult Pcp2cre/+;Kir2.1 Purkinje cells exhibited significantly\nlower simple spike firing rates and greater firing irregularity than controls, while complex spike\nrate and regularity were unchanged. Cerebellar nuclei neurons in adult mutant mice showed\nreduced firing rates, whereas firing regularity was unaffected (new Supplementary Fig. 5). We\nhave incorporated these new data into the revised Methods (pages 22-23, lines 534-562),\nResults (pages 5-8, lines 90-101, 120-125, 156-162), and Discussion (pages 14, 17, lines 311-\n315, 391-401) sections.\n2. How Kir2.1 affects Purkinje cell activity at young ages is demonstrated in Extended Figure 4.\nHowever, the authors do not focus on activity, but rather report membrane potential and the\namplitude of a current evoked at a negative holding potential. This seems counter-intuitive to me,\ngiven that we know that Purkinje cells fire action potentials at these young ages. Firing data would\n14\nbe much more convincing and informative in my opinion, since firing is what they are trying to\nmanipulate. I suggest that this is done as described above in point 2.The authors argue that the\neffects of Kir2.1 are mediated through the alteration in firing activity, so I think that needs to be\nshown directly. Without direct evidence of firing differences, it could be that Kir2.1 is having an\neffect by making the cells sicker or decreasing their Rin and making them leakier rather than\ndirectly through firing.\nResponse: To address the reviewer's concern, we quantified and included input resistance (Rin)\nvalues from our recordings. Kir2.1-overexpressing cells do indeed show a significantly reduced\nRin, consistent with the increased potassium conductance introduced by Kir2.1 (new\nSupplementary Fig. 8d, h, l). However, ex vivo recordings demonstrate that all cells can still fire\naction potentials. In vivo recordings confirm that this is not just a hypothetical scenario, but, in\nfact, all Purkinje cells show simple spike activity, ranging from 111 to 0.06 Hz (new Figure 1h).\nThus, the effect of synaptic input on the cell could be altered, but the main effect is a reduction in\nmembrane potential, leading to reduced intrinsic firing activity. Although morphological\ndevelopment is impaired (new Figure 2a-g), Calbindin staining revealed no evidence for\nneurodegeneration at any age, including in the adult mice used for behavioral testing. As noted\nabove, capacitance measurements are complex to interpret in developing Purkinje cells due to\nnon-isopotentiality and ongoing morphological changes. However, we plotted the capacitance\nvalues in Reviewers Fig. 3 and found a significant reduction in capacitance between control and\nmutant Purkinje cells at P7, P14 or P21.\nTo directly assess firing activity, we performed ex vivo recordings at each developmental stage.\nAfter achieving whole-cell configuration, no current was injected in current-clamp mode in order\nto measure intrinsic action potential firing and resting membrane potential. In control recordings,\n77%, 92%, and 81% of Purkinje cells exhibited spontaneous firing at P7, P14, and P21,\nrespectively. In contrast, only 6%, 0% and 8% of Pcp2creER;Kir2.1 Purkinje cells were intrinsically\nactive at the corresponding ages (new Supplementary Fig. 8a, e, i). These data directly\ndemonstrate that Kir2.1 effectively suppresses intrinsic firing throughout development and\nsupport our conclusion that the observed cellular and circuit effects arise from reduced intrinsic\nactivity rather than nonspecific cellular dysfunction. We have incorporated these new data and\n15\ntheir interpretation into the Methods (page 22, lines 513-528) and Results (pages 9-10, lines\n195-202) sections.\nReviewers Fig. 3 Overexpression of Kir2.1 decreases the capacitance of Purkinje cells at different\ndevelopmental ages. a Quantification of capacitance in control (n = 12 cells/3 mice) and Pcp2creER;Kir2.1\n(n = 12 cells/4 mice) Purkinje cells at P7. Mann-Whitney U test: ***P < 0.001. b Quantification of capacitance\nin control (n = 17 cells/2 mice) and Pcp2creER;Kir2.1 (n = 15 cells/5 mice) Purkinje cells at P14. Unpaired\nStudent's t-test: ***P < 0.001. c Quantification of capacitance in control (n = 26 cells/3 mice) and\nPcp2creER;Kir2.1 (n = 12 cells/5 mice) Purkinje cells at P21. Mann-Whitney U test: ***P < 0.001. Data related\nto Fig. 4.\n3. The manuscript focuses heavily on Purkinje cell structure, and relate this to behavior. Yet\nPurkinje cells are known to influence the development of other parts of the cerebellar circuit. It\nwould be useful if the authors could determine if other parts of the circuit are changed by this\nmanipulation of Purkinje cell activity. While I do not think it is required to characterize everything,\nat least the size of the lobules and the thickness of the granule cell layer (as a read-out of granule\ncell number), would be informative. The MLI number would also be interesting, since there are\ndendritic changes in Purkinje cells. This would help understand the extent of the changes in the\ncircuit and whether behavioral changes are likely to arise entirely from Purkinje-cell related\nchanges or may also involve other parts of the circuit. I think this is most important for the P1 and\nP7 injections since this is when granule cells and MLIs are still migrating.\nResponse: We appreciate the reviewer's suggestion to examine whether manipulating Purkinje\ncell activity alters additional components of the cerebellar circuit. To address this, we have now\n16\nanalysed cerebellar lobule size, granule cell layer and molecular layer thickness, and molecular\nlayer interneuron number in adult mice subjected to Kir2.1-overexpression from either P1 or P7\nthrough adulthood (new Supplementary Fig. 11). Because delaying the onset of Kir2.1-\noverexpression by just one week was sufficient to improve balance beam performance, we\nhypothesized that the early deficits observed in the most severe phenotype, where Kir2.1 was\noverexpressed from birth, might also impair the maturation of other cerebellar cell types whose\ndevelopment depends on proper Purkinje cell maturation, namely molecular layer interneurons\n(MLIs)14, 15 and granule cells (GCs)16, 17. To test this, we examined MLI density in adult\nPcp2cre/+;Kir2.1 mice and their respective controls. While parvalbumin (PV) is a known marker for\nboth Purkinje cells and MLIs18, it does not label all MLIs within the molecular layer19. Hence, we\nquantified the number of PV-positive (PV+) cells co-labeled with NeuroTrace (NeuT+), a\nfluorescent Nissl stain, within the molecular layer. Interestingly, the number of PV+ NeuT+ cells,\nbut not NeuT+ cells, was reduced in Pcp2cre/+;Kir2.1 mice compared with controls. The molecular\nlayer was also thinner than in controls. We next assessed MLI density in mice in which Purkinje\ncell activity was disrupted at P7, thereby sparing the first week of development from changes in\nactivity. The density of PV+ NeuT+ and NeuT+ MLIs, as well as the molecular layer thickness,\nwere unchanged between groups. Lastly, we analysed granule cell layer thickness (GCL) as a\nproxy for GC numbers and measured the size of vermal cerebellar lobules in Pcp2cre/+;Kir2.1\nmice and in P7-inducible Pcp2creER;Kir2.1 mice and their respective controls. There was no\ndifference in the GCL thickness of the cerebellar cortex between Pcp2cre/+;Kir2.1 or P7 inducible\nPcp2creER;Kir2.1 mice and controls. However, lobules I-II, IV-V, and IX were smaller in\nPcp2cre/+;Kir2.1 mice, while lobules IV-V and VI were reduced in the P7-inducible Pcp2creER;Kir2.1\nmice compared to controls. Together, these results indicate that early disruption of Purkinje cell\nintrinsic activity not only leads to more severe motor deficits but also more strongly interferes with\nthe proper maturation of cerebellar circuitry and structure. These new data help to further\ncharacterize the extent of circuit-wide changes induced by manipulating Purkinje cell activity and\nprovide important context for interpreting the behavioral changes observed in our experiments.\nWe have incorporated these analyses into the Methods (pages 25-26, lines 602-605, 631-641),\nResults (page 11, lines 229-251), and Discussion (page 16, lines 361-368) sections to include\nthis additional analysis.\n4. Finally, the authors state that they label only a subset of Purkinje cells with these methods. It\nwould be beneficial to understand approximately how many Purkinje cells are labeled and in which\n17\nlobules they tend to see labeling, if indeed there is some regionalization of expression of their\nKir2.1 transgene.\nResponse: We apologize for the lack of clarity regarding our experimental design in the original\nmanuscript. The goal of the experiments shown in new Figure 2a-g and Figure 4 was to\ninvestigate whether disrupting intrinsic activity impacted Purkinje cell morphological development.\nTo enable high-resolution dendritic reconstruction in a densely packed brain structure, we\ndeliberately employed a sparse labeling strategy using the Pcp2creER line with a single tamoxifen\ninjection (100mg/kg) at postnatal day 1. This approach generated sparse tdTomato or Kir2.1-\nmCherry expression in Purkinje cells, preventing overlap with neighboring cells and thereby\nallowing reliable quantification of individual dendritic arbors. To further validate the robustness of\nour findings, we obtained comparable results using a different approach for sparse labeling, i.e.,\nusing in utero electroporation, as shown in the new Supplementary Fig. 3. Representative\nimages of sparse labeling are now included in the revised manuscript in Supplementary Fig. 1a-\nd and Supplementary Fig. 7. While we have not quantified the precise number of labeled\nPurkinje cells, the representative images demonstrate broad and evenly distributed expression\nthroughout the cerebellar cortex. We did not observe any systematic regional bias, although some\nlobular variability in expression intensity is apparent, which is typical for Pcp2-driven expression.\nFor all other experiments, in vivo extracellular recordings, synaptic terminal quantification,\nbehavioral tests, and RNA sequencing, we employed a genetic strategy to achieve labeling of all\nPurkinje cells. This was accomplished either by modifying tamoxifen dosing and injection\nfrequency in the inducible Pcp2creER;Ai14 and Pcp2creER;Kir2.1 mice, or by using the non-inducible\nPcp2cre/+;Kir2.1 mouse model. Corresponding images of these protocols have been added to\nSupplementary Fig. 1e-g, Supplementary Fig. 9, and Supplementary Fig. 10.\nFor experiments requiring recombination from birth, while avoiding multiple injections in neonatal\npups, we used non-inducible Pcp2cre/+;Kir2.1 mice and their littermate controls. This ensured\nKir2.1-mCherry expression in all Purkinje cells from the earliest developmental stages. Previous\nstudies detected Pcp2 mRNA at P11 and GFP expression at E17 in a Pcp2-GFP transgenic mice2.\nWe found that in Pcp2cre/+(B6.Cg-Tg(Pcp2-cre)3555Jdhu/J)3 we could detect tdTomato or Kir2.1-\nmCherry at P1 in Pcp2cre/+;Ai14 and Pcp2cre/+;Kir2.1 mice, respectively (Reviewers Fig. 1 below).\nAt P1 it is possible to distinguish the parasagittal bands of tdTomato (Reviewers Fig. 1c) and\nKir2.1-mCherry (Reviewers Fig. 1c) expression characteristic of the Pcp2 gene at this age. By\nP7, Pcp2cre/+;Kir2.1 mice show labeling in all Purkinje cells, but with a higher intensity in the\nnodular regions of the cerebellum due to the Pcp2 gene developmental expression pattern (this\n18\ngene is first expressed in parasagittal bands overlapping Zebrin-positive cells, and then the\nexpression extends to all Purkinje cells) (new Supplementary Fig. 9a, b). Achieving labelling of\nall Purkinje cells in the inducible Pcp2creER;Kir2.1 mice at comparable stages required daily\ntamoxifen injections due to the expression pattern of Pcp2 gene, which significantly increased\nneonatal stress and mortality. An overview of all experimental designs is now provided in\nSupplementary Fig. 14. These additions clarify that labeling patterns are consistent across\nmouse strains and tamoxifen induction protocols, ensuring that the observed phenotypes are not\nattributable to differences in recombination efficiency. The corresponding text and images have\nbeen incorporated into the Methods section (pages 20-21, lines 475-488) and Supplementary\nInformation in the revised manuscript.\nReviewers Fig. 1 Specific labeling of Purkinje cells at P1 in Pcp2cre/+;Ai14 and Pcp2cre/+;Kir2.1\na Experimental design. Brain from conditional Pcp2cre/+;Ai14 was collected at P1. b Schematic illustrating\nthe genetic strategy used to express tdTomato specifically in Purkinje cells, showing that upon Cre-\nmediated recombination, it is an all-or-none event. c Coronal cerebellar sections at P1 from Pcp2cre/+;Ai14\nmice show labeling of tdTomato (magenta) and DAPI (grey). d Experimental design. Brain from conditional\nPcp2cre/+; Kir2.1 mice was collected at P1. e Schematic illustrating the genetic strategy used to express\nKir2.1-mCherry specifically in Purkinje cells. f Coronal cerebellar sections at P1 from Pcp2cre/+;Kir2.1 mice\nshow labeling of Kir2.1-mCherry (magenta) and DAPI (grey). Pcp2, Purkinje cell protein 2 promoter; cre,\nrecombinase; Rosa26, ubiquitous promoter/enhancer; STOP, sequence interrupting gene expression;\n19\ntdTomato, red fluorescent protein; Kir2.1, inward-rectifier potassium channel; mCherry, red fluorescent\nprotein. Scale bar, 500 µm (c, f).\nReferences\n1. Oberdick, J., Levinthal, F. & Levinthal, C. A Purkinje cell differentiation marker shows a partial DNA\nsequence homology to the cellular sis/PDGF2 gene. Neuron 1, 367-376 (1988).\n2. Tomomura, M., Rice, D.S., Morgan, J.I. & Yuzaki, M. Purification of Purkinje cells by fluorescence-\nactivated cell sorting from transgenic mice that express green fluorescent protein. Eur J Neurosci 14, 57-\n63 (2001).\n3. Zhang, X.M., et al. Highly restricted expression of Cre recombinase in cerebellar Purkinje cells.\nGenesis 40, 45-51 (2004).\n4. Zhou, H., et al. Cerebellar modules operate at different frequencies. Elife 3, e02536 (2014).\n5. Osorio, C., et al. Pre-ataxic loss of intrinsic plasticity and motor learning in a mouse model of SCA1.\nBrain 146, 2332-2345 (2023).\n6. Ebner, B.A., et al. Purkinje cell ataxin-1 modulates climbing fiber synaptic input in developing and\nadult mouse cerebellum. J Neurosci 33, 5806-5820 (2013).\n7. Furrer, S.A., et al. Reduction of mutant ataxin-7 expression restores motor function and prevents\ncerebellar synaptic reorganization in a conditional mouse model of SCA7. Hum Mol Genet 22, 890-903\n(2013).\n8. Lin, C.C., et al. Reduced cerebellar rhythm by climbing fiber denervation is linked to motor rhythm\ndeficits in mice and ataxia severity in patients. Sci Transl Med 17, eadk3922 (2025).\n9. Tempia, F., et al. Parallel fiber to Purkinje cell synaptic impairment in a mouse model of\nspinocerebellar ataxia type 27. Front Cell Neurosci 9, 205 (2015).\n10. Kurihara, H., et al. Impaired parallel fiber-->Purkinje cell synapse stabilization during cerebellar\ndevelopment of mutant mice lacking the glutamate receptor delta2 subunit. J Neurosci 17, 9613-9623\n(1997).\n11. Golowasch, J., et al. Membrane capacitance measurements revisited: dependence of capacitance\nvalue on measurement method in nonisopotential neurons. J Neurophysiol 102, 2161-2175 (2009).\n12. Balantic, K., Weiss, V.U., Allmaier, G. & Kramar, P. Calcium ion effect on phospholipid bilayers as\ncell membrane analogues. Bioelectrochemistry 143, 107988 (2022).\n13. Carter, T.D., Zupancic, G., Smith, S.M., Wheeler-Jones, C. & Ogden, D. Membrane capacitance\nchanges induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein. J\nPhysiol 513 ( Pt 3), 845-855 (1998).\n14. Telley, L., et al. Dual Function of NRP1 in Axon Guidance and Subcellular Target Recognition in\nCerebellum. Neuron 91, 1276-1291 (2016).\n15. Fleming, J.T., et al. The Purkinje neuron acts as a central regulator of spatially and functionally\ndistinct cerebellar precursors. Dev Cell 27, 278-292 (2013).\n16. Wechsler-Reya, R.J. & Scott, M.P. Control of neuronal precursor proliferation in the cerebellum by\nSonic Hedgehog. Neuron 22, 103-114 (1999).\n17. Lewis, P.M., Gritli-Linde, A., Smeyne, R., Kottmann, A. & McMahon, A.P. Sonic hedgehog signaling\nis required for expansion of granule neuron precursors and patterning of the mouse cerebellum. Dev Biol\n270, 393-410 (2004).\n18. Stichel, C.C., Kagi, U. & Heizmann, C.W. Parvalbumin in cat brain: isolation, characterization, and\nlocalization. J Neurochem 47, 46-53 (1986).\n19. Schilling, K. & Oberdick, J. The treasury of the commons: making use of public gene expression\nresources to better characterize the molecular diversity of inhibitory interneurons in the cerebellar cortex.\nCerebellum 8, 477-489 (2009).\nOsório et al.\nWe sincerely thank the reviewers for their insightful and constructive comments. Their feedback\nhas been invaluable in improving the clarity, rigor, and overall quality of our manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have convincingly addressed my main concerns. They performed additional\nexperiments and provided extensive analyses that clarify ambiguities present in the previous\nversion of the manuscript. The revised manuscript is also easier to read and more clearly\ndemonstrates the importance of Purkinje cell intrinsic excitability in cerebellar development.\nOverall, this work represents one of the most comprehensive datasets on this topic compared\nwith prior studies.\nNonetheless, the following related study should also be cited: Lorenzetto E, Caselli L, Feng G,\nYuan W, Nerbonne JM, Sanes JR, Buffelli M. *Genetic perturbation of postsynaptic activity\nregulates synapse elimination in developing cerebellum.* **Proc Natl Acad Sci U S A.** 2009 Sep\n22;106(38):16475–80.\nMy remaining concern is that the main Figure 5 should be extended to include the data currently\nshown in Supplementary Figure 10 (panels I–L). In its current form, Figure 5 does not clearly\nconvey that phenotype severity depends on the timing of induction. Adding these panels would\nstrengthen the figure and support this key conclusion.\nResponse: We thank the reviewer for their thoughtful and positive evaluation of our manuscript\nand for recognizing the breadth of the dataset and its contribution to the field. We are pleased\nthat the additional experiments and analyses have clarified the remaining ambiguities and\nimproved the clarity and impact of the manuscript. As suggested, we have now cited the relevant\nstudy by Lorenzetto et al. (PNAS, 2009), which provides important context regarding the role of\npostsynaptic activity in cerebellar synapse elimination. This reference has been added to the\nrevised manuscript in the Discussion section (pages 17-18, lines 413-419). In response to the\nreviewer’s comment regarding Figure 5, we have extended the main figure to include the data\npreviously presented in Supplementary Fig. 10 (panels i–l). Incorporating these panels into\ninduction, thereby strengthening the figure and directly supporting this key conclusion. Please find\nthese new changes in the revised manuscript in the Results section (pages 10-11, lines 216-228,\n1277-1291). We hope the reviewer finds that these changes provide more clarity to the results.\nReviewer #2 (Remarks to the Author):\nI am happy with the response to my questions, and feel that the changes incorporated in the\nrevised manuscript significantly strengthen it. I have no further concerns with the manuscript. I\nthink it will add to our understanding of cerebellar development, an important topic.\nResponse: We thank the reviewer for their positive and constructive evaluation of our manuscript.\nWe are pleased that the revisions have addressed their concerns and strengthened the study. We\nparticularly appreciate their recognition of the contribution of our findings to advancing\nunderstanding of cerebellar development and circuit maturation.\n2"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have convincingly addressed my main concerns. They performed additional experiments and provided\nextensive analyses that clarify ambiguities present in the previous version of the manuscript. The revised manuscript is also\neasier to read and more clearly demonstrates the importance of Purkinje cell intrinsic excitability in cerebellar development.\nOverall, this work represents one of the most comprehensive datasets on this topic compared with prior studies.\nNonetheless, the following related study should also be cited:\nLorenzetto E, Caselli L, Feng G, Yuan W, Nerbonne JM, Sanes JR, Buffelli M. *Genetic perturbation of postsynaptic activity\nregulates synapse elimination in developing cerebellum.* **Proc Natl Acad Sci U S A.** 2009 Sep 22;106(38):16475–80.\nMy remaining concern is that the main Figure 5 should be extended to include the data currently shown in Supplementary\nFigure 10 (panels I–L). In its current form, Figure 5 does not clearly convey that phenotype severity depends on the timing of\ninduction. Adding these panels would strengthen the figure and support this key conclusion.", "reviewer_2": "I am happy with the response to my questions, and feel that the changes incorporated in the revised manuscript significantly\nstrengthen it. I have no further concerns with the manuscript. I think it will add to our understanding of cerebellar\ndevelopment, an important topic."}, "author_response": "Response to reviewers for Nature Communications manuscript NCOMMS-24-63742A by\nOsório et al.\nWe sincerely thank the reviewers for their insightful and constructive comments. Their feedback\nhas been invaluable in improving the clarity, rigor, and overall quality of our manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have convincingly addressed my main concerns. They performed additional\nexperiments and provided extensive analyses that clarify ambiguities present in the previous\nversion of the manuscript. The revised manuscript is also easier to read and more clearly\ndemonstrates the importance of Purkinje cell intrinsic excitability in cerebellar development.\nOverall, this work represents one of the most comprehensive datasets on this topic compared\nwith prior studies.\nNonetheless, the following related study should also be cited: Lorenzetto E, Caselli L, Feng G,\nYuan W, Nerbonne JM, Sanes JR, Buffelli M. *Genetic perturbation of postsynaptic activity\nregulates synapse elimination in developing cerebellum.* **Proc Natl Acad Sci U S A.** 2009 Sep\n22;106(38):16475–80.\nMy remaining concern is that the main Figure 5 should be extended to include the data currently\nshown in Supplementary Figure 10 (panels I–L). In its current form, Figure 5 does not clearly\nconvey that phenotype severity depends on the timing of induction. Adding these panels would\nstrengthen the figure and support this key conclusion.\nResponse: We thank the reviewer for their thoughtful and positive evaluation of our manuscript\nand for recognizing the breadth of the dataset and its contribution to the field. We are pleased\nthat the additional experiments and analyses have clarified the remaining ambiguities and\nimproved the clarity and impact of the manuscript. As suggested, we have now cited the relevant\nstudy by Lorenzetto et al. (PNAS, 2009), which provides important context regarding the role of\npostsynaptic activity in cerebellar synapse elimination. This reference has been added to the\nrevised manuscript in the Discussion section (pages 17-18, lines 413-419). In response to the\nreviewer’s comment regarding Figure 5, we have extended the main figure to include the data\npreviously presented in Supplementary Fig. 10 (panels i–l). Incorporating these panels into\ninduction, thereby strengthening the figure and directly supporting this key conclusion. Please find\nthese new changes in the revised manuscript in the Results section (pages 10-11, lines 216-228,\n1277-1291). We hope the reviewer finds that these changes provide more clarity to the results.\nReviewer #2 (Remarks to the Author):\nI am happy with the response to my questions, and feel that the changes incorporated in the\nrevised manuscript significantly strengthen it. I have no further concerns with the manuscript. I\nthink it will add to our understanding of cerebellar development, an important topic.\nResponse: We thank the reviewer for their positive and constructive evaluation of our manuscript.\nWe are pleased that the revisions have addressed their concerns and strengthened the study. We\nparticularly appreciate their recognition of the contribution of our findings to advancing\nunderstanding of cerebellar development and circuit maturation.\n2"}]

Decision letter