Design and validation of a Kit conditional knockout mouse.

(A,B) Kit receptor tyrosine kinase (Kit) is enriched in parvalbumin positive GABAergic interneurons of the molecular layer (i.e. basket and stellate cells, MLIs) of the cerebellar cortex, where they synapse upon each other and upon Purkinje cells (PCs, Calbindin+), which express Kit Ligand (KL). Scale bar 10 microns. Expression pattern schematized in B.

(C) In humans and mice, Kit is encoded by up to 21 exons, which in mouse is encoded on the plus strand of chromosome 5 at 75,735,647-75,817,382 bp. We generated a Kit conditional knockout mouse in which Kit Exon 4 is flanked by LoxP sites.

(D) We generated Control mice homozygous for the Kit floxed allele Kittm1c(EUCOMM)Mirow which varied in Pax2-Cre)1Akg transgene status, with the goal of depleting Kit from MLIs in embryonic development.

(E) Male and Female, Control and Kit KO mice were born in equal ratios. KO mice were notably hypopigmented in hair and whiskers, though not eyes.

(F) Confocal microscopy of Kit immunoreactivity in age and sex matched Control and Kit KO littermates demonstrates the established enrichment of Kit in the molecular layer of the cerebellar cortex, and its loss in Kit KO. Scale Bar 500 microns top row, 50 microns inset.

(G) Utilizing a distinct assay and primary antibody, we confirm the detection of Kit immunoreactivity in Controls, and its loss in Kit KO litter mates of either sex by Western Blot of total protein lysates of cerebella. We affirmed equivalent protein loading by GAPDH and parvalbumin.

The knockout of Kit from Cerebellar Cortex Interneurons Impairs Inhibition of Purkinje Cells.

(A,B) Experimental design and example traces of postsynaptic currents in Purkinje Cells (PCs) from Control animals or from those with Kit KO from cerebellar cortex molecular layer interneurons (MLIs). Scale bar is 500 ms x 100 pA.

(C) A frequency distribution plot for individual sIPSC event amplitudes recorded in PCs as in A,B. A KS test reveals a significant difference in the distribution of these event amplitudes; p<0.0001, n in chart.

(D) For each PC, the average Frequency, Amplitude, and Duration of sIPSC was determined. There was a ∼50% decrease in sIPSC frequency p=0.0013, but no change in Amplitude or Duration.

(E-G) Experimental design: In separate experiments, we recorded miniature postsynaptic currents from PCs or from MLIs in Control and in Kit KO animals.

(H) Example traces of mIPSCs or (I) mEPSCs recorded in PCs, and example traces of mIPSC in MLIs, all from Control or from Kit KO animals. Scale for H and J is 50 pA, 25 pA for I, all 500 ms.

(K-M) Analysis of per cell average miniature event Frequency and Amplitude revealed that Kit KO reduced mIPSC frequency, but not amplitude, in PCs by >50%, p=0.013. mEPSCs in PCs and mIPSC in MLIs were not different between Control and Kit KO.

N in charts, refers to number of cells. Error bars are SEM. Statistical significance is by two-tailed t-test, with Welch’s correction as needed. NS indicates p≥0.05.

Knockout of Kit Ligand from Purkinje Cells decreases their inhibitory input.

(A,B) Experimental design and example traces of inhibitory postsynaptic currents detected in Purkinje Cells (PCs) from Control animals or from those with Kit Ligand knockout (KL KO) accomplished by a PCP2-Cre x KL Floxed strategy. Scale bar is 500 ms x 100 pA.

C) A frequency distribution plot for individual sIPSC event amplitudes recorded in PCs as in A,B. A KS test reveals a significant difference in the distribution of these event amplitudes; p<0.0001, n in chart.

(D) For each PC, the average Frequency, Amplitude, and Duration of sIPSC was determined. There was a ∼30-40%% decrease in sIPSC Frequency p=0.0078, Amplitude p=0.024 and Duration p=0.0006.

(E, F) The average mIPSC recorded in PC KL KO vs Control PCs had a >50% decreased Frequency p<0.0001 without a corresponding decrease in mIPSC Amplitude. The average mEPSC Frequency and Amplitude recorded in separate PCs did not differ between Control and KL KO.

N in charts, refers to number of cells. Error bars are SEM. Statistical significance is by two-tailed t-test, with Welch’s correction as needed. NS indicates p>=0.05.

Local levels of Kit Ligand influence the inhibition Purkinje cells receive.

(A) To accomplish in vivo mixtures of Control and Kit Ligand knockout (KL KO) Purkinje Cells (PCs), an AAV encoding Cre under the PC specific L7.6 promoter was co-injected with an AAV encoding a Cre-On mCherry cassette under the Ef1α promoter.

(B) To accomplish in vivo mixed population of Control and Kit Ligand overexpressing (KL OX) PCs, the L7.6 Cre AAV was co-injected with AAV expressing mCherry and (T2A) murine Kit Ligand isoform 2 under the Ef1α promoter.

(C,D) In animals injected at P18, PC KL KO neurons demonstrated a ∼70% decrease in sIPSC event frequency compared to adjacent Control PCs (p<0.0001) or PCs recorded from Sham control animals (p=0.0002). Control vs Sham sIPSC frequency was not significantly different. Similarly, the sIPSC event amplitude was significantly reduced by PC KL KO vs adjacent Control PCs (p=0.0009) or those from Sham animals (p=0.31). Control vs Sham sIPSC amplitude was not different.

(E, F) In animals injected at P18, PC KL OX neurons demonstrated an 8-fold increase in sIPSC Frequency vs local Control PCs (p<0.0001). The frequency of sIPSC events in KL OX PCs was not significantly different from PCs recorded in different Sham animals; this Sham PC sIPSC frequency (while comparable across studies) was 7-fold higher than Control PCs within the KL OX experimental animals (p<0.0001). A similar pattern was found for sIPSC amplitude, being significantly elevated (3-fold) in the KL OX PCs vs their neighboring Control PCs (p=0.0003), but not distinguishable from those recorded in Sham animals.

(G) Proposed model for a function of Purkinje Cell Kit Ligand and Molecular Layer Interneuron Kit. Under normal conditions, the balanced expression of PC KL mediates equivalent presynaptic function of MLI axons via Kit. In focal PC KL KO or PC KL OX, those PCs with the locally highest KL levels sustain nominal levels of MLI mediated inhibition at the expense of reduced functional input to out-competed PCs.

N in charts, refers to number of cells. Error bars are SEM. Statistical significance is by Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons test. NS indicates p>=0.05.

Kit receptor tyrosine kinase is a highly conserved gene affiliated with neurological impairment.

(A) KIT protein sequences were extracted from NCBI ortholog on March 2023 and aligned with MUSCLE. Conservation was placed on a 21 amino acid sliding window to calculate linear motifs, as done in PMID:3557896. The intracellular domain possesses a split cytoplasmic kinase motif demonstrating high conservation across 461 species.

(B) Alignment of Active Site and Post-Translational Motif (PTMs) to Kit amino acid conservation and gnomAD, ClinVar, and Geno2MP human variants.

(C) Human Genome Variation Society annotations for human DNA and corresponding encoded amino acid changes affiliated with their mutational class (Annotation), Site (AA), and Conservation across 461 species. Combined Annotation Dependent Depletion (CADD) score for deleteriousness of SNV or indel variants. Polymorphism Phenotyping v2 (PolyPhen2) annotations predict impact of amino acid substitution on Kit structure function. Allele counts in Gnomad and the number of profiles in the Human Phenotype Ontology associated with Nervous System Abnormality.

Kit knockout does not alter molecular layer interneuron number or distribution.

(A) Immunohistochemistry and confocal microscopy of Control and Kit KO mouse cerebellar cortex validates the normal enrichment and targeted depletion of Kit in Control and Kit KO animals respectively. Parvalbumin immunoreactivity labels Molecular Layer Interneurons and Purkinje Cells, which are also selectively labeled by Calbindin immunoreactivity.

(B) Quantification of the density of Parvalbumin positive and or Calbindin negative somas within the molecular layer reveals that the Density of MLIs is not reduced by Kit KO. Quantification of the average position of MLI somas between the basal (0) and distal (1) borders of the Molecular Layer reveals that the Control and Kit KO MLIs have comparable distributions within the molecular layer. Quantification of the thickness of the the molecular layer reveals no significant difference between Control and Kit KO conditions.

Statistical significance was evaluated by two-tailed t-test with Welch’s correction as necessary, except for Distribution which utilized Mann-Whitney. N in columns reveals the number of different animals whose comparable tissues were evaluated. NS reflects a p value >0.05.

Kit knockout does not alter intrinsic properties of molecular layer interneurons.

(A) Experimental design. Patch clamp recordings were performed on Molecular Layer Interneurons in acute cerebellar slice preparations from Control or from Kit KO animals.

(B,C) Patch clamp recordings of spontaneous action potentials and intrinsic properties in Control and Kit KO MLIs revealed no significant difference in firing Rate, or in membrane Capacitance or Input Resistance. Scale bar 100 ms x 10mV.

(D, E) Current steps were injected into Control or Kit KO MLIs, Example traces (D) and quantification (E) revealed no significant effect of genotype on firing rate. Scale bar 100 ms x 10mV.

(F) Experimental Design. Paired pulses were delivered via stimulating electrode placed in the Outer or the Inner molecular layer, and evoked Inhibitory Post Synaptic Currents were evaluated.

(G, H) The average strength of the first evoked response, nor the Paired Pulse Ratio, did not differ between Control and Kit KO, under either stimulation of the Outer or Inner molecular layer.

Statistical significance was evaluated by two-tailed t-test with Welch’s correction as necessary. N in columns reveals the number of different recorded cells per condition. NS reflects a p value >0.05.

Kit knockout reduces the size of molecular layer interneuron synaptic structures.

(A) Immunohistohemistry and confocal microscopy of presynaptic (VGAT) and postsynaptic (Gephyrin) markers of GABAErgic synapses upon Purkinje Cells (Calbindin), for both Control and Kit KO. An Inset from Control (Yellow Box, first Row Merge ROI), demonstrates example triple positive puncta. First two rows, scale bar 50 microns.

(B) In data not shown, the density of GABAergic synaptic puncta upon Purkinje Cells was not impacted by Kit KO, however, a KS test of the distribution of the sizes of individual puncta from Control vs Kit KO revealed a significant (P<0.0001) decrease. N in chart reflects number of puncta analyzed from 7 Control and from 7 Kit KO animals.

(C, D, E) Immunohistochemistry and confocal microscopy of markers of the pinceau formation of MLI axons upon PC soma and initial axon segments. For both Control and Kit KO, Calbindin and Parvalbumin immunoreactivity was determined in conjunction with pinceau markers Kit, Kv1.2, or PSD-95. As evidence by Parvalbumin positive calbindin negative pinceaux structures in Kit KO in C, pinceaux structures still exist in Kit KO, though appear smaller. This was affirmed by reduced area of Kv1.2 immunoreactivity, and by POSD-95 immuno-reactivity. Scale bars are 50 microns.

Sparse Acute Depletion of Kit Ligand Reduces GABAergic Input To Purkinje Cells.

(A, B, C) Lentivirus encoding mCherry-T2A-Cre under the Purkinje Cells specific promoter L7.6 was injected into postnatal day 7 mice homozygous for a Kit Ligand (KL) floxed allele at P7. Demonstrated here is an area of direct hit and high transduction. (B) Areas with sparse PC transduction were used to record from KL KO or from uninfected adjacent Control PCs, with an example of a IR-DIC identified PC (C).

(D, E). Quantification of the average sIPSCs recorded from P7 Control or sparse KL KO PCs revealed that KL KO reduced both the frequency (p=0.0008) and the amplitude (p=0.012) of GABAergic inhibition of PCs by ∼50%.

(F, G, H) AAV encoding Cre under the L7.6 promoter was co-injected with an AAV encoding Cre-On mCherry under the Ef1α promoter into the cerebellum of postnatal day 18 or 56 KL floxed homozygous mice. Demonstrated in F is a direct hit with high transduction, areas as in (G) with sparse transduction were selected for recordings of mCherry positive KL KO PCs and their adjacent uninfected Control PCs patched under IR-DIC (H).

(I,J,K, L). Analysis of the average sIPSC Frequency and Amplitude in Control and KL KO PCs revealed that at either P18 (p=0.005) or P56 (p=0.008), KL KO robustly reduced GABAergic event frequency in PCs by over 50%. The reduction in sIPSC amplitude reached significance in the P56 (p=0.0096), but not the P18 cohort, here (p=0.104).

Statistical significance was evaluated by two-tailed t-test with Welch’s correction as necessary. N in columns reveals the number of different cells recorded. NS reflects a p value >0.05.