Kit Ligand and Kit receptor tyrosine kinase sustain synaptic inhibition of Purkinje Cells

Reviewer #1 (Public Review):

This manuscript from Zaman et al., investigates the role of cKit and Kit ligand in inhibitory synapse function at molecular layer interneuron (MLI) synapses onto cerebellar Purkinje cells (PC). cKit is a receptor tyrosine kinase expressed in multiple tissues, including select populations of neurons in the CNS. cKIt is activated by Kit ligand, a transmembrane protein typically expressed at the membrane of connected cells. A strength of this paper is the use of cell-specific knockouts of cKit and Kit ligand, in MLIs and PCs, respectively. In both cases, the frequency of spontaneous or miniature (in the presence of TTX) IPSCs was reduced. This suggests either a reduction in the number of functional inhibitory release sites or reduced release probability. IPSCs evoked by electrical stimulation in the molecular layer showed no change in paired-pulse ratio, indicating release probability is not changed in the cKit KO, and favoring a reduction in the number of release sites. Changes in IPSC amplitude were more subtle, with some analyses showing a decrease and others not. These data suggest that disruption of the cKit-Kit ligand complex reduces the number of functional synapses with only minor changes in synapse strength. However, immunolabelling of inhibitory synapses in cKit KO mice using VGAT and Gephyrin antibodies revealed no change in the number of puncta, but reduced size of puncta. This result is more consistent with reduced synapse strength (size) without a change in synapse number. The apparent contradiction of these results is not resolved. It would be interesting to know if immunolabeling of inhibitory synapses in Kit ligand KO mice would produce similar results.

In separate experiments, the authors used viral expression of Cre (driven by the PC-specific L7 promotor) to sparsely KO Kit ligand in PCs. In recordings from neighboring Cre+ (Kit ligand KO) and Cre - (kit ligand intact) PCs, the spontaneous IPSC frequency and amplitude were reduced. Using a similar viral approach, they also overexpressed Kit ligand in wild-type PCs. Here the results are more difficult to interpret. The frequency and amplitude of spontaneous IPSCs were significantly greater in Cre+ PCs compared to Cre- cells. However, the effect appears to be primarily due to a drastic reduction in IPSC amplitude and frequency in the control Cre- cells rather than an increase in Cre+ cells. This puzzling result is interpreted as evidence that cKit influences the proportion of synapses that are functional for neurotransmission, but not the number of release sites. Though this interpretation is not described in detail.

Overall, this paper makes great use of genetic and viral approaches to examine the function of cKit and Kit ligand at MLI-PC synapses. Measurements are generally limited to immunolabeling and spontaneous IPSC recordings, a wider variety of approaches, such as EM imaging or recording from connected MLI-PC pairs would likely provide more detail on specific pre- or postsynaptic phenotypes and more clearly determine whether the number or strength of synapses is changing.

Reviewer #2 (Public Review):

In their study, Zaman et al. demonstrate that deletion of either the receptor tyrosine kinase Kit from cerebellar interneurons or the kit ligand (KL) from Purkinje cells reduces the inhibition of Purkinje cells. They delete Kit or KL at different developmental time points, illustrating that Kit-KL interactions are not only required for developmental synapse formation but also for synapse maintenance in adult animals. The study is interesting as it highlights a molecular mechanism for the formation of inhibitory synapses in Purkinje cells.

The tools generated, such as the floxed Kit mouse line and the virus for Kit overexpression, may have broader applications in neuroscience and beyond.

However, to enhance the publication's impact and strengthen its hypotheses, conclusions, and scientific rigor, it would be beneficial to include additional experimental details, data analyses (particularly regarding the quantification of electrophysiology data), as well as methodological and textual clarifications.

One general weakness is that Kit expression is not limited to molecular layer interneurons but also extends to the Purkinje layer and Golgi interneurons. Although this expression may not conflict with the reported results, as Purkinje layer interneurons form few or no synapses onto Purkinje cells, it should be highlighted in the text (introduction and/or discussion).

In summary, the data support the hypothesis that the interaction between Kit and KL between cerebellar Molecular Layer Interneurons and Purkinje Cells plays a crucial role in promoting the formation and maintenance of inhibitory synapses onto PCs. This study provides valuable insights that could inform future investigations on how this mechanism contributes to the dynamic regulation of Purkinje cell inhibition across development and its impact on mouse behavior.

Reviewer #3 (Public Review):

Summary:
Bidirectional transsynaptic signaling via cell adhesion molecules and cell surface receptors contributes to the remarkable specificity of synaptic connectivity in the brain. Zaman et al., investigate how the receptor tyrosine kinase Kit and its trans-cellular kit ligand regulate molecular layer interneuron (MLI)- Purkinje cell (PC) connectivity in the cerebellum. Presynaptic Kit is specific for MLIs, and forms a trans-synaptic complex with Kit ligand in postsynaptic PC cells. The authors begin by generating Kit cKOs via an EUCOMM allele to enable cell-type specific Kit deletion. They cross this Kit cKO to the MLI-specific driver Pax2-Cre and conduct validation via Kit IHC and immunoblotting. Using this system to examine the functional consequences of presynaptic MLI Kit deletion onto postsynaptic PC cells, they record spontaneous and miniature synaptic currents from PC cells and find a selective reduction in IPSC frequency. Deletion of Kit ligand from postsynaptic PC cells also results in reduced IPSC frequency, together supporting that this trans-synaptic complex regulates GABAergic synaptic formation or maturation. The authors then show that sparse Kit ligand overexpression in PCs decreases neighboring uninfected control sIPSCs in a potentially competitive manner.

Strengths:
Overall, the study addresses an important open question, the data largely support the authors' conclusions, the experiments appear well-performed, and the manuscript is well-written. I just have a few suggestions to help shore up the author's interpretations and improve the study.

Weaknesses:
The strong decrease in sIPSC frequency and amplitude in control uninfected cells in Figure 4 is surprising and puzzling. The competition model proposed is one possibility, and I think the authors need to do additional experiments to help support or refute this model. The authors can conduct similar synaptic staining experiments as in Fig S4 but in their sparse infection paradigm, comparing synapses on infected and uninfected cells. Additional electrophysiological parameters in the sparse injection paradigm, such as mIPSCs or evoked IPSCs, would also help support their conclusions.

The authors should validate KL overexpression and increased cell surface levels using their virus to support their overexpression conclusions.