GoKawiilSerial EM reconstructions
EM reconstructions were based on a single animal. We previously imaged and aligned a 770 × 750 × 53-μm3 volume of lobule V of the mouse cerebellum for EM reconstructions composed of 1,176 45-nm-thick parasagittal sections. We used automated image segmentation to generate neuron boundaries36. We used the neuron segmentation to reconstruct ten CFs that were identified based on their characteristic morphology such as their large axons projecting into the molecular layer and running along individual PCs. All of these CFs were reconstructed in their entirety within the molecular layer, and the dendritic arbours of the associated PCs were contained within the EM volume.
CF spillover contacts onto MLIs were identified by the physical contact of the CF bouton to an MLI dendrite. Vesicles are present in these boutons but they were not clustered in proximity to presynaptic active zones and PSDs were not associated with these contact sites. MLI contacts were analysed for ten CFs and subtyped into MLI1 or MLI2 on the basis of previous identification methods8. The surface areas of CF contacts onto MLIs were determined by tracing the contact area using annotation tools in Neuroglancer, retrieving their three-dimensional (3D) coordinates and through MATLAB calculating the 3D surface area. Parallel fibre synapses onto MLIs were determined by their small-diameter boutons and completely parallel morphology which was traced through the depth of the dataset.
CF–MLI1–PC and CF–MLI2–MLI1–PC synapses onto local and neighbouring PCs were identified using automated synapse prediction47. The neighbouring PC was determined to be the PC immediately adjacent to a PC that has an identified CF. A total of 608 CF–MLI1–PC synapses were made onto local PCs and 388 onto neighbouring PCs whereas a total of 2,799 CF–MLI2–MLI1–PC synapses were made onto local PCs and 2,583 were onto neighbouring PCs. Pathway analysis was done using Python. Reconstructions were used to estimate the fraction of GrC–MLI contacts that are influenced by CF contacts (Extended Data Table 1).
Slice experiments
Animal procedures were performed in accordance with the National Institutes of Health and Animal Care and Use Committee guidelines and protocols approved by the Harvard Medical School Standing Committee on Animals. C57BL/6 mice were obtained from Charles River Laboratories. Animals of either sex were randomly selected for experiments. Animals were housed on a normal light–dark cycle with an ambient temperature of 18–23 °C with 40–60% humidity.
Acute parasagittal slices (220-μm thick) were prepared from postnatal day (P)28–45 C57BL/6 mice. Mice were anaesthetized with an intraperitoneal injection of 100 mg kg−1 ketamine and 10 mg kg−1 xylazine and perfused transcardially with an ice-cold solution containing (in mM): 110 choline chloride, 7 MgCl 2 , 2.5 KCl, 1.25 NaH 2 PO 4 , 0.5 CaCl 2 , 25 glucose, 11.6 sodium ascorbate, 3.1 sodium pyruvate and 25 NaHCO 3 , equilibrated with 95% O 2 and 5% CO 2 . Slices were cut in the same solution, and then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 1.5 CaCl 2 and 25 glucose, equilibrated with 95% O 2 and 5% CO 2 . As indicated, some experiments were performed in elevated external calcium (2.5 mM). Following incubation at 34 °C for 30 min, the slices were kept up to 6 h at room temperature until recording.
Recordings were performed at 32 °C with an internal solution containing (in mM): 35 CsF, 110 CsCl, 10 HEPES, 10 EGTA and 2 QX-314 (pH adjusted to 7.2 with CsOH, osmolarity adjusted to 300 mOsm kg−1). Visually guided whole-cell recordings were obtained with patch pipettes of ∼3-MΩ resistance pulled from borosilicate capillary glass (BF150-86-10, Sutter Instrument). Electrophysiology data were acquired using a Multiclamp 700B amplifier (Axon Instruments), digitized at 20 kHz and filtered at 4 kHz using Igor Pro (Wavemetrics) running mafPC (courtesy of M. A. Xu-Friedman). Acquisition and analysis of slice electrophysiological data were performed using custom routines written in Igor Pro (Wavemetrics). The following receptor antagonists were added to the ACSF solution to block GABAergic and glycinergic synaptic currents (in μM): 10 SR95531 (gabazine), 1.5 CGP and 1 strychnine. All drugs were purchased from Abcam and Tocris. No power analysis or other statistical methods were used to pre-determine sample sizes. Sample sizes were similar to previous publications.
We recorded MLIs in voltage clamp at −65 mV. Recordings were made from lobules IV–V of the vermis. We recorded from MLIs in the inner two-thirds of the molecular layer and determined the identity of MLI1s and MLI2s by their characteristic electrical properties as previously described8. MLI1s and MLI2s are molecularly and functionally distinct interneuron classes intermingled in the molecular layer8,17. MLI2s have relatively uniform properties regardless of their location in the molecular layer, whereas MLI1s comprise a continuous molecular gradient spatially distributed from nearest to the PC layer to the top of the molecular layer. MLI1s and MLI2s identified by cell fills and post hoc in situ hybridization exhibited distinct electrical properties17. MLI1s express connexin 36, they are electrically coupled to each other and spontaneous action potentials in neighbouring MLI1s produce spikelets, whereas MLI2s do not express connexin 36, they are not electrically coupled to other cells and spikelets are not present17. We classified MLIs with spikelets as MLI1s. Although spikelets are present in most MLI1s, a lack of spikelets is insufficient to identify MLI2s because spikelets in MLI1s require intact spontaneous firing in neighbouring MLI1s, which can be disrupted by poor slice health or by slicing damaging MLIs near the surface of the slice. MLI1s have input resistances ranging from ∼200 MΩ near the PC layer to up to ∼1 GΩ near the top of the molecular layer, whereas most MLI2s have resistances of greater than 1 GΩ (refs. 8,17). We found that resistances of both MLI1s and MLI2s were slightly higher with our caesium-based internal solution than with the potassium-based internal solutions used in previous studies. MLI2s were identified by a lack of spikelets and input resistances of greater than 1 GΩ. Experiments were not performed on neurons that had resistances in the 500-MΩ to 1-GΩ range and lacked spikelets (fewer than 5% of all MLIs), because they could not be unambiguously categorized based on electrical properties alone. We avoided cells in the upper third of the molecular layer where MLI1s tend to have higher input resistances and more MLI1s lack spikelets. This approach yields high-confidence classification of MLI1s and MLI2s based on passive electrical properties in real time without needing post hoc in situ hybridization, but it excludes some MLI1s that lack spikelets, excludes a small number of MLI2s with intermediate resistances and excludes MLIs in the upper third of the molecular layer.
We used a theta glass stimulus electrode to stimulate CFs in the GrC layer with pairs of stimuli (350 µs in duration, 50-ms interstimulus interval, 10–100 μA, 10-s intertrial interval) at different locations in an area around 50 µm into the GrC layer and around 100 µm on each side of the soma of the MLI being recorded. We recorded more than ten sites per cell in search of inputs that were all-or-none and with marked PPR (PPR < 0.6). We then reduced the stimulus intensity to threshold to evaluate the all-or-none nature of the response and contamination from GrC inputs. Stimulation at the threshold for CF activation stochastically evokes successes and failures, and slight increases in stimulus intensity eliminate failures. This indicates it is a single all-or-none input. To evaluate whether the CF input was isolated, failure trials were examined for GrC responses. Traces shown are averages of ten trials. For analysis of the kinetics, we calculated the 20–80 rise and decay times. We bath-applied TBOA (50 μM) while stimulating CF inputs with single stimuli every 10 s. The access resistance and leak current of the MLIs were monitored continuously. Traces shown are averages of ten trials for baseline and in the presence of TBOA. Parallel fibres were stimulated in the molecular layer with pairs of stimuli (350 µs in duration, 140–250 μA, 20-ms interstimulus interval, 5-s intertrial interval) within about 100 µm of the soma of the MLI being recorded. Parallel fibre inputs with paired-pulse facilitation were found for all MLIs recorded. Analysis of properties of CF and GrC synapses onto MLI2s and MLI1s in Fig. 2 was restricted to cells with isolated paired-pulse responses for ten trials.
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