Roles of molecular layer interneurons in sensory information processing in mouse cerebellar cortex Crus II in vivo

Abstract

Background: Cerebellar cortical molecular layer interneurons (MLIs) play essential roles in sensory information processing by the cerebellar cortex. However, recent experimental and modeling results are questioning traditional roles for molecular layer inhibition in the cerebellum.

Methods and main results: Synaptic responses of MLIs and Purkinje cells (PCs), evoked by air-puff stimulation of the ipsilateral whisker pad were recorded from cerebellar cortex Crus II in urethane-anesthetized ICR mice by in vivo whole-cell patch-clamp recording techniques. Under current-clamp (I = 0), air-puff stimuli were found to primarily produce inhibition in PCs. In MLIs, this stimulus evoked spike firing regardless of whether they made basket-type synaptic connections or not. However, MLIs not making basket-type synaptic connections had higher rates of background activity and also generated spontaneous spike-lets. Under voltage-clamp conditions, excitatory postsynaptic currents (EPSCs) were recorded in MLIs, although the predominant response of recorded PCs was an inhibitory postsynaptic potential (IPSP). The latencies of EPSCs were similar for all MLIs, but the time course and amplitude of EPSCs varied with depth in the molecular layer. The highest amplitude, shortest duration EPSCs were recorded from MLIs deep in the molecular layer, which also made basket-type synaptic connections. Comparing MLI to PC responses, time to peak of PC IPSP was significantly slower than MLI recorded EPSCs. Blocking GABA(A) receptors uncovered larger EPSCs in PCs whose time to peak, half-width and 10-90% rising time were also significantly slower than in MLIs. Biocytin labeling indicated that the MLIs (but not PCs) are dye-coupled.

Conclusions: These findings indicate that tactile face stimulation evokes rapid excitation in MLIs and inhibition occurring at later latencies in PCs in mouse cerebellar cortex Crus II. These results support previous suggestions that the lack of parallel fiber driven PC activity is due to the effect of MLI inhibition.

Figure 1. Air-puff stimulation of the ipsilateral whisker pad evoked reliable spike firing in basket-type MLIs.

A, Left, whole-cell patch-clamp recording from a basket-type MLI in response to hyperpolarizing (−100 µA), followed by a series of depolarizing (+50 pA/step) current pulses. Right, enlarged trace from the quadrangle shown in the left panel. B, Left, under current-clamp (I = 0), superposition of 20 sequential traces (upper) and raster plot of spike firing (lower) showing the basket-type MLI in response to the air-puff stimulus (arrow, 30 ms). Right, enlarged traces (upper) and raster plot (lower) of left panel. Time point (0) denotes the onset of stimulus. C, Under voltage-clamp (V_hold = −70 mV) conditions, five consecutive traces demonstrate the air-puff stimulation (bar, 30 ms)-evoked EPSCs (right) in the basket cell. D, Left, a photomicrograph depicting the morphology of the basket-type MLI (asterisk) filled with biocytin. Note that the basket cell drops descending collaterals that wrap around at least five PCs soma (arrowheads) and is dye-coupled with a group of other MLIs (arrows). Right, magnified photomicrographs from the quadrangles in the left panel showing the dye-coupled stellate cells at different focal planes. PCL, Purkinje cell layer; ML, molecular layer.

Figure 2. Air-puff stimulation of the ipsilateral whisker pad evoked spike firing in stellate-type MLIs.

A, Whole-cell patch-clamp recording from a stellate-type MLI in response to hyperpolarizing pulses (−100 pA) followed by a series of depolarizing current pulses (+50 pA/step). B, Under current clamp (I = 0), superposition of 20 consecutive traces (upper) and a raster plot of spike firing (lower) showing the stellate-type MLI response to air-puff stimulation (black triangle, 30 ms). Time point 0 denotes the onset of the air-puff stimulation. C, Under voltage-clamp (V_hold = −70 mV), five sequential traces showing air-puff stimulation (bar, 30 ms)-evoked EPSCs in the stellate-type MLI. D, A photomicrograph depicting the morphology of the stellate-type MLI (asterisk) filled with biocytin and dye-coupled to several other MLI s (arrows). E, Under current-clamp conditions (n = 0), example traces (n = 5) showing the air-puff stimulation-evoked spike firing or spikelet discharge in a stellate-type MLI. Spikelet discharges are indicated by asterisks.

Figure 3. Relationships between properties of the evoked-EPSCs in MLIs and the depth of their somas location.

A, Representative currents traces show the air-puff stimulation-evoked EPSCs in a basket-type MLI (BC; red) and a stellate-type MLI (SC; green). The time point (0) indicates the onset of the responses. B-E, Plots show the amplitude (B), time to peak (C), half-width (D) and 10–90% rising time (E) of EPSCs versus the depth of the somas in the molecular layer, respectively. The stellate-type MLIs are indicated by green color dots, and the basket-type MLIs are indicated by red color dots. The solid lines indicate linear regression (R).

Figure 4. Comparison of the air-puff stimulation-evoked responses in a basket

-type MLI and a PC in the same mouse cerebellar Crus II. A, Under current-clamp (I = 0) conditions, air-puff stimulation (grey shadow) evoked spike firing in a basket-type MLI (lower), and an IPSP with a pause in spike firing in a PC (upper), in the same mouse cerebellar Crus II. B, Under voltage-clamp (V_hold = −70 mV), air-puff stimulation (grey shadow) evoked fast EPSCs in the basket-type MLI (lower) and IPSCs in the PC (upper). C, Enlarged current traces from (B) and the mean values (± SEM) of the time to peak for the current traces evoked by air-puff stimulation in the PC (black; n = 5) and the basket-type MLI (red; n = 5). D, Consecutive photomicrographs showing the basket-type MLI (white arrow; left) and the PC (white arrow; right) filled with biocytin. The two recorded cells were apart from ∼150 µm in coronal plane. PCL, PC layer; ML, molecular layer.

Figure 5. Comparison of the air-puff stimulation-evoked responses in stellate-type MLIs and PCs in the same mouse cerebellar Crus II.

A, Under current-clamp (I = 0) conditions, the air-puff stimulation (grey shadow) evoked spike firing in a stellate-type MLI (lower), and an IPSP with a pause of spike firing in a PC (upper), in the same mouse cerebellar Crus II. Asterisks indicate spikelets discharge. B, Under voltage-clamp (V_hold = −70 mV), tactile stimulation (grey shadow) evoked fast EPSCs in the stellate-type MLI (lower) and IPSCs in the PC (upper). C, Enlarged current traces from (B) and the mean values (± SEM) of the time to peak for the current traces evoked by the air-puff stimulation in the PCs (black; n = 5) and the basket-type MLIs (red; n = 5). D, Photomicrographs show the morphology of the cells in A-C. The left column shows an overview of the location of the biocytin-labeled stellate-type MLI, which is indicated with a black circle in the left photomicrograph. The middle column shows the detail of the biocytin-labeled stellate-type MLI. The right column shows the detail of the biocytin-labeled PC. The two recorded cells were apart from ∼300 µm in coronal plane. PCL, PC layer; ML, molecular layer.

Figure 6. Properties of the air-puff stimulation-evoked EPSCs in MLIs and PCs.

A, Representative currents traces showing the air-puff stimulation-evoked EPSCs in a MLI (MLI; red) and a PC (blue). The EPSCs in the PC were evoked in the presence of SR95531 (20 µM), a GABA_A selective antagonist. The time point (0) indicates the onset of the responses. B, Bar graph showing the mean amplitude of the EPSCs evoked by the air-puff stimulation in the MLIs (MLI; red; n = 10) and the PC (blue; n = 10). C, Summary of data showing the time to peak for the EPSCs in the MLIs (MLI; red; n = 10) and the PC (blue; n = 10). D, Pooled data showing the half-width of the EPSCs in the MLIs (MLI; red; n = 10) and the PC (blue; n = 10). E, Summary of data showing the 10–90% rising time of the EPSCs in the MLIs (MLI; red; n = 10) and the PC (blue; n = 10).