Differential maturation of climbing fiber innervation in cerebellar vermis

Abstract

Folding of the brain surface is a general morphological adaptation to maximize surface area in a limited cranial volume. Surface folding is present not only in the neocortex but also in the cerebellar cortex. This folding creates subdivisions of the cortical surface: the sulci, the gyri, and the straight bank region, which is interposed. Is cortical folding only the solution to a surface-volume problem or does it also confer functional differences on the subdivisions that are created by this geometry? Here we have used the innervation of Purkinje cells by climbing fibers as a model system to explore potential functional differences. Purkinje cells are innervated by multiple climbing fibers at birth but undergo an activity-dependent refinement, such that by postnatal day (P) 21, most are contacted by a single climbing fiber. Using whole-cell recording from slices of cerebellar vermis derived from juvenile (P18-25) or adult (P60-83) mice, we found that significantly more Purkinje cells in the sulcus were innervated by multiple climbing fibers than in the gyrus or bank subdivisions; however, the basic properties of climbing fiber-Purkinje cell EPSCs such as kinetics, amplitude, and paired-pulse ratio were similar across cortical subdivisions. To search for a morphological correlate of differential multiple climbing fiber innervation, we labeled climbing fibers and performed reconstructions of immunofluorescent images. These revealed that, unlike the bank-gyrus subdivisions, most of the climbing fibers in the sulcus do not innervate the superficial molecular layer. These findings suggest that the subdivisions of the cerebellar cortex produced by folding may create functionally distinct entities.

Figure 5.

Most of the CFs in sulcus do not innervate the superficial portion of the molecular layer. CFs were labeled with an Ab against dextran-conjugated tracer (green), and Purkinje cells were labeled with Ab against calbindin-d-28K (red). Representative confocal stacks show the CF innervation of the molecular layer in bank (A) and in sulcus (B). Scale bar, 100 μm. For quantification, the molecular layer was divided into four segments, and the degree of CF innervation in each segment was measured (see Materials and Methods). C, The degree of CF innervation in each segment in bank (closed column) and in sulcus (open column). Inset shows the thickness of molecular layer in both subdivisions. Data represent mean ± SEM. ^*p < 0.05; ^**p < 0.01.

Figure 1.

Whole-cell patch-clamp recordings from Purkinje cells reveal CF multiple innervation. CF-PC EPSCs were evoked by a stimulation pipette placed in the granule cell layer. A, B, Examples of the location of recorded Purkinje cells in bank (A) and in sulcus (B) are shown in images from transilluminated parasagittal slices. The recording pipette is indicated by “R” in each figure. Scale bar, 200 μm. C, D, Representative traces of CF-PC EPSCs innervated by single (C) and double (D) CFs, and corresponding plot of EPSC peak amplitude as a function of stimulus intensity. Holding potential was -18 mV.

Figure 2.

The proportion of Purkinje cells innervated by multiple CFs is greater in the sulcus subdivision than the bank subdivision in the cerebellar vermis. Percentage of Purkinje cells showing number of discrete steps of CF-PC EPSC in bank (closed column) and in sulcus (open column) in the cerebellar vermis. Data in A and B were obtained from parasagittal slices of juvenile mice (P18-25; 123 cells from 21 animals) and adult mice (P60-83; 115 cells from 19 animals), respectively. Numbers above the bars indicate percentage of Purkinje cells showing multiple CF innervation.

Figure 3.

Amplitude, paired-pulse depression, and kinetics of CF EPSCs are not different between bank and sulcus Purkinje cells. A, Representative traces showing single and double CF innervation recorded from Purkinje cells in bank and in sulcus in the cerebellar vermis of adult mice. Several traces at subthreshold and suprathreshold stimulus intensity are superimposed. B, Electrophysiological parameters of the CF EPSP in bank (closed circle; n = 38 for single innervation and n = 6 for double innervation) and in sulcus (open circle; n = 35 for single innervation and n = 12 for double innervation). Measurements for double-innervated cells were made using the lower threshold CF response. Averaged holding potential was -17.0 ± 3.1 mV in bank and -17.2 ± 3.1 mV in sulcus (mean ± SD). Data in the figure are represented as the mean ± SD.

Figure 4.

Difference in CF innervation pattern between bank and sulcus. CFs were labeled with dextran-conjugated green fluorescent tracer injected into the inferior olive in rats. Purkinje cells innervated by the labeled CFs were identified in living brain slices using a microscope equipped with both fluorescence and infrared gradient contrast optics and then labeled with a red fluorescent dye via a patch pipette. Projected confocal stacks show that CFs in sulcus (B, D) are restricted to more proximal portions of the dendritic tree of the target Purkinje cells than those in bank (A, C). Note that the relatively thick secondary dendritic branches of target Purkinje cells were not innervated by the CF in B. White dotted lines indicate the pial surface. Scale bar, 100 μm.

Figure 6.

Although the population of CFs is significantly more restricted to proximal portions of the molecular layer in sulcus than in bank, a small number of atypical fibers are in both regions. Examples of some atypical CF innervation patterns are shown. A, CF in bank fails to innervate superficial molecular layer. B, CF in sulcus innervates superficial molecular layer. Scale bar, 100 μm. C, Histogram showing the number of CFs as a function of degree of CF innervation in the first segment.

Figure 7.

Multiple innervation is similar in bank and sulcus subdivisions of the cerebellar hemispheres. Percentage of Purkinje cells showing number of discrete steps of CF-PC EPSC in bank (closed column) and in sulcus (open column) in the cerebellar hemispheres. Data shown in A-C were obtained from P18-25 sagittal slices (61 cells from 15 animals), P72-83 sagittal slices (59 cells from 15 animals), and P19-25 coronal slices (44 cells from 8 animals) of mouse cerebellar hemisphere, respectively. Numbers above the bars indicate percentage of Purkinje cells showing multiple CF innervation.