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17.05.2019 | Original Article Open Access

Quantitative organization of the excitatory synapses of the primate cerebellar nuclei: further evidence for a specialized architecture underlying the primate cerebellum

Brain Structure and Function
Haian Mao, Salah Hamodeh, Angelos Skodras, Fahad Sultan
Wichtige Hinweise

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The online version of this article (https://​doi.​org/​10.​1007/​s00429-019-01888-8) contains supplementary material, which is available to authorized users.

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The cerebellar intrinsic connectivity is of remarkable regularity with a similar build repeated many times over. However, several modifications of this basic circuitry occur that can provide important clues to evolutionary adaptations. We have observed differences in the wiring of the cerebellar output structures (the deep cerebellar nuclei, DCN) with higher dendritic length density in the phylogenetically newer DCN. In rats, we showed that an increase in wiring is associated with an increase in the presynaptic vesicular glutamate transporter 1 (vGluT1). In this study, we have extended our analysis to the rhesus monkey and can show similarities and differences between the two species. The similarities confirm a higher density in vGluT1+ boutons in the lateral (LN/dentate) and posterior interpositus nucleus compared to the phylogenetically older DCN. In general, we also observe a lower density of vGluT1 and 2+ boutons in the monkey, which however, yields a similar number of excitatory boutons per neuron in both species. The only exception is the vGlut1+ boutons in the macaque LN/dentate, which showed a significantly lower number of vGluT1+ boutons per neuron. We also detected a higher percentage of co-labelled vGluT1 and 2 boutons in the macaque than we found in the rat. In summary, these results confirm that the hyposcalled dendrites of the monkey LN/dentate also show a lower number of vGluT1+ boutons per neuron. These results provide further support of our model relating the dendritic morphology of the LN/dentate neurons to the morphology of the specially enlarged LN/dentate nucleus in primates.

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Figure legend S1: Preabsorption test for vGluT2. In the cerebellar cortex the original vGluT2 channel containing lipofuscin not only detects the beaded-like climbing fiber terminals in the molecular layer (small arrow), the glomeruli structures in the granule layer (small arrow) but also the lipofuscin signals from Purkinje cells layer (big arrow) (a). The autofluorescence in the cerebellar cortex is obtained in a separate channel using excitation at 405 nm and emission filters at 650-750 nm (b). After channel subtraction, the structures detected by vGluT2 antibody is confined to the molecular and granule layer (c). In contrast, after applying the vGluT2 antibody and the peptide antigen mixture we only detect the autofluorescence in the Purkinje cell layer (d and e). No signal was left after subtracting lipofuscin channel from the vGluT2 channel (f). In the DCN, bouton-like structures and big granules were detected in the vGluT2 channel (g). The autofluorescence was also present in DCN (h). Only puncta structures were left after channel subtraction (i). The preabsorption test also abolished the vGluT2 staining in the DCN (j-l). Scale bar in (f): 20 μm and applies also to (a-e). Scale bar in (l): 10um and applies to (g-k). Results of masking lipofuscin in vGluT1 and vGluT2 channels. We scanned unstained macaque cerebellar sections by exciting the tissue at 405 nm and by collecting the emission from 650-750 nm and detected fluorescent granule-like structures (Fig. S2a). When the specimens were scanned in lambda mode, these granule-like structures showed a broad emission spectral range from 450 to 750 nm and peaking at around 530 nm. This indicates that these structures are lipofuscin (Fig. S2b). Since the lipofuscin has not only a broad emission spectrum but also broad excitation spectra, granule-like structures were also obtained under the excitation of 633 nm laser (Fig. S2c). Again these structures have broad emission spectra under lambda mode (Fig. S2d). However, independent of the granules’ location (inside the neuronal soma ROI 3, Fig. S2a or outside the soma ROI 1,2 and 4, Fig. S2a), they share the same fluorescence emission properties, thus further endorsing the compositional homogeneity of these structures. By contrast, the intensity of tissue background fluorescence was lower than the lipofuscin fluorescence and dropped to baseline at longer wavelengths (ROI 5, Fig. S2a and b, ROI 2, Fig. S2c and d). The lipofuscin fluorescence can also be well segregated from the tissue background fluorescence from the emission range of 650-750 nm (light green rectangle in Fig. S2b and d). We then tested whether the lipofuscin fluorescence can be segregated from our staining. Alexa 405 was used to detect Purkinje cell axons in our quadruple staining with optimal excitation at 405 nm. The emission spectrum of the Alexa 405 and lipofuscin differed considerably. The emission range was 405-600 nm with the peak around 450 nm for Alexa 405. The Alexa 405 emission range was much narrower than that of lipofuscin, and most importantly, there was no emission from 650-750 nm, indicating that exciting the lipofuscin at 405 nm and obtaining its emission within the range 650-750 nm would result in a pure signal (Fig. S2e-f). This configuration ensured no bleed-through of fluorescence emission of the rest of the fluorochromes into the lipofuscin emission signal. In a next step, we needed to remove the lipofuscin staining from the vGluT1 and vGluT2 signal. We used the lipofuscin channel to produce a mask (Fig. S3a), by creating surfaces fitted to the lipofuscin granules (Fig. S3b). The quantification of lipofuscin volume in DCN from two monkeys was also obtained on the basis of the constructed surfaces. The amount of lipofuscin in the DCN of the 18-year-old D98 is about two-fold that of the 13-year-old H01. However, the amount of lipofuscin in the subnuclei does not differ (Fig. S4). The lipofuscin mask was subtracted from the vGluT1 and vGluT2 channels and we obtained the masked vGluT1 and vGluT2 channels, respectively (Fig. S3c, e). Finally, the lipofuscin-removed vGluT1 and vGluT2 channels were surface-rendered for quantification (Fig. S3d, f) (EPS 35179 kb)
Figure legend S2: Lipofuscin fluorescence detection and its spectral characteristics. The lipofuscin fluorescence in the unstained DCN was present both in the neuronal cytoplasm (ROI 3) and outside the neuron (ROI 1, 2, and 4) under excitation of 405 nm (a). The emission spectrum of each region of interest (ROI) selected is shown in (b). The ROI 5 was tissue background. The spectra of ROI 1-4 were widely spread from 411 nm to 752 nm, albeit with varying intensities. The maximum emission intensity under 405 nm excitation was around 530 nm (b). The unstained section can also be excited at 633 nm (c) and the spectra are shown in (d). The quadruple stained rhesus monkey section was scanned under 405 nm excitation in lambda mode (e). The fluorescence spectra from PCP2/Alexa 405 and lipofuscin differ considerably: ROI of 4 and 5 are from PCP2 stained structures and ROI 1-3 and 7-8 are from lipofuscin (e, f). In the emission region of 650-750 nm, the PCP2/Alexa 405 was reduced to baseline, while lipofuscin still emits a high-intensity signal (f). The light green columns in (b) and (d) indicate the emission range 650-750 nm. The ROI 6 was tissue background. Scale bar in (a), (c) and (e) is 10um (EPS 23883 kb)
Figure legend S3: Workflow to remove lipofuscin autofluorescence (AF) from vGluT1 and vGluT2 channels. The lipofuscin channel signal (a) was surface-rendered in Imaris (b). This mask was then used to subtract from the original vGluT1 and vGluT2 channels (c, e) to obtain the masked vGluT1 and vGluT2 channel (d, f). Scale bar in (f) and in all other images is 10um (EPS 3923 kb)
Figure legend S4: Lipofuscin quantification in DCN. The volume of the lipofuscin surfaces was obtained and compared in different nuclei of two monkeys (D98 (a) and H01 (b)). The lipofuscin volume fraction was higher in the older monkey (D98 i.e., a). Lipofuscin density did not differ significantly between the DCN (ANOVA, p = 0.28 for D98, p = 0.27 for H01). Examples of microscopic images with lipofuscin (red) is shown in (c) and (d) with vGluT1 channel (green) (EPS 8090 kb)
Figure legend S5: Quantile–quantile plots of the power transformed data (a–d). The Shapiro p values in (a) (c)and (d) are larger than 0.05, indicating that the transformed vGluT1 and 2 density and vGluT2 volume are derived from normally distributed populations. The vGluT1+ bouton volume data was not power transformed (b) (EPS 875 kb)
Supplementary material 6 (DOCX 12 kb)
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