3.1 Characteristic of tdTomato cells in GAD2-cre;ZsGreen-tdTomatofl/fl mice
Firstly, we used a Cre-loxP strategy to generate a conditional knock-in mice with specific fluorescence in GABAergic Purkinje cell (Fig. 1A). The Gad2-ires-Cre mice inserted a Cre recombinase sequence at Gad2 allele to make all GABAergic neurons containing Cre recombinase.
So in the ZsGreen-tdTomatofl/f mice, ZsGreen and tdTomato were knocked in and the loxP sites were buried on both sides of ZsGreen. After crossing of these two mice, all GABAergic neurons would transfer from expressing ZsGreen to expressing tdTomato due to Cre recombinase cutting loxP sites. The genotyping strategy used four sets of primers to produce four bands of 1465 bp (for ZsGreen-tdTomato with loxP site), 285 bp (for wild type site), 352 bp (for Cre recombinase), and 250 bp (for wild type site) (Fig. 1B).
Secondly, average body appearances were compared between control Gad2-cre mice and the Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 1C). Interestingly and apparently, Gad2-cre;ZsGreen-tdTomatofl/fl mice showed dazzling green light visible to the naked eye in external auricle skin (arrow), plantar skin (arrow), as well as perianal skin (arrow), although the body weights of both types of mice had no significant difference. Moreover, the brains of Gad2-cre;ZsGreen-tdTomatofl/fl mice showed green fluorescence, while the brains of control Gad2-cre mice showed normal pink, which made it very easy to distinguish the GABAergic knock-in mice.
Thirdly, the confocal microscope images were taken to show that Purkinje cells expressed tdTomato in the cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice, while non-GABAergic cells expressed ZsGreen (Figs. 1D and 1E). We could find that the neuronal bodies of Prukinje cells with red fluorescence (asterisk marked in Fig. 1E) were aligned like dominos stacked throughout the PCL (purkinje cell layer), also they had large dendritic arbors (Fig. 1E) like within the molecular layer (ML) and sent axons like bright flame out off cerebellar cortex (Fig. 1E), therefore they left the dark area of granular layer (GCL) which were occupied by green non-GABAergic cells (Fig. 1E).
Fourthly, the Imaris file was designed to allow better visualization of Purkinje cells specifically expressed tdTomato fluorescence in GAD2-cre;ZsGreen-tdTomatofl/fl mice (shown in Videos. 1-3). We could see that the neuronal bodies of Prukinje cells with red fluorescence were aligned like dominos stacked throughout the PCL, while the dark area of granular layer (GCL) were occupied by green non-GABAergic cells.
3.2 Double stainings verified tdTomato cells were Purkinje cells
Previous studies have shown that Cerebellar Purkinje cells could be marked by Calbinin (CB) [15], while the granular cells be marked by NeuN [16]. In addition, the astrocytes (as GFAP as marker) and microglia (as Iba1 as marker) were believed to be scattered throughout the cerebellar cortex [17,18]. So, in order to verify the cells expressing tdTomato fluorescence in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice were exclusively Purkinje cells, the present immunostainings with the four antibodies mentioned above were observed under the psudo blue color. The percentage of blue cells in the distinct cell subgroups was determined.
It could be indicated that dominos stacked - like tdTomato-positive cells (red) were 95.0% colocalized with CB (green) (Fig. 2A), so that they appeared the color of purple. On the contrary, immunostaing data showed few of tdTomato-positive cells were colocalized with NeuN, the marker of granular cells (Fig. 2B). Moreover, almost none of tdTomato-positive cells were colocalized either with astrocyte marker GFAP (Fig. 2C) or with microglia marker Iba1 (Fig. 2D).
At the same time, ZsGreen-positive cells were densely distributed cross GCL, scattered though PCL, and few in ML (Fig. 2). Double stainings showed almost none of these green cells were colocalized with Purkinje cell marker CB (Fig. 2A). On the contrary, these green cells were mainly consist of NeuN-positive granular cells (Fig. 2B). Our results also confirmed that ZsGreen-positive cells were not astrocyte (Fig. 2C) or microglia (Fig. 2D).
These data suggested that tdTomato-positive cells were primarily expressed in GABAergic Purkinje cells within cerebellar cortex. Then, RNAscope in situ hybridization was performed to examine the presences of seven mitochondrial proteins within the Purkinje cells of Gad2-cre;ZsGreen-tdTomatofl/fl mice.
3.3 Application of RNAscope in situ hybridization onto GAD2-cre;ZsGreen-tdTomatofl/fl mice for Purkinje cell - specific spatial analysis
Identification of mRNAs of mitochondrial fusion (Mfn2), calcium transporter (Mcu and Nclx) and uncoupling proteins (Ucp2 and Ucp4) in Purkinje cells in the cerebellar cortex of GAD2-cre;ZsGreen-tdTomatofl/fl mice with RNAscope probes. Red cells were Purkinje cells; green cells were non-GABAergic cells; blue dots were RNA fluorescence. Moreover, ACD scoring system was used to calculate the overall H scores of Dendritic Weighted Formula (DWF) and Soma Weighted Formula (SWF).
3.3.1 Mfn2 mRNA
Mitofusin 2 (Mfn2) control the fusion of the outer mitochondrial membrane (OMM), but the physiological function of Mfn2 in Purkinje cells remains unclear. Our data firstly demonstrated that Mfn2 mRNAs expression was evident in Prukinje cells in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 3A). Moreover, 76.5% dendrite shafts were ranked as Bin 1 because they only had 1-3 dots per shaft (Fig. 3B); On the contrary, 71.8% soma were ranked as Bin 4 because they had more than 15 dots per cell body (Fig. 3C). The overall H scores of DWF and SWF were calculated as 60 and 139, respectively (Table 4).
3.3.2 Mcu mRNA
Mitochondrial Ca2+ uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, located on the inner mitochondrial membrane (IMM). Our data confirmed the previous report [5] that few Mcu mRNAs expressions were present on Prukinje cells in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 3D). About Moreover, 57.1% dendrite shafts were ranked as Bin 0 because they had no dot per shaft (Fig. 3E); Similarly, 58.1% soma were also ranked as Bin 1 (Fig. 3F). The overall H scores of DWF and SWF were high as 70 and 22, respectively (Table 4).
3.3.3 Nclx mRNA
Conversely to MCU, Ca2+ release is under the control of the Na+/Ca2+ exchanger, encoded by the NCLX gene, located on IMM. Our data firstly demonstrated that Nclx mRNAs expression were only scattered on both soma and dendrites in Prukinje cells in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 3G). About 91.7% dendrite shafts were ranked as Bin 0 because they had no dot per shaft (Fig. 3H); Similarly, 58.3% soma were ranked as Bin 1 because they had only no more than 3 dots per cell body (Fig. 3I). The overall H scores of DWF and SWF were 4 and 31, respectively (Table 4).
3.3.4 Ucp2 mRNA
UCP2 are IMM proteins that may regulate mitochondrial energy metabolism and ROS generation. Our data firstly demonstrated that unexpectedly, few Ucp2 mRNAs expression was present in Prukinje cells in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 3J). Moreover, 75.0% dendrite shafts and 41.2% soma were ranked as Bin 0 because they had no dot (Figs. 3K and 3L). The overall H scores of DWF and SWF were only 15 and 27, respectively (Table 4).
3.3.5 Ucp4 mRNA
UCP4, another IMM protein for regulating mitochondrial energy metabolism and ROS generation, has been looked like a twin of UCP2. Our data confirmed that few Ucp4 mRNAs expression was present in dendritic shafts of Prukinje cells in cerebellum of Gad2-cre;ZsGreen-tdTomatofl/fl mice (Fig. 3M). However, 67.6% soma were ranked as Bin 3 because they had 10-15 dots per cell body (Figs. 3N and 3O). The overall H scores of DWF and SWF were only 14 and 103, respectively (Table 4).
3.4 Double RNAscope profiling of mitochondrial molecules in cerebellar cortex of normal mice
In order to verify the characterization of the molecules, we conducted double RNAscope assays to simultaneously detect two targets on the normal mice section.
3.4.1 Mfn1 (green) and Pcp2 (red)
Because our present Mfn1 probe was not suitable for the detection in the samples from GAD2-cre;ZsGreen-tdTomatofl/fl mice, we did double RNAscope profiling of Mfn1 mRNA (green) in Purkinje cells (red) which were distinguished by the red fluorescence with the mRNA probe of Purkinje cell protein-2 (pcp2) gene (Fig. 4A). Higher magnification images showed that within the PCL, some dots could be found in the soma of Purkinje cells (red) (Fig. 4B). However, within both the ML and the GCL, few green dots could be found in the dendrites or axons of Purkinje cells, respectively (Fig. 4B). ACD quantification confirmed the middle-level expression level of Mfn1 mRNAS in soma (Fig. 4C). The calculation of the percent of double Pcp2-Mfn1 stainings on single Pcp2-positive expressions confirmed the very low colocalization both in the ML and in the GCL (Fig. 4D). The data suggested Mfn1 mRNAs are present only in the soma of the Purkinje cells, instead of processes.
3.4.2 Drp1 (green) and Mcu (red)
Because our previous report had presented the detailed distribution of Drp1 mRNA in the cerebellar cortex of normal mice by in situ hybridization method, the present double RNAscope profiling was to verify the distribution of Mcu in Purkinje cells because these cells could be distinguished by Drp1 fluorescence (Fig. 5A). Higher magnification images confirmed that green Drp1-positive dots could outline the soma of Purkinje cells (Fig. 5B). Our results of Gad2-cre;ZsGreen-tdTomatofl/fl mice by in situ hybridization had showed that a number of dots of Mcu mRNAs were present in processes and soma of Prukinje cells (Fig. 3D). Here, the double RNAscope profiling was consistent with the previous distribution pattern. Unexpectedly and interestingly, almost none of dots of Drp1 mRNAs was co-localized with dots of Mcu mRNAs, wherever at ML, PCL or GCL (Fig. 5C). The data suggested the separation of Drp1 on OMM and Mcu on IMM even they were all abundant in the Purkinje cells.
3.4.3 Ucp2 (green) and Mfn2 (red)
Our results of Gad2-cre;ZsGreen-tdTomatofl/fl mice by RNAscope in situ hybridization had showed that a number of dots of Mfn2 mRNAs were present in soma of Prukinje cells, although this kind of high level did not happen in dendrites of Purkinje cells (Figs. 3A-3C). On the contrary, the dots of Ucp2 mRNAs were only scattered not only on the soma but also on the dendrites of Prukinje cells (Figs. 3J-3L). The present double RNAscope profiling confirmed the dense expressions of Mfn2 in soma of the Purkinje cells (Fig. 6A), so that in the higher magnification images these cells could be distinguished by Mfn2 red fluorescence (Fig. 6B). Moreover, unsurprisingly, almost none of dots of Ucp2 mRNAs was co-localized with dots of Mfn2 mRNAs (Fig. 6C). The data verified the presence of Mfn2 but the nonexistence of Ucp2 on the Purkinje cells.