Establishment of topographic circuit zones in the cerebellum of scrambler mutant mice - PubMed (original) (raw)
Establishment of topographic circuit zones in the cerebellum of scrambler mutant mice
Stacey L Reeber et al. Front Neural Circuits. 2013.
Abstract
The cerebellum is organized into zonal circuits that are thought to regulate ongoing motor behavior. Recent studies suggest that neuronal birthdates, gene expression patterning, and apoptosis control zone formation. Importantly, developing Purkinje cell zones are thought to provide the framework upon which afferent circuitry is organized. Yet, it is not clear whether altering the final placement of Purkinje cells affects the assembly of circuits into topographic zones. To gain insight into this problem, we examined zonal connectivity in scrambler mice; spontaneous mutants that have severe Purkinje cell ectopia due to the loss of reelin-disabled1 signaling. We used immunohistochemistry and neural tracing to determine whether displacement of Purkinje cell zones into ectopic positions triggers defects in zonal connectivity within sensory-motor circuits. Despite the abnormal placement of more than 95% of Purkinje cells in scrambler mice, the complementary relationship between molecularly distinct Purkinje cell zones is maintained, and consequently, afferents are targeted into topographic circuits. These data suggest that although loss of disabled1 distorts the Purkinje cell map, its absence does not obstruct the formation of zonal circuits. These findings support the hypothesis that Purkinje cell zones play an essential role in establishing afferent topography.
Keywords: cerebellum; circuitry; connectivity; disabled1; positional map; topography.
Figures
Figure 1
ZebrinII and PLCβ4 expressing Purkinje cells are organized into discrete domains in adult scrambler mutants. (A,B) The expression pattern of PLCβ4 is complementary to the expression pattern of zebrinII as observed on adult coronal tissue sections cut through the wild type mouse cerebellum. (C,D) In adult scrambler mutants, PLCβ4 and zebrinII zones are present in relatively normal configurations (i.e. complementarity between zones is maintained) but are organized into ectopic Purkinje clusters. The boundaries between zebrinII and PLCβ4 are poorly delineated and in some regions, we observed overlapping expression (arrowhead in D). Lobule numbers are indicated by Roman numerals. Abbreviations: pcl, Purkinje cell layer; ml, molecular layer; ePC, ectopic Purkinje cells; m, cerebellar medial; l, lateral. Scale bar in A = 1 mm; B = 100 μm (applies to D); C = 500 μm.
Figure 2
Spinocerebellar mossy fiber targeting into longitudinal zones is conserved in scrambler mutant mice. (A) VGLUT2 is a well-established pre-synaptic marker for mossy fiber terminals. Deconvolution microscopy demonstrates that VGLUT2 and WGA-Alexa 555 co-label a sub-population of spinocerebellar mossy fibers. (B) Image of an injection site after delivering WGA-Alexa 555 into the lower thoracic - upper lumbar region of the adult spinal cord. (C) The schematic illustrates the origin and termination of WGA-Alexa 555 labeled spinocerebellar neurons. (D) In wild type mice, WGA-Alexa 555 labeled mossy fibers terminals align with PLCβ4 immunoreactive Purkinje cells. (E) Similar to wild type mice, in scrambler mutant mice spinocerebellar fibers align with PLCβ4 immunoreactive Purkinje cell clusters. The dotted lines in panels E and G indicate the boundaries of PLCβ4 or zebrinII immunoreactive Purkinje cells. (F) In wild type mice, spinocerebellar fibers do not align with zebrinII immuoreactive Purkinje cells. (G) Ectopic spinocerebellar fibers in scrambler mutant mice do not innervate zebrinII immunoreactive Purkinje cell clusters. Abbreviations: ePC, ectopic Purkinje cells; m, midline; CN, cerebellar nuclei. Scale bar in A = 50 μm; in B = 500 μm; in D = 200 μm (applies to D–G).
Figure 3
Mossy fiber terminals within ectopic Purkinje cell clusters fail to form large grape-like glomeruli (rosettes). (A,C) Anterogradely transported WGA-Alexa 555 accumulates as punctate deposits in mossy fiber axons and terminals. (A,B) High magnification image of WGA-Alexa 555 traced mossy fiber glomeruli in the granule cell layer of wild type adult mice (boxed area in B). The arrowheads are highlighting typical large terminal rosettes. (C,D) In scrambler adult mice, mossy fibers fail to differentiate into the complex “grape-like” glomerular structure. Arrowheads are pointing to relatively normal mossy fiber terminals within the ectopic cluster from the boxed region in (D). Scale bar in B = 1 mm; C = 50 μm (applies to A,C); D = 500 μm.
Figure 4
Postnatal spinocerebellar afferents are topographically targeted in scrambler mutants. (A). In P4/5 wild type mice, WGA-Alexa 555 labeled mossy fiber terminals align with PLCβ4 immunoreactive Purkinje cells. (C) Similar to wild type mice, in P4/5 scrambler mutant mice spinocerebellar fibers align with PLCβ4 immunoreactive Purkinje cell clusters. The dotted line in panel C indicates the boundary of an ectopic PLCβ4 immunoreactive Purkinje cell cluster. (B,D) The schematic illustrates the termination of WGA-Alexa 555 labeled spinocerebellar mossy fibers in wild type (B) and scrambler (D) mice. Abbreviations: ePC, ectopic Purkinje cells; pcl, Purkinje cell layer; gl, granule cell layer. Scale bar in B = 750 μm; C = 200 μm (applies to A, C); D = 200 μm.
Figure 5
Mossy fiber terminal structure is established in postnatal scrambler mice. (A,C) Anterogradely transported WGA- Alexa 555 accumulates as punctate deposits in spinocerebellar mossy fiber axons and terminals in wild type P4/5 mice. (B,D) Punctate deposits were also observed in the terminals of P4/5 scrambler mutant mice. The images in panels (C,D) were taken from the boxed regions in panels (A,B), respectively. Scale bar in A = 750 μm; B = 200 μm; D = 100 μm (applies to C,D).
Figure 6
Npy-Gfp labeled climbing fiber topography is maintained in scrambler mutants. (A). VGLUT2 is a molecular marker known to label climbing fiber terminals. VGLUT2 and Npy-Gfp co-label a sub-population of climbing fibers (arrows). (B) Whole mount schematic illustrating the normal pattern of Npy-Gfp labeled climbing fibers innervating PLCβ4 immunoreactive Purkinje cell zones. (C,C') Npy-Gfp is expressed in two broad parasagittal zones that span ~500 μm on either side of the midline in adult wild type mice. (C) Npy-Gfp labeled climbing fiber zones overlap with PLCβ4 immunoreactive Purkinje cell zones. (C') Npy-Gfp labeled climbing fiber zones respect Purkinje cell stripe boundaries and do not invade the zebrinII immunoreactive Purkinje cell zones. (D,D′) Npy-Gfp is expressed in climbing fibers in scrambler:Npy-Gfp mutant mice. (D) Similar to wild type mice, in scrambler mice climbing fibers selectively innervate PLCβ4 immunoreactive Purkinje cell clusters. The dotted lines in panels (D,D′) indicate the boundaries of PLCβ4 and zebrinII immunoreactive Purkinje cells. (D') Npy-Gfp expressing climbing fibers do not target zebrinII labeled Purkinje cells in scrambler mutant mice. (C″,D″) High magnification images of boxed regions in (C,D) show that Npy-Gfp is expressed in climbing fiber zones that terminate upon PLCβ4 immunopositive Purkinje cells in both wild type and scrambler mutant mice. Scale bar in A = 60 μm; in C' = 250 μm (applies to C–C); in C″= 50 μm; in D = 500 μm (applies to D–D′); in D″ = 100 μm.
References
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