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Spatiotemporal dynamics of lesion-induced axonal sprouting and its relation to functional architecture of the cerebellum

View Article: PubMed Central - PubMed

ABSTRACT

Neurodegenerative lesions induce sprouting of new collaterals from surviving axons, but the extent to which this form of axonal remodelling alters brain functional structure remains unclear. To understand how collateral sprouting proceeds in the adult brain, we imaged post-lesion sprouting of cerebellar climbing fibres (CFs) in mice using in vivo time-lapse microscopy. Here we show that newly sprouted CF collaterals innervate multiple Purkinje cells (PCs) over several months, with most innervations emerging at 3–4 weeks post lesion. Simultaneous imaging of cerebellar functional structure reveals that surviving CFs similarly innervate functionally relevant and non-relevant PCs, but have more synaptic area on PCs near the collateral origin than on distant PCs. These results suggest that newly sprouted axon collaterals do not preferentially innervate functionally relevant postsynaptic targets. Nonetheless, the spatial gradient of collateral innervation might help to loosely maintain functional synaptic circuits if functionally relevant neurons are clustered in the lesioned area.

No MeSH data available.


Multicolour imaging of zebrin II zones and CFs.(a) Coronal section of cerebellar cortex from Aldoc-tdTomato tg crossed with Nefl-EGFP tg mouse. (a, left) Wide-field image of an unfixed, freshly prepared coronal section showing the sagittally oriented bands of zebrin II-expressing (+zone, red) and non-expressing (-zone) zones. (a, right) Magnified image of the area enclosed in the white square from the image on the left. This image was taken using a two-photon microscope to show that EGFP expressing CFs (cyan) can be visualized in both zebrin +zones (red) and –zones. White dashed line indicates the zonal boundary. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. Scale bar, 50 μm. (b) Representative images taken in vivo at the level of the PCL in lobule VIII at 1 and 13 weeks after 3-AP injection show the same PCs as zebrin II-expressing and non-expressing. Zebrin II expression in PCs was stable in all double-transgenic animals that were treated with 3-AP and imaged longer than 4 weeks (n=3 animals). Black and white asterisks indicate examples of zebrin II expressing and non-expressing PCs, respectively. Scale bar, 20 μm.
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f4: Multicolour imaging of zebrin II zones and CFs.(a) Coronal section of cerebellar cortex from Aldoc-tdTomato tg crossed with Nefl-EGFP tg mouse. (a, left) Wide-field image of an unfixed, freshly prepared coronal section showing the sagittally oriented bands of zebrin II-expressing (+zone, red) and non-expressing (-zone) zones. (a, right) Magnified image of the area enclosed in the white square from the image on the left. This image was taken using a two-photon microscope to show that EGFP expressing CFs (cyan) can be visualized in both zebrin +zones (red) and –zones. White dashed line indicates the zonal boundary. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. Scale bar, 50 μm. (b) Representative images taken in vivo at the level of the PCL in lobule VIII at 1 and 13 weeks after 3-AP injection show the same PCs as zebrin II-expressing and non-expressing. Zebrin II expression in PCs was stable in all double-transgenic animals that were treated with 3-AP and imaged longer than 4 weeks (n=3 animals). Black and white asterisks indicate examples of zebrin II expressing and non-expressing PCs, respectively. Scale bar, 20 μm.

Mentions: Post-lesion collateral sprouting yields new synaptic connections that are not present under normal circumstances. If postsynaptic targets of new collaterals are functionally relevant to the original targets of parent axons, collateral sprouting might contribute to functional recovery after lesion. On the other hand, if new collaterals randomly innervate any available postsynaptic target without regard to its functional relevance, this is likely to be detrimental to brain function. Therefore, examining the target selectivity of newly sprouted collaterals in relation to functional circuits is crucial for understanding potential consequences of post-lesion axonal remodelling and designing interventional strategies for future therapies. To study post-lesion CF collateral sprouting in relation to the functional architecture, as defined by zebrin II expression, we crossed the Nefl-EGFP tg mice with another line of transgenic mice in which tdTomato is expressed under the aldolase C promoter (Aldoc-tdTomato tg mice; aldolase C is the gene that encodes zebrin II)1824. As shown in Fig. 4a, tdTomato-positive and -negative zones are clearly visible, and two-photon multicolour imaging allowed simultaneous visualization of CFs and tdTomato-labelled functional zones in the double-transgenic mice. Boundaries between tdTomato-positive and -negative zones precisely match with the boundaries identified by zebrin II-expressing (positive) and -non-expressing (negative) zones in Aldoc-tdTomato tg mice18. This was also the case in our double-transgenic mice (Supplementary Fig. 3). Consistent with a previous study, which shows that Bergmann glia express zebrin II (ref. 25), we observed tdTomato signal in Bergmann glia (Supplementary Fig. 3). However, this did not interfere with unambiguously distinguishing zones of tdTomato-/zebrin II-positive and -negative PCs (functional zones) in vivo (Fig. 4b).


Spatiotemporal dynamics of lesion-induced axonal sprouting and its relation to functional architecture of the cerebellum
Multicolour imaging of zebrin II zones and CFs.(a) Coronal section of cerebellar cortex from Aldoc-tdTomato tg crossed with Nefl-EGFP tg mouse. (a, left) Wide-field image of an unfixed, freshly prepared coronal section showing the sagittally oriented bands of zebrin II-expressing (+zone, red) and non-expressing (-zone) zones. (a, right) Magnified image of the area enclosed in the white square from the image on the left. This image was taken using a two-photon microscope to show that EGFP expressing CFs (cyan) can be visualized in both zebrin +zones (red) and –zones. White dashed line indicates the zonal boundary. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. Scale bar, 50 μm. (b) Representative images taken in vivo at the level of the PCL in lobule VIII at 1 and 13 weeks after 3-AP injection show the same PCs as zebrin II-expressing and non-expressing. Zebrin II expression in PCs was stable in all double-transgenic animals that were treated with 3-AP and imaged longer than 4 weeks (n=3 animals). Black and white asterisks indicate examples of zebrin II expressing and non-expressing PCs, respectively. Scale bar, 20 μm.
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f4: Multicolour imaging of zebrin II zones and CFs.(a) Coronal section of cerebellar cortex from Aldoc-tdTomato tg crossed with Nefl-EGFP tg mouse. (a, left) Wide-field image of an unfixed, freshly prepared coronal section showing the sagittally oriented bands of zebrin II-expressing (+zone, red) and non-expressing (-zone) zones. (a, right) Magnified image of the area enclosed in the white square from the image on the left. This image was taken using a two-photon microscope to show that EGFP expressing CFs (cyan) can be visualized in both zebrin +zones (red) and –zones. White dashed line indicates the zonal boundary. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. Scale bar, 50 μm. (b) Representative images taken in vivo at the level of the PCL in lobule VIII at 1 and 13 weeks after 3-AP injection show the same PCs as zebrin II-expressing and non-expressing. Zebrin II expression in PCs was stable in all double-transgenic animals that were treated with 3-AP and imaged longer than 4 weeks (n=3 animals). Black and white asterisks indicate examples of zebrin II expressing and non-expressing PCs, respectively. Scale bar, 20 μm.
Mentions: Post-lesion collateral sprouting yields new synaptic connections that are not present under normal circumstances. If postsynaptic targets of new collaterals are functionally relevant to the original targets of parent axons, collateral sprouting might contribute to functional recovery after lesion. On the other hand, if new collaterals randomly innervate any available postsynaptic target without regard to its functional relevance, this is likely to be detrimental to brain function. Therefore, examining the target selectivity of newly sprouted collaterals in relation to functional circuits is crucial for understanding potential consequences of post-lesion axonal remodelling and designing interventional strategies for future therapies. To study post-lesion CF collateral sprouting in relation to the functional architecture, as defined by zebrin II expression, we crossed the Nefl-EGFP tg mice with another line of transgenic mice in which tdTomato is expressed under the aldolase C promoter (Aldoc-tdTomato tg mice; aldolase C is the gene that encodes zebrin II)1824. As shown in Fig. 4a, tdTomato-positive and -negative zones are clearly visible, and two-photon multicolour imaging allowed simultaneous visualization of CFs and tdTomato-labelled functional zones in the double-transgenic mice. Boundaries between tdTomato-positive and -negative zones precisely match with the boundaries identified by zebrin II-expressing (positive) and -non-expressing (negative) zones in Aldoc-tdTomato tg mice18. This was also the case in our double-transgenic mice (Supplementary Fig. 3). Consistent with a previous study, which shows that Bergmann glia express zebrin II (ref. 25), we observed tdTomato signal in Bergmann glia (Supplementary Fig. 3). However, this did not interfere with unambiguously distinguishing zones of tdTomato-/zebrin II-positive and -negative PCs (functional zones) in vivo (Fig. 4b).

View Article: PubMed Central - PubMed

ABSTRACT

Neurodegenerative lesions induce sprouting of new collaterals from surviving axons, but the extent to which this form of axonal remodelling alters brain functional structure remains unclear. To understand how collateral sprouting proceeds in the adult brain, we imaged post-lesion sprouting of cerebellar climbing fibres (CFs) in mice using in vivo time-lapse microscopy. Here we show that newly sprouted CF collaterals innervate multiple Purkinje cells (PCs) over several months, with most innervations emerging at 3–4 weeks post lesion. Simultaneous imaging of cerebellar functional structure reveals that surviving CFs similarly innervate functionally relevant and non-relevant PCs, but have more synaptic area on PCs near the collateral origin than on distant PCs. These results suggest that newly sprouted axon collaterals do not preferentially innervate functionally relevant postsynaptic targets. Nonetheless, the spatial gradient of collateral innervation might help to loosely maintain functional synaptic circuits if functionally relevant neurons are clustered in the lesioned area.

No MeSH data available.