Limits...
Abundant expression of guidance and synaptogenic molecules in the injured spinal cord.

Jacobi A, Schmalz A, Bareyre FM - PLoS ONE (2014)

Bottom Line: To assign the expression of these molecules to distinct populations of interneurons we labeled short and long propriospinal neurons by retrograde tracing and glycinergic neurons using a transgenically expressed fluorescent protein.Overall the expression pattern of guidance and synaptogenic molecules in the cervical spinal cord appeared to be stable over time and was not substantially altered following a midthoracic spinal cord injury.Taken together, our study indicates that many of the guidance and synaptogenic cues that regulate neuronal circuit formation in development are also present in the adult CNS and therefore likely contribute to the remodelling of axonal connections in the injured spinal cord.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Neuroimmunology, Ludwig-Maximilians University Munich, Munich, Germany.

ABSTRACT

Background: Spinal interneurons have emerged as crucial targets of supraspinal input during post-injury axonal remodelling. For example, lesioned corticospinal projections use propriospinal neurons as relay stations to form intraspinal detour circuits that circumvent the lesion site and contribute to functional recovery. While a number of the molecules that determine the formation of neuronal circuits in the developing nervous system have been identified, it is much less understood which of these cues are also expressed in the injured spinal cord and can thus guide growing collaterals and initiate synaptogenesis during circuit remodelling.

Methodology/principal findings: To address this question we characterized the expression profile of a number of guidance and synaptogenic molecules in the cervical spinal cord of healthy and spinal cord-injured mice by in situ hybridization. To assign the expression of these molecules to distinct populations of interneurons we labeled short and long propriospinal neurons by retrograde tracing and glycinergic neurons using a transgenically expressed fluorescent protein. Interestingly, we found that most of the molecules studied including members of slit-, semaphorin-, synCAM-, neuroligin- and ephrin- families as well as their receptors are also present in the adult CNS. While many of these molecules were abundantly expressed in all interneurons examined, some molecules including slits, semaphorin 7a, synCAM4 and neuroligin 1 showed preferential expression in propriospinal interneurons. Overall the expression pattern of guidance and synaptogenic molecules in the cervical spinal cord appeared to be stable over time and was not substantially altered following a midthoracic spinal cord injury.

Conclusions: Taken together, our study indicates that many of the guidance and synaptogenic cues that regulate neuronal circuit formation in development are also present in the adult CNS and therefore likely contribute to the remodelling of axonal connections in the injured spinal cord.

Show MeSH

Related in: MedlinePlus

In Situ hybridization pattern of Slit-1,-2,-3 in the cervical spinal cord.In situ hybridization of slit-1 (A-F), slit-2 (G-L), slit-3 (M-R) mRNA in the unlesioned cervical spinal cord. Strong signals for slit-1 (A) and slit-2 (G) are detected with the anti-sense probe in cervical interneurons and motoneurons while slit-3 (M) shows a weaker signal. No signals are detected with the sense probes for slit-1 (B), slit-2 (H) or slit-3 (N). (C, I, O) Epifluorescence images of double-labeled neurons in the ventral horn (NeuN: green; In situ signal: red). (D-F) Co-localization of slit-1 mRNA in glycinergic neurons (D; GlyT2: green; slit-1: red), LPSN (E; LPSN: green; slit-1: red; NT435: blue) and SPSN (F; SPSN: green; slit-1: red; NT435: blue) in the cervical spinal cord. (J-L) Co-localization of slit-2 mRNA in glycinergic neurons (J; GlyT2: green; slit-2: red), LPSN (K; LPSN: green; slit-2: red; NT435: blue) and SPSN (L; SPSN: green; slit-2: red; NT435: blue) in the cervical spinal cord. (P-R) Co-localization of slit-3 mRNA in glycinergic neurons (P; GlyT2: green; slit-3: red), LPSN (Q; LPSN: green; slit-3: red; NT435: blue) and SPSN (R; SPSN: green; slit-3: red; NT435: blue) in the cervical spinal cord. (S-U) Quantification of the proportion of glycinergic neurons, LPSN and SPSN expressing slit-1 (S), slit-2 (T) and slit-3 (U) in the unlesioned and lesioned cervical spinal cord. Scale bars in A,B,G,H,M,N, 250 µm; Scale bars in C,I,O, 25 µm; Scale bars in D-F,J-K,P-R, 25 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3921160&req=5

pone-0088449-g001: In Situ hybridization pattern of Slit-1,-2,-3 in the cervical spinal cord.In situ hybridization of slit-1 (A-F), slit-2 (G-L), slit-3 (M-R) mRNA in the unlesioned cervical spinal cord. Strong signals for slit-1 (A) and slit-2 (G) are detected with the anti-sense probe in cervical interneurons and motoneurons while slit-3 (M) shows a weaker signal. No signals are detected with the sense probes for slit-1 (B), slit-2 (H) or slit-3 (N). (C, I, O) Epifluorescence images of double-labeled neurons in the ventral horn (NeuN: green; In situ signal: red). (D-F) Co-localization of slit-1 mRNA in glycinergic neurons (D; GlyT2: green; slit-1: red), LPSN (E; LPSN: green; slit-1: red; NT435: blue) and SPSN (F; SPSN: green; slit-1: red; NT435: blue) in the cervical spinal cord. (J-L) Co-localization of slit-2 mRNA in glycinergic neurons (J; GlyT2: green; slit-2: red), LPSN (K; LPSN: green; slit-2: red; NT435: blue) and SPSN (L; SPSN: green; slit-2: red; NT435: blue) in the cervical spinal cord. (P-R) Co-localization of slit-3 mRNA in glycinergic neurons (P; GlyT2: green; slit-3: red), LPSN (Q; LPSN: green; slit-3: red; NT435: blue) and SPSN (R; SPSN: green; slit-3: red; NT435: blue) in the cervical spinal cord. (S-U) Quantification of the proportion of glycinergic neurons, LPSN and SPSN expressing slit-1 (S), slit-2 (T) and slit-3 (U) in the unlesioned and lesioned cervical spinal cord. Scale bars in A,B,G,H,M,N, 250 µm; Scale bars in C,I,O, 25 µm; Scale bars in D-F,J-K,P-R, 25 µm.

Mentions: The process of axon pathfinding is mediated by a number of guidance molecules, among them slits (slit-1,-2,-3) and their receptors (robo-1,-2,-3), which have been shown to have a repulsive effect on axons during development [22]–[23] and have been proposed to restrict axonal growth at the lesion site following spinal cord injury [24]. To assess whether slits can also regulate axon growth during axonal remodelling distant from the lesion site we first investigated the expression of slit family members in the unlesioned mouse spinal cord by in situ hybridization. Hybridization with the anti-sense probe shows that slit-1, slit-2 and slit-3 mRNAs are detected in all laminae of the cervical gray matter (Fig. 1A,G,M) while hybridization with the sense probe showed no signal (Fig. 1B,H,N). In particular, slit-1 and slit-2 show high expression levels throughout the spinal gray matter and in particular in the ventral horn (Table 2) while slit-3 mRNA seems to be expressed at a lower level. Analysis after counterstaining with NeuN suggests that slits are expressed by neurons including by interneurons and motoneurons (Fig. 1C,I,O). To better understand which populations of spinal interneurons express slit mRNA, we visualized glycinergic interneurons using a transgenic label (Fig. 1D,J,P) and LPSN (Fig. 1E,K,Q) and SPSN (Fig. 1F,L,R) using retrograde labelling . Our results show that, 60 to 80% of all propriospinal neurons but only about 30% of glycinergic interneurons express slit-1, -2 and -3 (Fig. 1S-U). While overall this expression pattern is rather unchanged in the cervical spinal cord of mice at 3 and 12 weeks after injury, there is a moderate but significant increase in the proportion of propriospinal neurons that expressed slit-1 or slit-3 after injury (Fig. 1 S-U).


Abundant expression of guidance and synaptogenic molecules in the injured spinal cord.

Jacobi A, Schmalz A, Bareyre FM - PLoS ONE (2014)

In Situ hybridization pattern of Slit-1,-2,-3 in the cervical spinal cord.In situ hybridization of slit-1 (A-F), slit-2 (G-L), slit-3 (M-R) mRNA in the unlesioned cervical spinal cord. Strong signals for slit-1 (A) and slit-2 (G) are detected with the anti-sense probe in cervical interneurons and motoneurons while slit-3 (M) shows a weaker signal. No signals are detected with the sense probes for slit-1 (B), slit-2 (H) or slit-3 (N). (C, I, O) Epifluorescence images of double-labeled neurons in the ventral horn (NeuN: green; In situ signal: red). (D-F) Co-localization of slit-1 mRNA in glycinergic neurons (D; GlyT2: green; slit-1: red), LPSN (E; LPSN: green; slit-1: red; NT435: blue) and SPSN (F; SPSN: green; slit-1: red; NT435: blue) in the cervical spinal cord. (J-L) Co-localization of slit-2 mRNA in glycinergic neurons (J; GlyT2: green; slit-2: red), LPSN (K; LPSN: green; slit-2: red; NT435: blue) and SPSN (L; SPSN: green; slit-2: red; NT435: blue) in the cervical spinal cord. (P-R) Co-localization of slit-3 mRNA in glycinergic neurons (P; GlyT2: green; slit-3: red), LPSN (Q; LPSN: green; slit-3: red; NT435: blue) and SPSN (R; SPSN: green; slit-3: red; NT435: blue) in the cervical spinal cord. (S-U) Quantification of the proportion of glycinergic neurons, LPSN and SPSN expressing slit-1 (S), slit-2 (T) and slit-3 (U) in the unlesioned and lesioned cervical spinal cord. Scale bars in A,B,G,H,M,N, 250 µm; Scale bars in C,I,O, 25 µm; Scale bars in D-F,J-K,P-R, 25 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921160&req=5

pone-0088449-g001: In Situ hybridization pattern of Slit-1,-2,-3 in the cervical spinal cord.In situ hybridization of slit-1 (A-F), slit-2 (G-L), slit-3 (M-R) mRNA in the unlesioned cervical spinal cord. Strong signals for slit-1 (A) and slit-2 (G) are detected with the anti-sense probe in cervical interneurons and motoneurons while slit-3 (M) shows a weaker signal. No signals are detected with the sense probes for slit-1 (B), slit-2 (H) or slit-3 (N). (C, I, O) Epifluorescence images of double-labeled neurons in the ventral horn (NeuN: green; In situ signal: red). (D-F) Co-localization of slit-1 mRNA in glycinergic neurons (D; GlyT2: green; slit-1: red), LPSN (E; LPSN: green; slit-1: red; NT435: blue) and SPSN (F; SPSN: green; slit-1: red; NT435: blue) in the cervical spinal cord. (J-L) Co-localization of slit-2 mRNA in glycinergic neurons (J; GlyT2: green; slit-2: red), LPSN (K; LPSN: green; slit-2: red; NT435: blue) and SPSN (L; SPSN: green; slit-2: red; NT435: blue) in the cervical spinal cord. (P-R) Co-localization of slit-3 mRNA in glycinergic neurons (P; GlyT2: green; slit-3: red), LPSN (Q; LPSN: green; slit-3: red; NT435: blue) and SPSN (R; SPSN: green; slit-3: red; NT435: blue) in the cervical spinal cord. (S-U) Quantification of the proportion of glycinergic neurons, LPSN and SPSN expressing slit-1 (S), slit-2 (T) and slit-3 (U) in the unlesioned and lesioned cervical spinal cord. Scale bars in A,B,G,H,M,N, 250 µm; Scale bars in C,I,O, 25 µm; Scale bars in D-F,J-K,P-R, 25 µm.
Mentions: The process of axon pathfinding is mediated by a number of guidance molecules, among them slits (slit-1,-2,-3) and their receptors (robo-1,-2,-3), which have been shown to have a repulsive effect on axons during development [22]–[23] and have been proposed to restrict axonal growth at the lesion site following spinal cord injury [24]. To assess whether slits can also regulate axon growth during axonal remodelling distant from the lesion site we first investigated the expression of slit family members in the unlesioned mouse spinal cord by in situ hybridization. Hybridization with the anti-sense probe shows that slit-1, slit-2 and slit-3 mRNAs are detected in all laminae of the cervical gray matter (Fig. 1A,G,M) while hybridization with the sense probe showed no signal (Fig. 1B,H,N). In particular, slit-1 and slit-2 show high expression levels throughout the spinal gray matter and in particular in the ventral horn (Table 2) while slit-3 mRNA seems to be expressed at a lower level. Analysis after counterstaining with NeuN suggests that slits are expressed by neurons including by interneurons and motoneurons (Fig. 1C,I,O). To better understand which populations of spinal interneurons express slit mRNA, we visualized glycinergic interneurons using a transgenic label (Fig. 1D,J,P) and LPSN (Fig. 1E,K,Q) and SPSN (Fig. 1F,L,R) using retrograde labelling . Our results show that, 60 to 80% of all propriospinal neurons but only about 30% of glycinergic interneurons express slit-1, -2 and -3 (Fig. 1S-U). While overall this expression pattern is rather unchanged in the cervical spinal cord of mice at 3 and 12 weeks after injury, there is a moderate but significant increase in the proportion of propriospinal neurons that expressed slit-1 or slit-3 after injury (Fig. 1 S-U).

Bottom Line: To assign the expression of these molecules to distinct populations of interneurons we labeled short and long propriospinal neurons by retrograde tracing and glycinergic neurons using a transgenically expressed fluorescent protein.Overall the expression pattern of guidance and synaptogenic molecules in the cervical spinal cord appeared to be stable over time and was not substantially altered following a midthoracic spinal cord injury.Taken together, our study indicates that many of the guidance and synaptogenic cues that regulate neuronal circuit formation in development are also present in the adult CNS and therefore likely contribute to the remodelling of axonal connections in the injured spinal cord.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Neuroimmunology, Ludwig-Maximilians University Munich, Munich, Germany.

ABSTRACT

Background: Spinal interneurons have emerged as crucial targets of supraspinal input during post-injury axonal remodelling. For example, lesioned corticospinal projections use propriospinal neurons as relay stations to form intraspinal detour circuits that circumvent the lesion site and contribute to functional recovery. While a number of the molecules that determine the formation of neuronal circuits in the developing nervous system have been identified, it is much less understood which of these cues are also expressed in the injured spinal cord and can thus guide growing collaterals and initiate synaptogenesis during circuit remodelling.

Methodology/principal findings: To address this question we characterized the expression profile of a number of guidance and synaptogenic molecules in the cervical spinal cord of healthy and spinal cord-injured mice by in situ hybridization. To assign the expression of these molecules to distinct populations of interneurons we labeled short and long propriospinal neurons by retrograde tracing and glycinergic neurons using a transgenically expressed fluorescent protein. Interestingly, we found that most of the molecules studied including members of slit-, semaphorin-, synCAM-, neuroligin- and ephrin- families as well as their receptors are also present in the adult CNS. While many of these molecules were abundantly expressed in all interneurons examined, some molecules including slits, semaphorin 7a, synCAM4 and neuroligin 1 showed preferential expression in propriospinal interneurons. Overall the expression pattern of guidance and synaptogenic molecules in the cervical spinal cord appeared to be stable over time and was not substantially altered following a midthoracic spinal cord injury.

Conclusions: Taken together, our study indicates that many of the guidance and synaptogenic cues that regulate neuronal circuit formation in development are also present in the adult CNS and therefore likely contribute to the remodelling of axonal connections in the injured spinal cord.

Show MeSH
Related in: MedlinePlus