Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice

Neuroscience Letters 365 (2004) 97–101

Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice Shohei Oshima a,∗ , Keiko Yamada b , Tetsuo Shirakawa a , Masahiko Watanabe b a

Department of Pediatric Dentistry, Hokkaido University School of Dentistry, Sapporo 060-8586, Japan b Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan Received 12 August 2003; received in revised form 19 April 2004; accepted 22 April 2004

Abstract The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: High-affinity choline transporter; Neuronal tracer; Motoneuron; Axotomy; Nerve regeneration; Mouse

Acetylcholine (ACh) acts as a neurotransmitter at the neuromuscular junction of skeletal muscles. Recently, the high-affinity choline transporter CHT1 was identified and its specific expression in cholinergic neurons, including motoneurons, has been demonstrated [10]. After ACh hydrolysis by acetylcholinesterase (AChE), CHT1 works for the uptake of choline, which is utilized for ACh synthesis by choline acetyltransferase (ChAT) in the presynaptic terminals. Then, ACh is packaged into synaptic vesicles by vesicular transporter VAChT. It has been shown that motoneurons are able to survive after axotomy and regenerate their axons, and that expressions of ChAT and VAChT are drastically reduced in motoneurons after axotomy [8]. However, it is unclear how the expression of CHT1 is affected by axotomy and how it is influenced by the regeneration of impaired motor nerve. To address the issues, we followed the changes of CHT1 expression in axotomized hypoglossal neurons by in situ hybridization with special reference to their axonal reconnection to the target.

∗ Corresponding author. Tel.: +81-11-706-4292; fax: +81-11-706-4307. E-mail address: [email protected] (S. Oshima).

Adult C57BL/6J male mice (8–9 weeks) were used in this study and they were treated according to the Guide for the Care and Use of Laboratory Animals of Hokkaido University School of Medicine. Under anesthesia with pentobarbital (50 mg/kg of body weight), the right hypoglossal nerve was cut under the digastric muscle with a pair of iridectomy scissors and the nerve edges were placed closely to each other to facilitate regeneration. The mice were perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) before or at 1, 2, 3 or 4 weeks after axotomy (n = 3 at each point). Two days before the fixation, 0.25 mg of Fast Blue (Sigma, St. Louis, MO, USA) dissolved in 30 ␮l of distilled water was injected singly into the apex of the tongue by using syringe with a 30 G needle. Paraffin sections of the hypoglossal nucleus in the coronal plane (5 ␮m in thickness) were prepared. Four paraffin sections at an interval of 200 ␮m were chosen from the rostro-caudal extent of each mouse in for in situ hybridization. Digoxigenin (DIG)-labeled sense and antisense CHT1 cRNA probes were prepared by in vitro transcription using linearized Bluescript SK(−) plasmid [13] containing nucleotide residues 224–1963 of rat CHT1 cDNA [10]. Hybridization was performed as previously described [13]. The hybridization reaction was visualized by using DIG Nucleic

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Acid detection Kit and HNPP Fluorescent Detection Set (Roche, Manheim, Germany). For quantitative analyses, profiles of motoneurons larger than 20 ␮m in diameter were exclusively analyzed for obtaining the ratio of motoneurons expressing CHT1 mRNA or labeled with Fast Blue in the lesioned side relative to those in the control side. For immunohistochemistry, frozen sections of the tongue were prepared in the coronal plane (50 ␮m in thickness) and reacted with anti-VAChT antibody (Chemicon, Temecula, CA), anti-PGP9.5 antibody (Ultraclone, Willows, UK) and FITC-labeled ␣-bungarotoxin (Molecular Probes, Eugene, OR) and examined by confocal laser scanning microscopy, as previously described [13]. In the control side, virtually all of the hypoglossal neurons were labeled intensely with antisense probe for CHT1 mRNA at 1, 2, 3 and 4 weeks after axotomy (Fig. 1A, C and E), similarly to those of the mice without axotomy (data not shown). In adjacent sections, many hypoglossal neurons in the control side were retrogradely labeled with Fast Blue injected into the tongue (Fig. 1B, D and F). In contrast, marked changes were noted in the lesioned side. One week after unilateral axotomy, hybridizing signals for CHT1 mRNA were very low or negative (Fig. 1A) and none of hypoglossal neurons were labeled with Fast Blue (Fig. 1B) in the lesioned side. The percentages of hypoglossal neurons expressing CHT1 mRNA and those labeled with Fast Blue in the lesioned side relative to those in the control side were 3.5 ± 4.8% and 0 ± 0%, respectively (mean ± S.D., n = 3, Fig. 1G) at 1 week after axotomy. Two weeks after axotomy, a few labeled neurons appeared in the lesioned side (Fig. 1C and D), and the percentages increased to 36.0 ± 18.2% for CHT1 mRNA and 29.7 ± 15.6% for Fast Blue (Fig. 1G). They remarkably increased at 3 weeks after axotomy, being 86.1 ± 5.9% for CHT1 mRNA and 80.4 ± 15.0% for Fast Blue (Fig. 1G). Four weeks after axotomy, most hypoglossal neurons in the lesioned side recovered intense expression of CHT1 mRNA (94.0 ± 2.1%) and intense retrograde labeling with Fast Blue (92.4 ± 5.8%) (Fig. 1G), resulting in comparable images between the lesioned side and the control side (Fig. 1E and F). The specificity of hybridization was confirmed by blank signals with a use of the sense probe (data not shown). These results suggest that CHT1 mRNA expression is swiftly downregulated in axotomized hypoglossal neurons, and that its re-expression correlates temporally with reconnection between the hypoglossal nucleus and tongue. To clarify the relationship between CHT1 mRNA re-expression and axonal reconnection at the cellular level, double fluorescence for CHT1 mRNA and Fast Blue was applied to the same sections at 2 and 4 weeks after axotomy (Fig. 2A–D). Due to the technical limitation that all of the reconnected hypoglossal neurons are not retrogradely labeled by local injection of Fast Blue into the tongue, the number of CHT1 mRNA-positive hypoglossal neurons exceeded that of Fast Blue-labeled ones (Fig. 2A versus B and C versus D). Nevertheless, Fast Blue-labeled neurons in the lesioned side were very few in number 2 weeks after

axotomy, when the intensity of Fast Blue varied considerably from motoneuron to motoneuron: in some it was as intense as in neurons intact in the control side (arrowheads in Fig. 2B), while in others it was slightly distinguishable from the background (arrows). Four weeks after axotomy, however, the number of hypoglossal neurons with intense Fast Blue-labeling increased markedly (arrowheads in Fig. 2D). Importantly, all of these Fast Blue-labeled hypoglossal neurons, irrespective of their fluorescence intensities, expressed CHT1 mRNA at high levels at 2 and 4 weeks after axotomy (Fig. 2A and C), which indicated that all of Fast Blue-labeled motoneurons were positive for CHT mRNA. Therefore, CHT1 was selectively re-expressed in those motoneurons with successful reconnection to their targets. Furthermore, strong signals for CHT1 mRNA in motoneurons with low Fast Blue labeling suggest that the re-expression starts when their reconnection is immature or their retrograde axoplasmic transport is yet inefficient. Finally, we examined the time-course of CHT1 re-expression in the hypoglossal nucleus in relation with the reorganization of neuromuscular junctions by double fluorescence analysis for VAChT (marker for cholinergic synaptic vesicles), PGP9.5 (marker for nerve fibers) and ␣-bungarotoxin (marker for ACh receptor sites on muscle fibers) (Fig. 2E–P). Before axotomy, the three markers well overlapped with each other on muscle fibers of the tongue (Fig. 2E, I and M), indicating the apposition of presynaptic terminals to postsynaptic specialization at the neuromuscular junction. One week after axotomy, signals for presynaptic markers (VAChT and PGP9.5) disappeared in the lesioned side of the tongue, leaving postsynaptic specialization alone on muscle fibers (Fig. 2F, J and N). Two weeks after axotomy, VAChT and PGP9.5 appeared in the tongue, but they were fragmental and were localized around postsynaptic sites (Fig. 2G, K and O). Four weeks after axotomy, the majority of the ␣-bungarotoxin-bound postsynaptic specialization was tightly apposed to the clustered immunofluorescence for VAChT and PGP9.5 at the neuromuscular junctions (Fig. 2H, L and P). It is well known that motoneurons in the lower brainstem and spinal cord can survive after peripheral nerve injury and regrow their axons to reinnervate target muscles [14]. Upon and after motor nerve injury, changes in expressions of various molecules occur in motoneurons. In general, injured motoneurons downregulate molecules related to the neurotransmission [11,15], whereas they upregulate cytoskeletal proteins [16,18], trophic factors and their receptors [4–6], growth-associated proteins [16], and molecules related to anti-neurotoxicity [7,17]. Matsuura et al. have demonstrated that ChAT and VAChT mRNAs in hypoglossal neurons are dramatically reduced 1 week after axotomy and recovered gradually thereafter [8]. In the present study, CHT1 is shown to be one of such neurotransmission molecules downregulated temporally after nerve injury. Taking the fact that high-affinity choline uptake in presynaptic terminals is the rate-limiting step in ACh synthesis, the simultaneous

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Fig. 1. Temporal changes of CHT1 mRNA expression (A, C, E) and Fast Blue labeling (B, D, F) in the hypoglossal neurons after the right hypoglossal nerve axotomy. Sections were prepared at 1 week (A, B), 2 weeks (C, D), and 4 weeks (E, F) after axotomy. (*) The central canal in the medulla oblongata; (Con) the control side of hypoglossal nucleus; (Les) the lesioned side of hypoglossal nucleus. Scale bar, 100 ␮m. A histogram in G indicates the ratios of hypoglossal neurons which expressed CHT1 mRNA (black bars) or were labeled with Fast Blue (white bars) in the lesioned side relative to those in the control side. Vertical lines above each bar indicate S.D.

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Fig. 2. Double fluorescence histochemistry of axotomized hypoglossal neurons and neuromuscular junctions. (A–D) Fluorescent in situ hybridization for CHT1 mRNA (A, C) and retrograde labeling with Fast Blue (B, D) in hypoglossal neurons at 2 weeks (A, B) and 4 weeks (C, D) after axotomy. Arrowheads indicate neurons highly expressing CHT1 mRNA with intense Fast Blue labeling, and arrows indicate those with faint Fast Blue labeling. (E–P) Double fluorescence for VAChT (red) and ␣-bungarotoxin (green) (E–H), PGP9.5 (red) and ␣-bungarotoxin (␣-BT, green) (I–L) and VAChT (green) and PGP9.5 (red) (M–P) at neuromuscular junctions before axotomy (E, I, M) and at 1 week (F, J, N), 2 weeks (G, K, O) and 4 weeks (H, L, P) after axotomy. Scale bars, D, 100 ␮m; Q, 10 ␮m.

S. Oshima et al. / Neuroscience Letters 365 (2004) 97–101

downregulation of CHT1, ChAT and VAChT implies that, when the connection of motor pathway is disrupted, molecular machineries for ACh synthesis and release are all shut off at the transcriptional level by a common mechanism. Matsuura et al. have demonstrated that the percentage of hypoglossal neurons expressing ChAT or VAChT mRNAs in the lesioned side relative to those in the control side was 40–80% at 4 weeks after axotomy [8]. In the present study, the percentage of hypoglossal neurons expressing CHT1 mRNA in the lesioned side relative to that in the control side was 94.0 ± 2.1% at 4 weeks after axotmy. As long as the percentage of the mRNA levels of these genes is compared, the expression of CHT1 mRNA recovered faster than those of ChAT and VAChT mRNAs after axotomy. As the speed of nerve regeneration varies depending on the damage of the lesioned site, the discrepancy of the recovery of the mRNA expressions may be due to the difference of surgical strategies for axotomy. To clarify the relationship between CHT1 mRNA expression and axon regeneration, we examined for the first time their relationship by injecting a retrograde tracer in the tongue. As revealed by the presence of fluorescent signals of Fast Blue, the first sign of motor nerve reconnection was observed 2 weeks after axotomy. At this time point, all of the reconnected hypoglossal neurons strongly expressed CHT1 mRNA, although they were few in number and still low in intensity for Fast Blue. Regenerating nerves expressing VAChT and PGP9.5 appeared in the tongue at 2 weeks after axotomy, but they were fragmental and less concentrated around postsynaptic sites. Four weeks after axotomy, reconnection of the hypoglossal neurons was found at the level more than 90% of that in the control side and most of them highly expressed CHT1 mRNA. Intense VAChT accumulation was also observed at the neuromuscular junctions. Taken together, molecular machineries essential for cholinergic transmission are upregulated concomitantly and coordinately with motor nerve regeneration. A recent finding of CHT1 localization on a subpopulation of cholinergic synaptic vesicles and its activity-dependent trafficking to presynaptic membrane further suggests functional importance of CHT1 re-expression at regenerating neuromuscular junctions [1]. During development, initial contact of motor nerves with their target muscles occurs without presence of ACh [9], but neuronal activity is necessary for the subsequent maturation of the neuromuscular junctions [2,3,12]. Therefore, cholinergic transmission re-established in link with motor nerve reconnection may facilitate structural and functional maturation of regenerating neuromuscular junctions.

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