Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Morcuende S, Matarredona ER, Benítez-Temiño B, Muñoz-Hernández R, Pastor AM, de la Cruz RR. Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion. J Comp Neurol. 2011; 519(12): 233552, which has been published in final form at https://dx.doi.org/10.1002/cne.22630. This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." Differential Regulation of the Expression of Neurotrophin Receptors in Rat Extraocular Motoneurons After Lesion Sara Morcuende, Esperanza R. Matarredona, Beatriz Be n ´ ı tez-Temi n ˜ o, Roc´ ı o M un ˜ oz-Her n ´ a ndez, A ´ ngel M. Pastor, * and Rosa R. de la Cruz Departamento de Fisiolog´ıa y Zoolog´ıa, Facultad de Biolog´ıa, Universidad de Sevilla, 41012 Sevilla, Spain ABSTRACT Neurotrophins acting through high-affinity tyrosine kinase receptors (trkA, trkB, and trkC) play a crucial role in regulating survival and maintenance of specific neuronal functions after injury. Adult motoneurons supplying extraocular muscles survive after disconnection from the target, but suffer dramatic changes in morphological and physiological properties, due in part to the loss of their trophic support from the muscle. To investigate the dependence of the adult rat extraocular motoneurons on neurotrophins, we examined trkA, trkB, and trkC mRNA expression after axotomy by in situ hybridization. trkA mRNA expression was detectable at low levels in unlesioned motoneurons, and its expression was downregulated 1 and 3 days after injury. Expression of trkB and trkC mRNAs was stronger, and after axotomy a simultaneous, but inverse regulation of both receptors was observed. Thus, whereas a considerable increase in trkB expression was seen about 2 weeks after axotomy, the expression of trkC mRNA had decreased at the same post-lesion period. Injured extraocular motoneurons also experienced an initial induction in expression of calcitonin gene-related peptide and a transient downregulation of cholinergic characteristics, indicating a switch in the phenotype from a transmitter-specific to a regenerative state. These results suggest that specific neurotrophins may contribute differentially to the survival and regenerative responses of extraocular motoneurons after lesion. INDEXING TERMS: axotomy; trk receptor; ChAT; CGRP; oculomotor; abducens Neurotrophins are a family of growth factors that play essential roles, not only in neuron survival and the establishment of intercellular communication during development, but also in the maintenance and plasticity of the adult nervous system and in the neuronal response to lesion (for review, see Sofroniew et al., 2001; Chao, 2003; Allen and Dawbarn, 2006). Consequently, neuronal dependence on trophic support continues throughout the entire lifespan. In mammals, the neurotrophin family includes nerve growth factor (NGF; Levi-Montalcini, 1982), brain-derived neurotrophic factor (BDNF; Leibrock et al., 1989), neurotrophin-3 (NT-3; Jones and Reichardt, 1990), and neurotrophin-4/5 (NT-4/5; Ip et al., 1992). Their survival-promoting effects have been shown to be mediated by high-affinity receptors of the trk family of tyrosine kinases (Sendtner et al., 1996), which include trkA, trkB, and trkC receptors (Barbacid, 1994). Typically, NGF binds to and activates trkA, BDNF and NT-4/5 interact with trkB, and NT-3 interacts with trkC (Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001). Besides these specific receptors, all neurotrophins bind the lowaffinity receptor p75. Trophic supply in the adult central nervous system may be interrupted by several types of insult, including axotomy, excitotoxicity, ischemia, or neurodegenerative diseases (Giehl et al., 1998; Venero et al., 2000, Boyd and Gordon, 2003). Indeed, both the neurotrophin requirement and the expression of different neurotrophin receptors are usually modified in lesioned neurons (Koliatsos Grant sponsor: Ministerio de Ciencia y Tecnolog´ıa, Fondo Europeo de Desarrollo Regional (MYCT-FEDER); Grant numbers: BFI2006-08414 and BFU2009-07121; Junta de Andaluc´ıa; Grant numbers: CVI-4617 and CVI6053; Grant num ber: P09-CVI-4617; Gran t sponsor: Fundacio ´ n Eugenio Rodr´ıguez Pascual. *CORRESPONDENCE TO: Dr A ´ ngel M. Pastor, Dept. de Fisiolo g´ ı a y Zoolog´ıa, Facultad de Biolog´ıa, Avda. Reina Mercedes, 6, 41012 Sevilla, Spain. E-mail: ampas[email protected] et al., 1991; Connor et al., 1996; Canals et al., 1999; Duprey-D´ıaz et al., 2002). Motoneurons represent one of the most widely used models for the study of trophic dependence. Motoneurons are known to receive NGF, BDNF, and NT-3 from the muscle (DiStefano et al., 1992). In the adult, motoneurons survive after target disconnection, but with altered morphological and physiological properties, probably due to the lack of trophic factors supplied from the muscle. However, alternative sources of trophic support may arise through autocrine or paracrine pathways (Kobayashi et al., 1996). Injury can also cause neuronal dedifferentiation and switching of the neuronal phenotype from a transmitterspecific to a regenerative state (Navarro et al., 2007), with the induction or upand downregulation of numerous molecules that are related to neuronal survival and axonal regeneration (Costigan et al., 2002; Kubo et al., 2002; Schmitt et al., 2003; Yang et al., 2004). In motoneurons, a reduction in choline acetyltransferase (ChAT) expression, an enzyme involved in the synthesis of the neurotransmitter acetylcholine, often occurs after nerve damage (Lams et al., 1988; Matsuura et al., 1997; Wang et al., 1997). In contrast, other molecules like calcitonin gene-related peptide (CGRP), a protein related to rearrangement of synaptic connections, increase their expre ssion after lesion (Calder o ´ et al., 1992; Borke et al., 1993; Piehl et al., 1993; Fukuoka et al., 1999). The oculomotor system constitutes a good model for the study of plastic changes induced by target loss; first, because it is a well-characterized system both anatomica ll y and ph y s i o l o g ic a ll y ( B¨ u ttner - Enneve r , 2006 ) , and s ec - ond because data are available that were obtained after different types of insult to both extraocular motoneurons and preoculomotor neurons. The motoneurons that innervate the eye muscles are located in three brainstem nuclei: 1) the oculomotor nucleus, innervating four of the extraocular muscles (the ipsilateral medial rectus, inferior rectus, and inferior oblique muscles, and the contralateral superior rectus muscle); 2) the trochlear nucleus innervating the contralateral superior oblique muscle; and 3) the abducens muscle innervating the ipsilateral lateral rectus muscle (G l i ck s man , 1980 ; B¨ u ttner - Ennever , 2006 ) . T h e a bducens nucleus also contains a group of premotor cells, the abducens internuclear neurons. In cats, these cells exhibit marked changes in firing properties after target removal, but return to normal after 1 month. This is likely due to the reestablishment of synaptic connections with novel targets within their terminal area (de la Cruz et al., 1994a,b, 1996, 2000; Pastor et al., 2000), although this also occurs with im p l an t e d neu r o n s a f t e r e mb ry o n i c g ra ft ( B e n ´ ı t e zTe m i n ˜ o e t al . , 2002 ; f o r re v i ew , s ee B e n ´ ı t e z-T e m i ˜ n o e t a l . , 200 5) . The functional recovery of these neurons has been reported to be causally linked to the neurotrophic supply arising from the new targets. In line with this, the exogenous administration of different neurotrophins (BDNF, NT-3, or NGF), for which oculomotor nuclei possess receptors (Be n ´ ı tez-T emi n ˜o et al., 2004), to the proximal stump of cat axotomized abducens motoneurons has been described to differentially restore the electrophysiological and morphological changes induced by lesion (Davis-L o ´ pez de Carrizo sa et al., 2009, 2010). In the present work, we have used the extraocular motor nuclei of adult rats as the anatomical substrate to investigate the changes induced by axotomy in the expression of several proteins that play a relevant role in the neural response to lesion. Our lesioning model consisted of enucleation, a procedure that involves both the injury to the extraocular motor nerves and the removal of target tissues within the orbit. In particular, we have characterized axotomyinduced changes in expression of the neurotrophin receptors trkA, trkB, and trkC, and of proteins related to either neurotransmission (ChAT) or regeneration (CGRP). This information will allow a better understanding of the process of structural and functional reorganization taking place in motoneurons after lesion, especially with respect to changes in trophic sensitivities and in the synthesis of distinct proteins whose functional role may reflect different operating modes of the motoneuron. MATERIALS AND METHODS Animals and surgical procedures Wistar rats were used in accordance with the guidelines of the European Union (86/609/EU) and Spanish law (R.D. 120/2005 BOE 252/34367-91, 2005) for the use and care of laboratory animals. Protocols used in this study were approved by the ethics committee of the Universidad de Sevilla. Adult rats (n ¼ 75) of either sex were monocularly enucleated under general anesthesia (sodium pentobarbital, 35 mg/kg, i.p.) as a method to axotomize extraocular motoneurons (Morcuende et al., 2005). The left eyeball was removed, the intraorbital tissues were eliminated, and the eyelids were sutured to close the cavity. Therefore, besides axotomy, the enucleation procedure also removed the target structures that could trophically supply the motoneurons. The analgesic pirazolone (0.1 g/kg, i.m.) was administered for 2 days, and the animals were monitored daily until healing for potential signs of distress. To analyze the time course of axotomy-induced changes in the expression of neurotrophin receptor mRNAs and in the expression of ChAT and CGRP proteins in extraocular motoneurons, animals were separated into five groups of different survival times after lesion (1, 3, 15, 30, and 60 days). Each experimental group comprised three to five animals. trk mRNAs expression by in situ hybridization In situ hybridization with oligonucleotide probes was performed as described by Wisden and Morris (2002). Oligonucleotide probes complementary to parts of mRNA encoding trkA, trkB, and trkC were synthesized (Genosys, Sigma, Saint Louis, MO) and radiolabeled with a - 35 S-dATP (Amersham, Piscatawa y, NJ) at the 3 0 end by using terminal deoxyribonucleotidyl transferase (Promega, Madison, WI). The specific activities of labeled probes used ranged from 200,000 to 300,000 cpm/ l l. The following antisense DNA oligonucleotides were used: for rat trkA mRNA 5 0 -AGG GTT GAA CTC AAA AGG GTT GTC CAT AAA GGC AGC CAT GAT G-3 0 (accession number M85214, bases 1,184–1 ,231; Meakin et al., 1992); rat trkB mRNA 5 0 -CTG CGA CTG CGT CAG CTC GGT GGG CGG GTT ACC CTC TGC CAT-3 0 (accession number M55291, bases 2,576–2,617; Middlem as et al., 1991); rat trkC mRNA 5 0 -GCT CCT TAA GGA AGT GGC CGT TGA TGG TCT GGT TGG CTG TGC CCA G-3 0 (accession number L03813, bases 1,201–1,246; Merlio et al., 1992). The trkB probe was complementary to mRNA encoding the tyrosine kinase domain of full-length trkB. The use of all these probes in the adult rat brain has been described elsewhere (Merlio et al., 1992; Piehl et al., 1994), and they generated the expected pattern. Rat brains were dissected out and frozen on dry ice-isopentane. Serial coronal sections (12 l m) throughout the brainstem were cut on a Leica CM 1850 cryostat and mounted on poly-L-lysine-coated slides, fixed for 5 minutes in 4% paraformaldehyde, dehydrated through a graded ethanol series, and stored in 95% ethanol. Parallel sections were processed for in situ hybridization with the different a 35 S-dATP 3 0 -end-l abeled probes. Sections tha t were to undergo in situ hybridization were air-dried for 1– 2 hours. Hybridizations were performed in a buffer containing 50% formamide, 4X sodium chloride-sodium citrated buffer (SSC), 10% dextran sulfate, 5X Denhardt’s solution, 0.2 mg/ml salmon sperm DNA, 0.1 mg/ml polyadenilic acid, 0.025 M sodium phosphate (pH 7.0), and 1 mM sodium pyrophosphate. Sections were hybridized at 42 ◦ C in a humidified chamber for 16–18 hours with 100 l l of hybridization buffer, 0.02 M dithiothreitol (DTT), and 1 l l of the radioactive probe (5 ng/ l l) per slide. Following hybridization, sections underwent three washes of decreasing stringency (two washes of 1X SSC at 55 ◦ C for 30 minutes and one wash of 0.1X SSC at room temperature for 1 minute), dehydrated in ethanol (70% and 95%, 1 minute each), and air-dried. Sections were exposed to a radiosensitive Kodak Biomax MR X-ray film for 1 week in darkness, and afterward dipped in Amersham LM1 photoemulsion, exposed for 6 weeks at 4 ◦ C, developed in Kodak D-19 developer, fixed with Kodak Rapid Fixer, and counterstained with cresyl violet. Hybridization signals were regarded as positive when accumulation of silver grains clearly above background levels (statistical support discussed below) were found located over the soma of individual cells. Controls for the specificity of the in situ labeling were demonstrated by a concentration-dependent depletion of the signal, provided by a 35 S-dATP-labeled probe, when the same unlabeled oligonucleotide was added in excess (100 fold) to the hybridization cocktail. Data analysis of in situ hybridization signals Tissues were visualized and analyzed by brightfield optic microscopy (Zeiss Axiophot, Oberkochen, Germany). The density of silver grains over the cytoplasm of each identified neuron was determined by quantifying the number of silver grains and measuring the area of each soma analyzed. Counts in the abducens, trochlear, and oculomotor nuclei were performed by using a 40 × objective. For each section, background signal level was determined by averaging grain counts over three areas of neuropil lacking positively labeled cell bodies, and background labeling density was subtracted from the cell labeling. For each trk receptor and survival time, motoneurons were sampled from the abducens nucleus (n ¼ 38–52), the trochlear nucleus (n ¼ 20–38), and the rostral two-thirds of the oculomotor nucleus (n ¼ 85–106) because the caudal third preferentially contains the contralaterally projecting superior rectus motoneurons. Values for the lesioned side were expressed as percentages relative to the unlesioned (control) side. Statistical analysis for significant differences between the control and lesioned sides were performed by Student’s t-test and between survival times by one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons. In all cases, the level of significance was P < 0.05. ChAT and CGRP immunostaining To prepare the tissue for immunocytochemistry, rats were perfused transcardially under deep anesthesia (sodium pentobarbital, 50 mg/kg, i.p.) with 100 ml of physiological saline followed by 250 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The brainstem was removed and cryoprotected by immersion in a solution of 30% sucrose in sodium phosphate-buffered saline (PBS) until it sank. The brainstem was then cut coronally at 40l m-thick sections on a cryostat. Sections were divided in two adjacent series to be processed for ChAT or CGRP immunostaining, respectively. Motoneurons in the abducens, trochlear, and oculomotor nuclei were identified by using an antibody against ChAT (polyclonal goat anti-ChAT, 1:100, AB144P, Chemicon, Temecula, CA; Table 1). Endogenous peroxidase activity was inactivated by a 10-minute incubation in a TABLE 1. Primary Antibodies Used in This Study Antigen Immunogen Host/type Manufacturer Dilution ChAT Human placental ChAT enzyme Goat polyclonal Chemicon (Temecula, CA), 1:100 CGRP Synthetic peptide (H-Ser-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-HisArg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-AspAsn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Glu-Ala-Phe-NH 2 ). cat. no. AB144P Rabbit polyclonal Bachem/Peninsula (San Carlos, CA), cat. no. T-4032. 1:6,000 Abbreviations: ChAT, choline acetyltransferase; CGRP, calcitonin gene-related peptide. solution containing 10% methanol and 2% H 2 O 2 in PBS. After washing with PBS, sections were incubated in blocking solution (3% normal rabbit serum in PBS with 0.1% Triton X-100, 40 minutes). Tissue was then incubated overnight in the primary antibody solution prepared in blocking solution with 0.05% sodium azide. After washing, tissue was exposed for 2 hours to the secondary antibody solution (biotinylated rabbit anti-goat, 1:250, Vector, Burlingame, CA). After rinsing, tissue was then incubated for 90 minutes in an avidin-biotin-horseradish peroxidase (HRP) complex (ABC, Vector). Motoneurons were finally made visible by using 3,3 0 -diaminobenzidine tetrahydrochloride (DAB) at 0.05% with 0.08% H 2 O 2 diluted in PBS. Sections were mounted on glass slides, dehydrated, cleared and coverslipped. For CGRP immunohistochemistry, the same protocol as above was used, but some additional steps were included. Nonspecific binding sites were blocked with 0.2 M glycine and lysine, 0.2% bovine serum albumin (BSA), 0.1% gelatin, and 10% normal goat serum in PBS with 0.1% Triton X-100. After 4 hours, tissue was incubated in a solution containing an antibody raised against CGRP (polyclonal rabbit anti-CGRP, 1:6000; T-4032, Peninsula/ Bachem, San Carlos, CA [previously available from Peninsula Laboratories, Belmont, CA, as ICH 6006]; Table 1) and then in the secondary antibody solution (biotinylated goat anti-rabbit, 1:250, Vector). After analysis for CGRP staining, coverslips were removed, and tissue was rehydrated in alcohols of decreasing concentrations, and then counterstained with toluidine blue in order to perform total cell counts in each nuclei. Antibody characterization The goat polyclonal anti-ChAT antibody was raised against human placental ChAT, which is identical to the brain enzyme (Bruce et al., 1985), and was affinity-purified (manufacturer’s technical information). The antibody stained a single band of 68–70 kDa corresponding to the predicted molecular weight of ChAT on Western blot from rat tissue (Corcoran et al., 2004; Brunelli et al., 2005). Specificity has been previously demonstrated by using the blocking peptide ( M ´ a rquez-Ruiz et al., 2007 ). All staining was abolished when the primary antibody was preincubated with 15 l g/ml of the blocking peptide (choline acetyltransferase, rat recombinant protein, Chemicon, cat. no. AG220) for 3 hours prior to being used on the tissue sections from rat and cat brain. In the present study, we have used this antibody as a marker of brainstem motoneurons in the III, IV, and VI nuclei, as it has been described to label cranial nerve motoneurons selectively (Geerling et al., 2010). With the same antibody, Wang and Morales (2009) observed a 100% correspondence between ChAT immunolabeling and ChAT mRNA labeling in the pedunculo-pontine nucleus. The rabbit polyclonal antibody against CGRP was raised against a synthetic peptide (H-Ser-Cys-Asn-ThrAla-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-ArgSer-Gly-Gly-Val-Val-Lys-Asp-Asn-Phe-Val-Pro-Thr-Asn-ValGly-Ser-Glu-Ala-Phe-NH 2 ). This antibody reacts 100% with rat a -CGRP and 79% with rat b -CGRP, but does not crossreact with rat amylin or calcitonin, as confirmed by radioimmunoassay (Peninsula Laboratories data sheet). It detects a single band of 4 kDa on Western blots of mouse trigeminal ganglion corresponding to the CGRP 32-amino acid peptide, and preadsorption with a -CGRP eliminated this band (Kosaras et al., 2009). Its specificity has been also tested by preabsorption with CGRP peptide, completely abolishing the immunostaining in rat nerve (Ma and Quirion, 2006) and rat brainstem (Wang et al., 2006). Cell counting and analysis of immunostaining data Cells immunolabeled for ChAT in the three extraocular motor nuclei (abducens, trochlear, and oculomotor nuclei) were visualized, and all motoneurons were counted by using a light microscope (Olympus BX61, Tokyo, Japan). Abercrombie’s correction was then applied (Abercrombie, 1946). Photomicrographs were taken by brightfield optic microscopy (Olympus BX61). Pictures were adjusted for brightness and contrast with Adobe Photoshop 7.0 (San Jose, CA). For the abducens and trochlear nuclei, the ipsilateral and contralateral nucleus were the respective lesioned sides. For the oculomotor nucleus, however, it was necessary to apply another correction due to the presence of a contralaterally projecting group of motoneurons (Morcuende et al., 2005). Figure 1. Modulation of trkA mRNA expression after axotomy. Images show the decrease in trkA mRNA in abducens and oculomotor neurons by in situ hybridization a short time after unilateral enucleation. Note the lower silver grain staining in lesioned neurons of both nuclei compared with control neurons. A: Image of control abducens neurons showing the silver grain labeling within the motoneuron c ytoplasm. Cells were counterstained with cresyl violet for identification. B: Image of abducens neurons on the lesioned side showing a reduced level of trkA mRNA signal 1 day after axotomy. C,D: Neurons in the control oculomotor nucleus (C) also exhibited levels of trkA mRNA labeling higher than on the injured side (D) 3 days after lesion. Abbreviations: ABD, abducens nucleus; OCM, oculomotor nucleus. Scale bar ¼ 50 lm in D (applies to A–D). Because the oculomotor nucleus contains four subnuclei of motoneurons, three ipsilateral and one contralateral for a given eye (Glicksman, 1980; B¨ u ttner-Ennever, 2006) with approximately equal number of motoneurons (Glicksman, 1980; Miyazaki, 1985), it was necessary to solve the following two equations to determine the number of lesioned and control cells: 3x þ y ¼ n 1 and 3y þ x ¼ n 2 , where n 1 and n 2 are the ipsilateral and contralateral number of oculomotor nucleus motoneurons, respectively. Thus, we determined x and y, where x is the number of ChAT-immunoreactive cells per lesioned subnucleus (three groups ipsilateral and one contralateral) and y is the number of ChAT-immunoreactive cells per control subnucleus. To evaluate the effects of axotomy, the number of lesioned ChAT-labeled cells was expressed as the percentage relative to the number of control labeled cells. Comparisons between groups were carried out by using one-way ANOVA followed by post hoc multiple comparisons. When the comparison was performed between two groups, Student’s paired t-test was used. In all cases, the level of significance was P < 0.05. Values were expressed as mean 6 SEM, and as percentages relative to the unlesioned side. For CGRP immunostaining, all labeled motoneurons were counted, and Abercrombie’s (1946) correction was then applied. In the oculomotor nucleus, the contralateral motoneurons expressing CGRP were considered to belong to the superior rectus group. No staining against CGRP was found in control motoneurons, so we could not express these data as percentages relative to the unlesioned side. Therefore, once CGRP counting was performed, tissue was counterstained by using toluidine blue, and all abducens, trochlear, and oculomotor Nisslstained neurons were counted and corrected by using Abercrombie’s method. Data were then expressed as the percentage of the total Nissl-stained cells that expressed CGRP. Statistical analysis and comparisons at different time points for each nucleus were carried out by using one-way ANOVA followed by post hoc multiple comparisons. In all cases, the level of significance was P < 0.05. RESULTS Expression of mRNAs for neurotrophin receptors of the trk family in the extraocular motor nuclei In situ hybridization against the mRNA of the three trk receptors was performed to assess their presence in the motoneurons that control the eye muscles. Positive labeling for trk mRNAs was found in the cell bodies of the neurons located in abducens, trochlear, and oculomotor nuclei of the unlesioned (control) side. The signal was mainly found over large cell bodies, most likely motoneurons (Figs. 1, 3, 5). The in situ hybridization labeling revealed clearly that the mRNA for trkB, the receptor for BDNF, was the most highly expressed trk neurotrophin receptor in control extraocular motoneurons. trkC mRNA and trkA mRNA were expressed in a lower degree, the latter being the trk receptor with the lowest signal expression (Figs. 1A,C, 3A,C,E, 5A,C,E). After enucleation and removal of the orbital contents, the trk receptor mRNA expression was analyzed at different time points after injury (1, 3, 15, 30, and 60 days) in the extraocular motoneurons. Modulation of trkA mRNA expression in extraocular motoneurons after lesion In the extraocular motoneurons, in situ hybridization signal for trkA mRNA decreased significantly, reaching approximately 65% of control values on days 1 and 3 after lesion in the abducens nuclei (day 1: 65.60 6 6.87 %, P < 0.05; day 3: 72.23 6 5.62%, P < 0.01; day 30: 58.13 6 11.39%, P < 0.05) and oculomotor nuclei (day 1: 63.79 6 8.03%, P < 0.01; day 3: 68.10 6 9.40%, P < 0.05; Figs. 1, 2). A similar decay was also observed in trochlear motoneurons in this period, but this decrement did not reach statistical significance compared with the control side (e.g., day 3: 57.61 6 14.63%, P > 0.05; Fig. 2B). The decrease observed in trkA mRNA after lesion was transient because trkA expression in the oculomotor and trochlear nuclei recovered to control values between 1 and 2 months after the lesion (Fig. 2). Therefore, injury of extraocular nerves produced a short-term decrease in the mRNA expression of NGF receptor in the axotomized neurons, despite the scarce but evident basal expression of trkA mRNA in control extraocular motoneurons. Figure 2. A–C: Time course of changes in the percentage of trkA mRNA expression in lesioned neurons with respect to control in the abducens (A), trochlear (B), and oculomotor (C) nuclei. The control value (100%) is represented by the dashed horizontal line. Data are mean 6 SEM. Asterisks represent statistically significant differences compared with the control side (*, P < 0.05; **, P < 0.01; Student’s t-test). l and † represent statistically significant differences compared with day 1 and day 3, respectively ( P < 0.05, ANOVA test). Figure 3. Modulation of trkB mRNA expression after axotomy. Images show the increase in trkB mRNA expression in abducens, trochlear, and oculomotor neurons by in situ hybridization 15 days after unilateral enucleation. Note the high levels of signal for trkB mRNA expression in the control neurons and even higher levels in axotomized neurons, particularly in the oculomotor nucleus. A,C,E: Neurons in the control abducens (A), trochlear (C), and oculomotor (E) nuclei. B,D,F: Images corresponding to their respective lesioned side. Abbreviations: ABD, abducens nucleus; OCM, oculomotor nucleus; TRO, trochlear nucleus. Scale bar ¼ 50 l m in F (applies to A–F). Modulation of trkB mRNA expression in extraocular motoneurons after lesion As illustrated in Figure 3, labeling of trkB was conspicuous in the motoneurons of the abducens, trochlear, and oculomotor nuclei of the control side. Hybridization with the trkB mRNA probe demonstrated a strong, but transient, increase in labeling over the extraocular neurons after lesion compared with control neurons. In the abducens nucleus, a significant elevation in trkB mRNA signal was detected 3 days after lesion, and was about 150% of control values on day 15. However, it returned to control levels by 1 month post lesion (day 3: 124.61 6 9.39%, P < 0.05; day 15: 157.53 6 10.36%, P < 0.01; Figs. 3A,B, 4A). Similarly, in trochlear neurons the change in trkB mRNA expression was significantly different from the control side at 3 and 15 days after axotomy (day 3: 149.90 6 28.01%, P < 0.05; day 15: 144.33 6 10.51%, P < 0.01; Figs. 3C,D, 4B), but was at control level by 2 months. However, in oculomotor neurons, the increase in labeling was delayed and smaller compared with the other two nuclei. Thus, we did not find any change in trkB expression at short time intervals after lesion (1–3 days), but a significant increase was measured between 15 days and 1 month (day 15: 118.86 6 7.15%, P < 0.05; day 30: 128.24 6 9.36%, P < 0.05), returning to control levels by 2 months (Figs. 3E,F, 4C). These results showed a significant upregulation in the expression of the BDNF receptor, trkB, in the motoneurons innervating the eye muscles of the adult rat after axotomy, which was consistently found in the three extraocular motor nuclei by 2 weeks after lesion. Control levels of expression were recovered in all these structures by 2 months. Modulation of trkC mRNA expression in extraocular motoneurons after lesion A noticeable downregulation in trkC mRNA expression was observed in the three motor nuclei by 15 days after axotomy (Figs. 5, 6). In the abducens nucleus, we could observe only a 54.27 6 3.01% level of expression in the experimental side compared with the expression observed in control abducens neurons (P < 0.001; Fig. 6A). At the same time point, the oculomotor neurons showed an equally significant diminution. This loss was sustained for longer, as was evident, but not significant, on day 3, and was still present on day 30 (day 15: 69.45 6 3.55%, P < 0.001; day 30: 87.97 6 3.23%, P < 0.05; Fig. 6C). The expression of trkC mRNA in lesioned trochlear motoneurons was also depressed on days 3–30, but not at 15 days post lesion (day 3: 63.34 6 4.47%, P < 0.001; day 15: 68.21 6 9.89%, P > 0.05; day 30: 72.43 6 8.48%, P < 0.05; Fig. 6B). In all cases, the signal regained normal values by 2 months (Fig. 6). Thus, the in situ hybridization revealed that trkC mRNA levels in the extraocular motor nuclei of the adult rat decreased between 3 and 30 days after axotomy in a nucleusdependent fashion. ChAT expression was downregulated transiently in extraocular motoneurons after lesion Motoneuronal ChAT expression can be altered by several types of insult, including axotomy. We analyzed this point in the adult rat extraocular motoneurons by means of immunostaining following enucleation. No change was Figure 4. A–C: Time course of changes in the percentage of trkB mRNA expression in lesioned neurons with respect to controls (100%, dashed horizontal line) in abducens (A), trochlear (B), and oculomotor (C) nuclei. Data are mean 6 SEM. Asterisks represent statistically significant differences compared with the control side (*, P < 0.05; **, P < 0.01; Student’s t-test). ● , †, and ‡ represent statistically significant differences compared with day 3, day 15, and day 30, respectively ( P < 0.05, ANOVA test). Figure 11. Schematic diagram indicating alterations in extraocular motoneurons after axotomy. A: Time course of changes in the expression of trk receptors, choline acetyltransferase (ChAT), and calcitonin gene-related peptide (CGRP) in extraocular motoneurons after axotomy. B-1: The connected neuron shows a normal phenotype in afferent synaptic activity and firing rate. B-2: The axotomized neuron has reduced levels of firing rate and afferent synaptic activity. B-3: The neuron switches from a transmitter to a regenerative phenotype whereby the autocrine/paracrine and anterograde trophic support gains relative importance with respect to the retrograde trophic support. Appropriate levels of retrograde trophic factors delivered from different sources would encourage restoration of normal values in the altered molecules. inputs that show a high degree of dependence on target reinnervation for their recovery from the axotomized state (Delgado-Garc´ıa et al., 1988; de la Cruz et al., 1996; Pastor et al . , 2000 ; B e n ´ ı te zTe mi ˜ n o e t a l. , 200 5) . Induction of CGRP expression by axotomy The upregulation of CGRP, a protein involved in the formation and maintenance of neuromuscular junctions and axonal regeneration, seems to be a common response of cranial motoneurons following axotomy (Fukuoka et al., 1999). Its expression is regulated in an activity-dependent manner (Gon z ´ a lez-F orero et al., 2002 ). We have observed and quantified a clear induction of its expression in the extraocular motor nuclei at short time intervals (1 and 3 days) after lesion. This early induction has been suggested to exert a trophic influence on the lesioned neurons in severe environmental insult (Dumoulin et al., 1992). Thirty days after lesion, we observed a new peak in CGRP expression (Fig. 10). Chang et al. (2004) obtained similar results, and argued a relation between the expression of this molecule and innervation of a new target. In line with this, CGRP has been involved in the formation of synaptic processes (Sala et al., 1995). Increased CGRP staining has been shown to persist for a long time, and to be still evident when nearly complete reinnervation has been achieved, even when levels of neurotrophin receptors have returned to control levels (Piehl et al., 1993). Therefore, the fact that CGRP protein was expressed in extraocular motoneurons after lesion might suggest that a process of axonal regrowth and reinnervation has been at least initiated (Fig. 11A). Nevertheless, in our lesion model (i.e., enucleation), the chances for reinnervating a new target are scarce, and this could explain the slow, albeit incomplete, reduction in CGRP we observed over 2 months. In conclusion, the present study shows that differential regulation of trk receptor expression occurs in extraocular motoneurons after injury, with the most significant change being the robust increase in trkB expression. We can hypothesize that a period of axonal regrowth might take place shortly after axotomy, suggested by the induction in CGRP protein. Injured motoneurons may be less dependent on NGF and NT-3, as indicated by the decrease in trkA and trkC expression. 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