Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development

Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development
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  Regular Article Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development  Sara Morcuende, Beatriz Benı´tez-Temin˜o, Marı´a Luisa Pecero,Angel M. Pastor, Rosa R. de la Cruz*  Departamento de Fisiologı´a y Zoologı´a, Facultad de Biologı´a, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012-Sevilla, Spain Received 18 November 2004; revised 7 April 2005; accepted 4 May 2005Available online 1 June 2005 Abstract The highly specific projection of abducens internuclear neurons onto medial rectus motoneurons in the oculomotor nucleus is a goodmodel to evaluate the dependence on target cells for survival during development and in the adult. Thus, the procedure we chose toselectively deprive abducens internuclear neurons of their natural target was the enucleation of postnatal day 1 rats to induce the death of medial rectus motoneurons. Two months later, we evaluated both the extent of reduction in target size, by immunocytochemistry against choline acetyltransferase (ChAT) and Nissl counting, and the percentage of abducens internuclear neurons surviving target loss, by calretininimmunostaining and horseradish peroxidase (HRP) retrograde tracing. Firstly, axotomized oculomotor motoneurons died in a high percentage( ¨ 80%) as visualized 2 months after lesion. In addition, we showed a transient (1 month) and reversible down-regulation of ChATexpressionin extraocular motoneurons induced by injury. Secondly, 2 months after enucleation, 61.6% and 60.5% of the population of abducensinternuclear neurons appeared stained by retrograde tracing and calretinin immunoreaction, respectively, indicating a significant extent of celldeath after target loss (38.4% or 39.5%). By contrast, in the adult rat, neither extraocular motoneurons died in response to axotomy nor abducens internuclear neurons died due to the loss of their target motoneurons induced by the retrograde transport of toxic ricin injected inthe medial rectus muscle. These results indicate that, during development, abducens internuclear neurons depend on their target motoneuronsfor survival, and that they lose this dependence with maturation. D  2005 Elsevier Inc. All rights reserved.  Keywords:  Oculomotor system; Axotomy; Injury-induced cell death; Neonatal rats; ChAT immunoreactivity; Enucleation; Ricin Introduction According to the trophic theory of neural connections,neurons are dependent on target cells for survival and for theexpression and maintenance of the normal phenotype(Purves, 1990). Target cells are the source of neurotrophicfactors, which seem to mediate this dependence. Thesemolecules are internalized by afferent neurons whereby theyregulate multiple electrophysiological and metabolic aspects(Lewin and Barde, 1996; McAllister et al., 1999). During embryonic and postnatal development, target dependenceseems to be maximal since any manipulation that interruptsthe normal flow between a neuronal population and itstarget cells leads to retrograde cell death in a significant  proportion of the afferent neurons (Lowrie and Vrbova´,1992; Moran and Graeber, 2004; Purves, 1990; Snider et al.,1992). In the mature nervous system, however, neuronssurvive target loss, although they exhibit several morpho-logical and physiological alterations in the absence of target contact, indicating that although target-derived factors arenot critical for survival, they are important in regulating thenormal neuronal function (reviewed in de la Cruz et al.,1996; Titmus and Faber, 1990; Vicario-Abejo´n et al., 2002).Many of the experimental studies directed at evaluatingthe role played by target cells on innervating neurons have been carried out following the transection of the pathway or  0014-4886/$ - see front matter   D  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2005.05.003* Corresponding author. Fax: +34 95 4233480.  E-mail address: (R.R. de la Cruz).Experimental Neurology 195 (2005) 244 –   nerve linking the two groups of cells (Moran and Graeber,2004; Snider et al., 1992; Titmus and Faber, 1990).However, axotomy, other than target disconnection, repre-sents also a cellular lesion. Therefore, it seems that a more precise approach to analyze target dependence would be theselective removal of target cells without the injury to the presynaptic neuron. However, there are few works of target deprivation performed by this type of procedure (see,however, Cooper et al., 1996; de la Cruz et al., 1996;Sofroniew et al., 1993). In the present study, we have usedthe oculomotor system as a model to analyze the con-sequences of selective target deprivation on cell survival.The projection of abducens internuclear neurons onto themedial rectus motoneurons of the oculomotor nucleus waschosen, as it offers two major advantages for the study of trophic interactions in the CNS. First, abducens internuclear neurons are well-characterized premotor neurons that connect very precisely with medial rectus motoneurons(de la Cruz et al., 1994a; Highstein et al., 1982; Nguyen et al., 1999). These neurons are located intermingled with themotoneurons of the abducens nucleus in the pons. Their axons travel through the contralateral medial longitudinalfascicle to terminate on the medial rectus subdivision of themesencephalic oculomotor nucleus (Evinger, 1988). Sec- ond, since these neurons project on motoneurons, target cells can be destroyed from the periphery without interferingwith the integrity of the CNS. Therefore, we questioned theimportance of neuron–target interactions in regulating thesurvival of developing abducens internuclear neurons. For this purpose, newborn rats were enucleated monocularly asthe procedure to kill the extraocular motoneurons. Twomonths later, cell survival of abducens internuclear neuronswas assessed by immunocytochemistry against calretinin, aselective marker of this neuronal population (de la Cruz et al., 1998) and by retrograde transport of horseradish peroxidase (HRP). The percentage of cell death induced by enucleation in the oculomotor motoneurons was alsoevaluated to estimate the extent of reduction in target size. A parallel study was performed in adult rats to compare theresponse. Preliminary results have been presented in abstract form (de la Cruz et al., 2004). Materials and methods  Neonatal and adult Wistar rats were used in accordancewith the guidelines of the European Union (86/609/EU) andthe Spanish legislation (BOE 67/8509-12, 1988) for the useand care of laboratory animals. Choline acetyltransferase immunostaining after enucleationin adult animals Adult rats ( n  = 12) were monocularly enucleated under general anesthesia (sodium pentobarbital, 35 mg/kg ip) as amethod to axotomize extraocular motoneurons. The right eye was removed through an incision made in the superior eyelid and intraorbital tissues were eliminated; finally, theorbital cavity was sutured. To study the time course of axotomy-induced changes in the cholinergic phenotype of extraocular motoneurons, animals were separated in four groups of different survival times following enucleation (1,4, 6, and 8 weeks). Animals of 1 and 4 weeks of survivaltime suffered as well the transection of the right facial nerveon the same day of the enucleation. The facial nerve wastransected at the level of the foramen stylomastoideum andthe proximal stump was ligated to prevent regeneration.Facial axotomy has been previously reported to induce adown-regulation in the expression of choline acetyltransfer-ase (ChAT) and therefore was used as a reference for our experiments in extraocular motoneurons (Yan et al., 1994). To prepare tissue for immunocytochemistry, rats were perfused transcardially under deep anesthesia (sodium pentobarbital, 50 mg/kg ip) with 100 ml of physiologicalsaline followed by 250 ml of 4% paraformaldehyde in 0.1M sodium phosphate buffer, pH 7.4 (PB). The brainstemwas removed and cryoprotected by immersion in a solutionof 30% sucrose in sodium phosphate-buffered saline (PBS)until sinking. Tissue was then cut coronally in 50- A m-thick sections on a cryostat and motoneurons in the oculomotor,trochlear, and abducens nuclei were identified using anantibody against ChAT (polyclonal goat anti-ChAT, 1:1000,Chemicon, Temecula, CA). Sections were incubated for 40min in a blocking solution containing 7% of normal rabbit serum (NRS) in PBS with 0.1% Triton X-100 (PBS-T).Tissue was then incubated overnight in the primaryantibody solution prepared in PBS-T containing 0.05%sodium azide and 3% NRS. After washing, tissue wasexposed for 90 min to the secondary antibody solutioncontaining biotinylated rabbit anti-goat IgG (1:250, Vector Laboratories, Burlingame, CA). Following rinsing, tissuewas incubated for 90 min in the avidin–biotin–HRPcomplex (ABC, Vector). Motoneurons were visualizedusing 3,3  V -diaminobenzidine tetrahydrochloride (DAB) at 0.05% with 0.01% hydrogen peroxide diluted in PBS.Sections were mounted on glass slides, dehydrated, cleared,and coverslipped. Control sections were processed in thesame way but the primary antibody was omitted or substituted by non-immune serum. In either case, noimmunostaining was observed. Cell survival experiments in neonatal animals One-day-old neonatal rats (postnatal day 1, P1) wereanesthetized by ether inhalation, and their right eye wasenucleated. This procedure was used to kill the medialrectus motoneurons by axotomy and therefore to deprivedeveloping abducens internuclear neurons of their target.After surgery and recovery from anesthesia, pups weretaken back to their mothers. Two months after enucleation,animals were separated in different groups for tissuetreatment and cell identification. S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256   245  A first group of animals ( n  = 3) was perfused using 4% paraformaldehyde in PB. Two different antibodies wereused for motor and internuclear neuron identification.Motoneurons were stained using the antibody against ChAT.Abducens internuclear neurons were identified with theantibody against calretinin (rabbit polyclonal anti-calretinin,1:6000, Swant, Bellinzona, Switzerland) since this proteinhas been proved to be a good and selective marker of thiscell type. A previous study in the cat has demonstrated that the majority of abducens internuclear neurons projecting tothe oculomotor nucleus (80.7%) contains calretinin, and that the labeling is selective since abducens motoneurons do not express this calcium-binding protein (de la Cruz et al.,1998). For calretinin detection, we followed the sameimmunocytochemistry protocol described above for ChAT, but with normal goat serum (NGS) instead of NRS, andusing as the secondary antibody a biotinylated goat anti-rabbit IgG (1:250, Vector).In a second group of animals ( n  = 3), abducensinternuclear neurons were identified by HRP injection inthe oculomotor nucleus. We performed bilateral injectionsof the tracer. First, the left (uninjured) side was identified by electrophysiological criteria, and then the right (enucleated) side was located using this landmark as areference. Anesthetized animals (sodium pentobarbital, 35mg/kg ip) were situated in a stereotaxic frame and a pair of hook-like stimulating electrodes were inserted into the left medial rectus muscle. By using a glass micropipette filledwith a 2-M NaCl solution, we identified the medial rectussubdivision of the oculomotor nucleus by the recording of the antidromic field potential induced after electricalstimulation (<0.1 mA, 50  A s) of the muscle. Then a glassmicropipette beveled to a tip size of 20  A m and filled with20% HRP solution in 0.05 M Tris–HCl, pH 7.4, and 0.05M NaCl was used to inject the tracer. The HRP solutionwas infused by using a pressure injection device ( ¨ 0.05  A lof volume). The right oculomotor nucleus was alsoinjected with HRP after displacing the glass pipette 500 A m laterally, which is the distance between the centers of  both oculomotor nuclei in the adult rat. Previous exami-nation of the right oculomotor nucleus in enucleatedanimals revealed no shrinkage and therefore a normalseparation of 500  A m between both nuclei. After 24 h,animals were deeply anesthetized and perfused using1.25% glutaraldehyde and 1% paraformaldehyde in PB.The brainstem was removed, cryoprotected, and cut coronally in 50- A m-thick sections in a cryostat. Sectionswere rinsed in PBS and incubated in 0.05% DAB in PBSfor 20 min. HRP reaction was then revealed by adding0.01% hydrogen peroxide.In a third group of animals ( n  = 5), fixed tissue wassectioned as described above after the perfusion of the ratswith 4% paraformaldehyde in PB and brainstem sectionswere mounted and stained with toluidine blue. Two addi-tional control (unoperated) animals were used for Nisslstaining of the abducens nucleus. Selective removal of target motoneurons in adult animals Since axotomy (e.g., by enucleation) is not followed byretrograde cell death in adult motoneurons (Delgado-Garcı´aet al., 1988), we killed the motoneurons innervating themedial rectus muscle in adult rats following the intra-muscular injection of the cytotoxic lectin of   Ricinuscommunis  agglutinin II (RCA60, namely ricin; Sigma, St.Louis, MO). In this way, we left adult abducens internuclear neurons deprived of target. Under general anesthesia, themuscle was isolated and ricin was injected using a Hamiltonsyringe at a dose of 1.1  A g dissolved in a final volume of 2 A l with physiological saline. This dose has been previouslyshown to be effective in inducing the death of medial r ectusmotoneurons in adult cats (de la Cruz et al., 1994a,b). The remaining extraocular muscles that are innervated ipsilat-erally from the oculomot or nucleus (i.e., the inferior rectusand the inferior oblique; Evinger, 1988) were also injected with the same dose of ricin to simulate as much as possiblethe experiments of enucleation performed in neonates.Animals ( n  = 3) were perfused 2 months after ricin injectionwith 4% paraformaldehyde in PB. After cutting the brainstem coronally at 50- A m-thick sections, motoneuronsin the oculomotor nucleus were stained using the antibodyagainst ChAT and the abducens internuclear neurons wereidentified by calretinin immunocytochemistry as describedabove.  Analysis of data Sections were visualized using a Zeiss Axiophot micro-scope (Carl Zeiss, Jena, Germany) and images obtained witha digital camera (Coolpix 995, Nikon, Tokyo, Japan). For cell countings, all sections obtained after cutting the wholeoculomotor or abducens nuclei were considered. Cellscomputed were those with the presence of the nucleus. Toevaluate the effects of lesion and target loss, the means of labeled cells were expressed as percentages relative to thecontrol side. Comparisons between groups were carried out  by using the analysis of variance (ANOVA) followed by post hoc multiple comparisons (Duncan’s method). Whenthe contrast was performed between two groups, theStudent’s  t   test was used. In all cases, the level of significance was  P   < 0.05. Results  Enucleation in the adult leads to a transient ChAT down-regulation in extraocular motoneurons We used ChATexpression as a marker of motoneurons toassess their survival after enucleation. The level of ChATexpression in the motoneurons innervating the extraocular eye muscles was evaluated by immunocytochemistry at different time intervals after monocular enucleation in adult  S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256  246  rats. According to previous works in other axotomized brainstem motoneurons, there is a transient down-regulationin ChAT expression that recover s normal values byapproximately 6 weeks after injury (Matsuura et al., 1997;Okura et al., 1999; Wang et al., 1997). Therefore, we aimedto determine in extraocular motoneurons, first, the timecourse of axotomy-induced changes in ChAT immunostain-ing, and second whether ChAT expression returned tonormal values in the long term and therefore could serve asa good marker for counting the number of extraocular motoneurons surviving axotomy.The immunoreactivity against ChAT revealed a dramaticdecrease in the number of labeled motoneurons in the right oculomotor, left trochlear, and right abducens nuclei 1 week after enucleation of the right eye, as compared with theuninjured side (Figs. 1A–C). A progressive recovery inChAT immunostaining was observed at longer time intervalsafter enucleation (Figs. 1D–L). Thus, the appearance of ChAT immunostaining in the extraocular motor nuclei wassimilar between both sides by 4, 6, and 8 weeks after injury.We quantified the number of ChAT-immunoreactive cellsin these three brainstem motor nuclei and at different timeintervals after lesion. The numbers were expressed as percentages relative to the control side (Fig. 2). Thus, inthe lesioned abducens nucleus, 1 week after enucleation,only 5.3% of the motoneurons appeared immunoreactiveagainst ChAT as compared with the control side. In particular, there were 288.0  T  26.3 (mean  T  SEM) labeledmotoneurons on the control (left) abducens nucleus versus15.3  T  3.2 immunoreactive cells on the lesion (right) side,t he dif ference being significant (ANOVA test;  P   < 0.001;Fig. 2A). The number of ChAT-immunoreactive motoneur-ons was recovered to 79.4% of the control value by 4 weeksafter lesion (control side: 234.7  T  39.1; lesion side: 186.3  T 3.2), a difference that was not significant. Recovery of thecholinergic phenotype was even more evident by 6 weeks(control side: 252.0  T  34.4; lesion side: 254.0  T  31.0;recovery to 100.8%) and 8 weeks (control side: 267.7  T 39.2; lesion side: 248.0  T  27.2; recovery to 92.6%) after lesion.In two groups of animals, the ones to survive for 1 and 4weeks, the right facial nerve was also transected. This procedure was done as a control since facial axotomy has been reported to produce a drastic down-regulation in ChAT Fig. 1. ChAT immunocytochemistry in the oculomotor system following right eye enucleation in the adult stage. The expression of ChAT in the threeextraocular motor nuclei dropped rapidly during the first week after lesion (A–C). Note the decrease in the number of ChAT-immunoreactive motoneurons inthe right abducens nucleus (ABD; A), left trochlear nucleus (TRO; B) and right oculomotor nucleus (OCM; C). ChAT immunostaining recovered progressivelyduring the following weeks (D–L), being similar to control by 8 weeks post-surgery (J–L). The dashed lines in panel A represent the boundaries of theabducens nuclei. Other abbreviations: III, IV, VI, oculomotor, trochlear, and abducens nuclei, respectively; VIIg, genu of the facial nerve. Scale bar: (in panel L)A–L, 500  A m. S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256   247  expression, mainly 1 week after lesion (Yan et al., 1994). Our results confirmed this reduction in the cholinergic phenotype of facial motoneurons (data not shown), as can be observed in the lack of ChAT immunostaining in thegenu of  the facial nerve (right side) by 1 and 4 weeks post-lesion (Figs. 1A and D).Since trochlear motoneurons innervate the superior oblique muscle of the contralateral eye, the trochlear nucleus on the left side was the one affected after right eye enucleation. In the affected trochlear nucleus, only10.1% of the motoneurons expressed ChAT 1 week after lesion (control side: 213.7  T  23.6; lesion side: 21.7  T  2.0;ANOVA test;  P   < 0.001) (Fig. 2B). Four weeks after enucleation, ChAT expression had risen to 90.9% of thecontrol level (control side: 223.7  T  37.6; lesion side: 203.3  T 45.7), being similar to control. The recovery in ChATimmunostaining was maintained at longer time intervals post-lesion (control side: 217.0  T  72.3, lesion side: 208  T 81.2, with 95.9% of recovery by 6 weeks; and control side:228.3  T  37.3, lesion side: 219.0  T  27.1, with 95.9% of recovery by 8 weeks).The oculomotor nucleus is composed of four subdivi-sions, containing the motoneurons innervating four of theextraocular muscles: the medial rectus, the inferior rectus,the inferior oblique (all of them ipsilateral), and the superior rectus (contralateral) (Evinger, 1988; Glicksman, 1980). Therefore, only three of the four subdivisions of theipsilateral (right) nucleus were affected by the enucleation;in addition, one subdivision of the contralateral (left)nucleus was also affected. Considering that the number of motoneurons per subnucleus is similar (Glicksman, 1980;Miyazaki, 1985), and that the projection of the superior rectus is contralateral, we corrected our countings so as toestimate the percentage of ChAT-labeled motoneuronsexpressed per subdivision of the right oculomotor nucleus,in comparison with the left (‘‘control’’) side. In this nucleus,the immunostaining against ChAT 1 week post-lesionyielded also a significantly (ANOVA test;  P   < 0.001) lower number of immunoreactive motoneurons on the side of theenucleation (control side: 762.7  T  43.7, lesion side: 321.0  T 39.3; Fig. 2C). At this time point, only 10.2% of theoculomotor motoneurons were immunoreactive against ChAT. Recovery of the cholinergic phenotype was seen by4 weeks after lesion (control side: 951.3  T  56.3; lesion side:788.3  T  54.7), when we found on the enucleated side 68.4%of the control expression per oculomotor subdivision, thedifference being non-significant. Six weeks after lesion,80% of the oculomotor motoneurons expressed ChAT(control side: 1024.3  T  55.9; lesion side: 916.7  T  70.3)and 95.5% of them expressed this enzyme 8 weeks after enucleation (control side: 963.7  T  25.8; lesion side:941.6  T  16.9).Consequently, since ChAT expression had returned tonormality by 8 weeks post-lesion, we used ChAT as amarker for motoneurons to assess their survival 2 monthsafter neonatal enucleation, as shown below. Moreover, these Fig. 2. Histograms comparing the percentage of ChAT-immunoreactivemotoneurons with respect to control at different time intervals after enucleation in adult rats for abducens motoneurons (A), trochlear motoneurons (B), and oculomotor motoneurons (C). Data are shown for each nucleus (abducens or trochlear) or subdivision (oculomotor). Oneweek after lesion, ChAT expression was significantly lower in all thesemotoneuronal groups, falling to 5.3%, 10.1%, and 10.2% in the abducens(A), trochlear (B), and oculomotor (C) nuclei, respectively. Differences inChAT expression decreased with time so that by 4 weeks post-lesion the percentages of ChAT-immunoreactive motoneurons returned to normalvalues, which were maintained at 6 and 8 weeks. Data represent means  T SEM;  n  = 3 animals per group; ANOVA test; *  P   < 0.001. S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256  248
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