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Experimental Neurology 1

Regular Article

Abducens internuclear neurons depend on their target motoneurons for

survival during early postnatal development

Sara Morcuende, Beatriz Benıtez-Temino, Marıa Luisa Pecero,

Angel M. Pastor, Rosa R. de la Cruz*

Departamento de Fisiologıa y Zoologıa, Facultad de Biologıa, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012-Sevilla, Spain

Received 18 November 2004; revised 7 April 2005; accepted 4 May 2005

Available online 1 June 2005

Abstract

The highly specific projection of abducens internuclear neurons onto medial rectus motoneurons in the oculomotor nucleus is a good

model to evaluate the dependence on target cells for survival during development and in the adult. Thus, the procedure we chose to

selectively 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 calretinin

immunostaining 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 ChAT expression

in extraocular motoneurons induced by injury. Secondly, 2 months after enucleation, 61.6% and 60.5% of the population of abducens

internuclear neurons appeared stained by retrograde tracing and calretinin immunoreaction, respectively, indicating a significant extent of cell

death 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 in

the medial rectus muscle. These results indicate that, during development, abducens internuclear neurons depend on their target motoneurons

for 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 the

expression and maintenance of the normal phenotype

(Purves, 1990). Target cells are the source of neurotrophic

factors, which seem to mediate this dependence. These

molecules are internalized by afferent neurons whereby they

regulate multiple electrophysiological and metabolic aspects

(Lewin and Barde, 1996; McAllister et al., 1999). During

embryonic and postnatal development, target dependence

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: rmrcruz@us.es (R.R. de la Cruz).

seems to be maximal since any manipulation that interrupts

the normal flow between a neuronal population and its

target 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, neurons

survive target loss, although they exhibit several morpho-

logical and physiological alterations in the absence of target

contact, indicating that although target-derived factors are

not critical for survival, they are important in regulating the

normal neuronal function (reviewed in de la Cruz et al.,

1996; Titmus and Faber, 1990; Vicario-Abejon et al., 2002).

Many of the experimental studies directed at evaluating

the role played by target cells on innervating neurons have

been carried out following the transection of the pathway or

95 (2005) 244 – 256

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256 245

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 the

selective 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 used

the oculomotor system as a model to analyze the con-

sequences of selective target deprivation on cell survival.

The projection of abducens internuclear neurons onto the

medial rectus motoneurons of the oculomotor nucleus was

chosen, 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 the

motoneurons of the abducens nucleus in the pons. Their

axons travel through the contralateral medial longitudinal

fascicle to terminate on the medial rectus subdivision of the

mesencephalic oculomotor nucleus (Evinger, 1988). Sec-

ond, since these neurons project on motoneurons, target

cells can be destroyed from the periphery without interfering

with the integrity of the CNS. Therefore, we questioned the

importance of neuron–target interactions in regulating the

survival of developing abducens internuclear neurons. For

this purpose, newborn rats were enucleated monocularly as

the procedure to kill the extraocular motoneurons. Two

months later, cell survival of abducens internuclear neurons

was assessed by immunocytochemistry against calretinin, a

selective 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 also

evaluated to estimate the extent of reduction in target size. A

parallel study was performed in adult rats to compare the

response. 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 accordance

with the guidelines of the European Union (86/609/EU) and

the Spanish legislation (BOE 67/8509-12, 1988) for the use

and care of laboratory animals.

Choline acetyltransferase immunostaining after enucleation

in adult animals

Adult rats (n = 12) were monocularly enucleated under

general anesthesia (sodium pentobarbital, 35 mg/kg ip) as a

method to axotomize extraocular motoneurons. The right

eye was removed through an incision made in the superior

eyelid and intraorbital tissues were eliminated; finally, the

orbital 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 survival

time suffered as well the transection of the right facial nerve

on the same day of the enucleation. The facial nerve was

transected at the level of the foramen stylomastoideum and

the proximal stump was ligated to prevent regeneration.

Facial axotomy has been previously reported to induce a

down-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 physiological

saline followed by 250 ml of 4% paraformaldehyde in 0.1

M sodium phosphate buffer, pH 7.4 (PB). The brainstem

was removed and cryoprotected by immersion in a solution

of 30% sucrose in sodium phosphate-buffered saline (PBS)

until sinking. Tissue was then cut coronally in 50-Am-thick

sections on a cryostat and motoneurons in the oculomotor,

trochlear, and abducens nuclei were identified using an

antibody against ChAT (polyclonal goat anti-ChAT, 1:1000,

Chemicon, Temecula, CA). Sections were incubated for 40

min 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 primary

antibody solution prepared in PBS-T containing 0.05%

sodium azide and 3% NRS. After washing, tissue was

exposed for 90 min to the secondary antibody solution

containing biotinylated rabbit anti-goat IgG (1:250, Vector

Laboratories, Burlingame, CA). Following rinsing, tissue

was incubated for 90 min in the avidin–biotin–HRP

complex (ABC, Vector). Motoneurons were visualized

using 3,3V-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 the

same way but the primary antibody was omitted or

substituted by non-immune serum. In either case, no

immunostaining was observed.

Cell survival experiments in neonatal animals

One-day-old neonatal rats (postnatal day 1, P1) were

anesthetized by ether inhalation, and their right eye was

enucleated. This procedure was used to kill the medial

rectus motoneurons by axotomy and therefore to deprive

developing abducens internuclear neurons of their target.

After surgery and recovery from anesthesia, pups were

taken back to their mothers. Two months after enucleation,

animals were separated in different groups for tissue

treatment and cell identification.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256246

A first group of animals (n = 3) was perfused using 4%

paraformaldehyde in PB. Two different antibodies were

used for motor and internuclear neuron identification.

Motoneurons were stained using the antibody against ChAT.

Abducens internuclear neurons were identified with the

antibody against calretinin (rabbit polyclonal anti-calretinin,

1:6000, Swant, Bellinzona, Switzerland) since this protein

has been proved to be a good and selective marker of this

cell type. A previous study in the cat has demonstrated that

the majority of abducens internuclear neurons projecting to

the 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 same

immunocytochemistry protocol described above for ChAT,

but with normal goat serum (NGS) instead of NRS, and

using as the secondary antibody a biotinylated goat anti-

rabbit IgG (1:250, Vector).

In a second group of animals (n = 3), abducens

internuclear neurons were identified by HRP injection in

the oculomotor nucleus. We performed bilateral injections

of the tracer. First, the left (uninjured) side was identified

by electrophysiological criteria, and then the right

(enucleated) side was located using this landmark as a

reference. Anesthetized animals (sodium pentobarbital, 35

mg/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 filled

with a 2-M NaCl solution, we identified the medial rectus

subdivision of the oculomotor nucleus by the recording of

the antidromic field potential induced after electrical

stimulation (<0.1 mA, 50 As) of the muscle. Then a glass

micropipette beveled to a tip size of 20 Am and filled with

20% HRP solution in 0.05 M Tris–HCl, pH 7.4, and 0.05

M NaCl was used to inject the tracer. The HRP solution

was infused by using a pressure injection device (¨0.05 Alof volume). The right oculomotor nucleus was also

injected with HRP after displacing the glass pipette 500

Am 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 enucleated

animals revealed no shrinkage and therefore a normal

separation of 500 Am between both nuclei. After 24 h,

animals were deeply anesthetized and perfused using

1.25% glutaraldehyde and 1% paraformaldehyde in PB.

The brainstem was removed, cryoprotected, and cut

coronally in 50-Am-thick sections in a cryostat. Sections

were rinsed in PBS and incubated in 0.05% DAB in PBS

for 20 min. HRP reaction was then revealed by adding

0.01% hydrogen peroxide.

In a third group of animals (n = 5), fixed tissue was

sectioned as described above after the perfusion of the rats

with 4% paraformaldehyde in PB and brainstem sections

were mounted and stained with toluidine blue. Two addi-

tional control (unoperated) animals were used for Nissl

staining of the abducens nucleus.

Selective removal of target motoneurons in adult animals

Since axotomy (e.g., by enucleation) is not followed by

retrograde cell death in adult motoneurons (Delgado-Garcıa

et al., 1988), we killed the motoneurons innervating the

medial rectus muscle in adult rats following the intra-

muscular injection of the cytotoxic lectin of Ricinus

communis agglutinin II (RCA60, namely ricin; Sigma, St.

Louis, MO). In this way, we left adult abducens internuclear

neurons deprived of target. Under general anesthesia, the

muscle was isolated and ricin was injected using a Hamilton

syringe at a dose of 1.1 Ag dissolved in a final volume of 2

Al with physiological saline. This dose has been previously

shown to be effective in inducing the death of medial rectus

motoneurons in adult cats (de la Cruz et al., 1994a,b). The

remaining extraocular muscles that are innervated ipsilat-

erally from the oculomotor nucleus (i.e., the inferior rectus

and the inferior oblique; Evinger, 1988) were also injected

with the same dose of ricin to simulate as much as possible

the experiments of enucleation performed in neonates.

Animals (n = 3) were perfused 2 months after ricin injection

with 4% paraformaldehyde in PB. After cutting the

brainstem coronally at 50-Am-thick sections, motoneurons

in the oculomotor nucleus were stained using the antibody

against ChAT and the abducens internuclear neurons were

identified by calretinin immunocytochemistry as described

above.

Analysis of data

Sections were visualized using a Zeiss Axiophot micro-

scope (Carl Zeiss, Jena, Germany) and images obtained with

a digital camera (Coolpix 995, Nikon, Tokyo, Japan). For

cell countings, all sections obtained after cutting the whole

oculomotor or abducens nuclei were considered. Cells

computed were those with the presence of the nucleus. To

evaluate the effects of lesion and target loss, the means of

labeled cells were expressed as percentages relative to the

control side. Comparisons between groups were carried out

by using the analysis of variance (ANOVA) followed by

post hoc multiple comparisons (Duncan’s method). When

the contrast was performed between two groups, the

Student’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 ChAT expression as a marker of motoneurons to

assess their survival after enucleation. The level of ChAT

expression 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) 244–256 247

rats. According to previous works in other axotomized

brainstem motoneurons, there is a transient down-regulation

in ChAT expression that recovers normal values by

approximately 6 weeks after injury (Matsuura et al., 1997;

Okura et al., 1999; Wang et al., 1997). Therefore, we aimed

to determine in extraocular motoneurons, first, the time

course of axotomy-induced changes in ChAT immunostain-

ing, and second whether ChAT expression returned to

normal values in the long term and therefore could serve as

a good marker for counting the number of extraocular

motoneurons surviving axotomy.

The immunoreactivity against ChAT revealed a dramatic

decrease 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 the

uninjured side (Figs. 1A–C). A progressive recovery in

ChAT immunostaining was observed at longer time intervals

after enucleation (Figs. 1D–L). Thus, the appearance of

ChAT immunostaining in the extraocular motor nuclei was

similar between both sides by 4, 6, and 8 weeks after injury.

We quantified the number of ChAT-immunoreactive cells

in these three brainstem motor nuclei and at different time

Fig. 1. ChAT immunocytochemistry in the oculomotor system following right

extraocular motor nuclei dropped rapidly during the first week after lesion (A–C).

the right abducens nucleus (ABD; A), left trochlear nucleus (TRO; B) and right ocu

during the following weeks (D–L), being similar to control by 8 weeks post-su

abducens nuclei. Other abbreviations: III, IV, VI, oculomotor, trochlear, and abduce

A–L, 500 Am.

intervals after lesion. The numbers were expressed as

percentages relative to the control side (Fig. 2). Thus, in

the lesioned abducens nucleus, 1 week after enucleation,

only 5.3% of the motoneurons appeared immunoreactive

against ChAT as compared with the control side. In

particular, there were 288.0 T 26.3 (mean T SEM) labeled

motoneurons on the control (left) abducens nucleus versus

15.3 T 3.2 immunoreactive cells on the lesion (right) side,

the difference 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 weeks

after lesion (control side: 234.7 T 39.1; lesion side: 186.3 T3.2), a difference that was not significant. Recovery of the

cholinergic 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 T39.2; 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 4

weeks, 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

eye enucleation in the adult stage. The expression of ChAT in the three

Note the decrease in the number of ChAT-immunoreactive motoneurons in

lomotor nucleus (OCM; C). ChAT immunostaining recovered progressively

rgery (J–L). The dashed lines in panel A represent the boundaries of the

ns nuclei, respectively; VIIg, genu of the facial nerve. Scale bar: (in panel L)

Fig. 2. Histograms comparing the percentage of ChAT-immunoreactive

motoneurons 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). One

week after lesion, ChAT expression was significantly lower in all these

motoneuronal groups, falling to 5.3%, 10.1%, and 10.2% in the abducens

(A), trochlear (B), and oculomotor (C) nuclei, respectively. Differences in

ChAT expression decreased with time so that by 4 weeks post-lesion the

percentages of ChAT-immunoreactive motoneurons returned to normal

values, 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) 244–256248

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 the

genu 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, only

10.1% 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 the

control level (control side: 223.7 T 37.6; lesion side: 203.3 T45.7), being similar to control. The recovery in ChAT

immunostaining was maintained at longer time intervals

post-lesion (control side: 217.0 T 72.3, lesion side: 208 T81.2, 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 the

extraocular 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 the

ipsilateral (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 to

estimate the percentage of ChAT-labeled motoneurons

expressed per subdivision of the right oculomotor nucleus,

in comparison with the left (‘‘control’’) side. In this nucleus,

the immunostaining against ChAT 1 week post-lesion

yielded also a significantly (ANOVA test; P < 0.001) lower

number of immunoreactive motoneurons on the side of the

enucleation (control side: 762.7 T 43.7, lesion side: 321.0 T39.3; Fig. 2C). At this time point, only 10.2% of the

oculomotor motoneurons were immunoreactive against

ChAT. Recovery of the cholinergic phenotype was seen by

4 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, the

difference 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 to

normality by 8 weeks post-lesion, we used ChAT as a

marker for motoneurons to assess their survival 2 months

after neonatal enucleation, as shown below. Moreover, these

Table 1

Effects of enucleation at P1 on oculomotor motoneurons (OCM Mns) and

abducens internuclear neurons (ABD Ints)

OCM Mns ABD Ints

ChAT Nissl HRP CR

Control

side

856.1 T 47.0 658.0 T 56.2 142.3 T 16.5 145.6 T 12.2

Affected

side

441.0 T 44.6** 326.8 T 58.2* 87.7 T 12.3* 88.0 T 8.6**

Countings of ChAT- and Nissl-stained cells in the oculomotor nucleus and

HRP- and calretinin (CR)-labeled internuclear neurons in the abducens

nucleus 2 months after right eye enucleation in neonatal rats (P1). The

affected side corresponds to the right oculomotor nucleus (enucleated) and

the left abducens nucleus (target deprived). Data represent mean T SEM; n =

3 animals in ChAT, HRP, and CR experiments; n = 5 in Nissl staining.

* P < 0.05, paired Student’s t test.

** P < 0.001, paired Student’s t test.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256 249

experiments demonstrated that extraocular motoneurons

survive axotomy in the adult rat.

Oculomotor motoneurons die after postnatal enucleation

Neonatal rats were enucleated at P1 to induce the death

of oculomotor motoneurons. Two months after enucleation

of the right eye, we quantified the number of motoneurons

surviving lesion by two procedures: ChAT immunoreactiv-

ity and Nissl staining. Two months after P1 enucleation, the

number of motoneurons that appeared immunolabeled

against ChAT in the side ipsilateral to the lesion was

markedly lower than in the contralateral side (Fig. 3A). We

found 856.1 T 47.0 motoneurons immunoreactive for ChAT

in the left oculomotor nucleus, and 441.0 T 44.6 labeled

motoneurons in the right nucleus (the lesion side; Table 1).

After correcting these numbers due to the contralateral

location of superior rectus motoneurons and expressing the

results per subdivision, we obtained a 21.9% of survival in

the population of motoneurons of the oculomotor nucleus 2

months after P1 enucleation (Fig. 4A). Therefore, the extent

of motoneuronal cell death was of 78.1% in the oculomotor

nucleus (Fig. 4A), as assessed by ChAT immunostaining.

Nissl staining of the oculomotor nucleus 2 months after

P1 enucleation also revealed a dramatic extent of cell loss

on the side ipsilateral to the lesion (Fig. 3B). By this

Fig. 3. Death of oculomotor motoneurons 2 months after right eye

enucleation in the postnatal (P1) stage. Images showing two coronal

sections through the oculomotor nucleus after ChAT immunolabeling (A) or

Nissl staining with Toluidine blue (B). Note the loss of motoneurons on the

right side. Scale bar: (in panel A) A–B, 300 Am.

technique, we found 658.0 T 56.2 motoneurons in the left

oculomotor nucleus and 326.8 T 58.2 motoneurons in the

right nucleus (Table 1). After applying the same correction,

we obtained a 19.4% of survival in each subdivision.

Therefore, the percentage of cell death in the population of

oculomotor motoneurons was of 80.6% (Fig. 4B), similar to

that obtained by ChAT immunostaining.

Fig. 4. Histograms representing the motoneuronal death in the oculomotor

nucleus 2 months after enucleation at P1. ChAT immunoreaction showed

78.1% of cell death per oculomotor subdivision (A), and Nissl staining

revealed a very similar degree of motoneuronal death, 80.6% (B). Values

are means T SEM; n = 3 animals in panel A; n = 5 animals in panel B;

paired Student’s t test; *P < 0.01, **P < 0.001.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256250

Surviving motoneurons showed normal morphological

features, as observed after both ChAT immunolabeling and

Nissl staining, and presented a normal soma size. Thus, the

average soma diameter (calculated as the mean of the

shortest and longest diameters of the cell body) of ChAT-

immunoreactive motoneurons surviving lesion was similar

to that of the control side (20.9 T 3.1 Am and 20.2 T 2.4 Am,

respectively).

Abducens internuclear neurons are dependent on their

target motoneurons during the postnatal period

The loss of ¨80% of oculomotor motoneurons induced

by neonatal enucleation led the population of preoculomotor

abducens internuclear neurons deprived of target. Therefore,

we questioned whether target loss at postnatal stages

affected the survival of developing central neurons. For this

purpose, we labeled abducens internuclear neurons 2

months after right eye enucleation by two different

procedures. Firstly, since abducens internuclear neurons

project to the contralateral medial rectus motoneurons of the

oculomotor nucleus, we identified them in a retrograde

manner following the injection of HRP into the oculomotor

nucleus. HRP was injected bilaterally to label also the

control neurons. After the histochemical detection of the

HRP, we found a higher number of labeled cells in the right

(control) abducens nucleus (Fig. 5B) as compared with the

left (target-deprived) side (Fig. 5A). The countings showed

a mean of 142.3 T 16.5 labeled cells in the control right side

whereas only 87.7 T 12.3 cells appeared retrogradely labeled

Fig. 5. Abducens internuclear neurons 2 months after right eye enucleation at P1.

bilateral injection of HRP in the oculomotor nucleus. Note the low number of intern

to those projecting to the control side (B). (C–D) Calretinin immunostaining of the

ipsilateral (i.e., control; in D) to the lesioned oculomotor nucleus. The dashed lin

genu of the facial nerve. Scale bar: (in panel D) A–D, 200 Am.

in the affected left abducens nucleus (i.e., the targetless

side; Table 1). Therefore, 38.4% of the population of

abducens internuclear neurons died following the loss of

nearly 80% of their target motoneurons during the

postnatal period (Fig. 6A).

The second procedure used to label the internuclear

neurons of the abducens nucleus was the immunostaining

against calretinin (de la Cruz et al., 1998). Using this

technique, a higher number of immunoreactive cells was

also observed in the control right abducens nucleus (Fig.

5D) as compared with the left targetless side (Fig. 5C). On

average, 145.6 T 12.2 immunopositive cells appeared in the

control nucleus versus 88.0 T 8.6 in the affected side (Table

1). The percentage of cell death obtained with calretinin

immunostaining (39.5%) was therefore similar to that

obtained after HRP retrograde labeling (Fig. 6B).

To corroborate the HRP and calretinin measurements,

countings of Nissl-stained sections through the abducens

nucleus were also performed. We found a significant

difference in the number of Nissl-stained cells between the

control and target-deprived abducens nuclei (Student’s t

test; P < 0.05; n = 3), but of lower magnitude (16.5%) as

compared with the other two procedures (i.e., HRP or

calretinin). This smaller percentage was expected since

Nissl staining also labels the motoneurons of the abducens

nucleus which constitute approximately two thirds of the

abducens cell population (Evinger, 1988).

There were no differences in average soma diameter of

the internuclear neurons (calretinin immunoreactive) that

survived in the affected abducens nucleus as compared with

(A–B) HRP retrograde labeling of the abducens internuclear neurons after

uclear neurons projecting to the lesioned oculomotor nucleus (A) compared

abducens internuclear neurons contralateral (i.e., target deprived; in C) and

e in panels A–D represents the boundaries of the abducens nucleus. VIIg,

Fig. 6. Histograms representing the percentage of abducens internuclear

neuron survival 2 months after P1 enucleation. (A) After bilateral HRP

injection in the oculomotor nucleus, 61.6% of abducens internuclear

neurons appeared labeled on the left side (i.e., projecting to the lesioned

oculomotor nucleus), as compared to the control (right) abducens nucleus.

(B) Percentage of calretinin (CR)-positive cells in the abducens nucleus.

The data showed the labeling of 60.4% of the population of abducens

internuclear neurons in the affected side, as compared to the control side.

Values are means T SEM; n = 3 animals per group; paired Student’s t test;

*P < 0.05, **P < 0.001.

Fig. 7. Effect of target depletion on abducens internuclear neurons in the

adult stage. (A) ChAT immunostaining in the oculomotor nucleus showed

the absence of the medial rectus subdivision (arrowheads) in the right side

after the injection of ricin in the right medial rectus muscle. (B) Calretinin

immunostaining of the abducens nuclei showing no differences in the

number of labeled abducens internuclear neurons between both sides

despite the unilateral depletion of their target motoneurons. The dashed

lines in panel B represent the boundaries of the abducens nuclei. (C–D)

Images showing at higher magnification the left (target deprived, C) and

right (control, D) abducens nuclei corresponding to the same section as in

panel B to illustrate the calretinin immunostaining of abducens internuclear

neurons. The arrows point to some labeled cells. MLF, medial longitudinal

fascicle; VIIg, genu of the facial nerve. Scale bar: (D) A, 300 Am; (D) B,

500 Am; (D) C–D, 250 Am.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256 251

the unaffected side (control side: 15.2 T 2.4 Am; targetless

side: 14.8 T 2.4 Am).

Abducens internuclear neurons survive the loss of their

target motoneurons in the adult

We also pursued the fate of abducens internuclear

neurons after target deprivation in adult animals. Since

enucleation in the adult was not followed by the retrograde

cell death of extraocular motoneurons (see above), we used

the cytotoxic lectin from R. communis agglutinin II (known

as ricin) as the method to kill the target medial rectus

motoneurons. Following its injection into a muscle, ricin is

internalized by axonic terminals and transported retro-

gradely towards the soma, killing the cell body by

inactivation of protein synthesis (Olsnes et al., 1974; Wiley,

1992). Therefore, we injected ricin (1.1 Ag in 2 Al) into the

(right) medial rectus muscle to produce the death of the

medial rectus motoneurons and leave the (left) abducens

internuclear neurons deprived of target. The remaining

oculomotor motoneurons innervating ipsilaterally their

muscles (i.e., the inferior rectus and the inferior oblique)

were also eliminated by intramuscular injection of ricin to

mimic as much as possible the situation of neonatal

enucleation. Two months after ricin injection, a drastic

reduction in the number of motoneurons was observed in

the right oculomotor nucleus, as assessed by ChAT

immunostaining. The column where medial rectus moto-

neurons are located appeared clearly devoid of ChAT-

immunoreactive cells (Fig. 7A; arrowheads). There were

265.7 T 9.8 ChAT-positive cells per subdivision in the

control side, whereas only 26.2 T 24.7 cells appeared

Fig. 8. (A) Histogram representing the percentage of cell death in the

oculomotor nucleus after the injection of ricin in the right medial rectus

muscle of adult rats. ChAT-immunoreactive motoneurons were counted in

both oculomotor nuclei, expressed per subdivision, and normalized to

control. (B) Histogram representing the lack of cell death in the population

of abducens internuclear neurons in response to target depletion in the adult

stage. There was no significant difference in the number of calretinin (CR)-

immunoreactive internuclear neurons between both abducens nuclei. Values

are means T SEM; n = 3; paired Student’s t test; *P < 0.05.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256252

immunoreactive against ChAT in the injected side (Fig. 8A).

The percentage of cell death in the oculomotor motoneurons

2 months after ricin injection in adult rats was therefore of

90.1%.

To study the effect of target depletion on abducens

internuclear neurons, we labeled selectively this cell type by

calretinin immunostaining (Fig. 7B). Despite the drastic

reduction in target size, there was no statistical difference

between the number of calretinin-immunoreactive cells in

the affected abducens nucleus (Fig. 7C) versus the control

side (Fig. 7D). We counted a mean of 117.5 T 2.5

internuclear neurons in the control (right) abducens nucleus,

and 112.5 T 3.5 in the (left) abducens nucleus projecting to

the depleted medial rectus subdivision of the oculomotor

nucleus (Fig. 8B).

Discussion

The present experiments demonstrate, in the oculomotor

system, that the degree of dependence of neurons on their

target cells varies with the stage of maturation of the CNS.

During the early postnatal period, target depletion resulted

in a significant extent of cell death in the population of

abducens internuclear neurons whereas in the adult these

cells survived the loss of their target. Since medial rectus

motoneurons are the target cells of this group of central

neurons, it was possible to remove selectively the moto-

neurons from the periphery by using lesioning procedures

that left the afferent cells and axons intact. We also found

that the response of extraocular motoneurons to disconnec-

tion from their target muscles was age dependent. Thus,

enucleation performed in neonates (P1) led to a dramatic

loss of motoneurons, whereas the same type of insult in the

adult was not followed by cell death. Axotomized extra-

ocular motoneurons showed a transient down-regulation in

the expression of ChAT that recovered by 4 weeks post-

lesion, likely in the absence of target reinnervation.

ChAT down-regulation in axotomized extraocular

motoneurons is reversible

Because ChAT is the biosynthetic enzyme of the neuro-

transmitter acetylcholine, the immunoreactivity against

ChAT has been widely used to identify and localize

cholinergic neurons, including motor neurons (Friedman et

al., 1995; Houser et al., 1983; Lams et al., 1988; Tuszynski

et al., 1996). The cholinergic phenotype, however, critically

changes after axotomy. Previous studies in hypoglossal,

facial, and spinal motoneurons have shown that, after

transection of the corresponding peripheral nerve, the

injured motoneurons lose immunoreactivity for ChAT and

show low levels of the transcript, indicating a substantial

down-regulation of the enzyme that takes place as early as 1

day and persists for more than 2 weeks after lesion

(Friedman et al., 1995; Tuszynski et al., 1996; Wang et

al., 1997; Yan et al., 1994). In the present experiments we

have demonstrated a similar response in the motoneurons

that innervate the extraocular eye muscles in the adult rat.

Thus, 1 week after axotomy, the number of ChAT-

immunoreactive motoneurons in the abducens, trochlear

and oculomotor nuclei had decreased dramatically to 5–

10% of the control number.

The decrease in ChAT immunoreactivity is, however,

transient and recovers with time to its original level. Thus,

motoneurons of the oculomotor system recovered ChAT

expression completely by 4 weeks, according to the

countings of ChAT-immunoreactive cells. The re-expression

of ChAT immunoreactivity has also been described in other

brainstem motor neurons (Matsuura et al., 1997; Okura et

al., 1999; Wang et al., 1997) and occurs at a similar time

interval after axotomy, i.e., by 4–8 weeks.

The loss of cholinergic phenotype in axotomized

motoneurons can be prevented by the administration of

neurotrophic factors. In particular, the ligands for the TrkB

receptors, brain-derived neurotrophic factor and neuro-

trophin-4/5, have been proven to exert a protective effect

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256 253

against the loss of acetylcholine-related enzymes, including

ChAT and acetylcholinesterase, in injured hypoglossal,

facial, and spinal motoneurons (Fernandes et al., 1998;

Friedman et al., 1995; Tuszynski et al., 1996; Yan et al.,

1994). Therefore, it could be argued that the reduction in

ChAT immunoreactivity observed in axotomized extraocu-

lar (present results) as well as other brainstem or spinal

motoneurons (Friedman et al., 1995; Tuszynski et al., 1996;

Wang et al., 1997; Yan et al., 1994) is due to the loss of

trophic molecules from the target muscle. However, the later

recovery in ChAT expression occurs even after ligation of

the peripheral nerve (Matsuura et al., 1997; Okura et al.,

1999; Wang et al., 1997). Also, if reinnervation into muscle

is allowed, the timing of ChAT recovery after axotomy is

not coincident with substantial reinnervation of target

muscle (Borke et al., 1993). In the present experiments,

extraocular motoneurons were axotomized by enucleation.

This procedure left no muscular tissue available to be

reinnervated by the sectioned motor axons, and therefore

ChAT resumed to normality without muscle reconnection.

Altogether it seems that the return of this neurotransmitter-

synthesizing enzyme to normal levels is regulated by factors

not derived from target muscle. A likely source of trophic

factors that could mediate this recovery are the Schwann

cells of the peripheral nerve. Indeed, after nerve transection

Schwann cells up-regulate the synthesis of several neuro-

trophic molecules, including nerve growth-factor (Johnson

et al., 1988), brain-derived neurotrophic factor (Meyer et al.,

1992), ciliary neurotrophic factor (Sendtner et al., 1992b),

and glial cell-line-derived neurotrophic factor (Trupp et al.,

1995). Alternatively, or in addition, neurotrophic delivery

from autocrine and/or paracrine pathways could also be

involved in the recovery of ChAT expression (Acheson et

al., 1995; Miranda et al., 1993).

The fact that ChAT immunoreactivity in extraocular

motoneurons regained normal levels by 4 weeks post-

axotomy, and that these were maintained at least up to 8

weeks (maximum time studied), validated the use of this

tool as a good marker for injured oculomotor motoneurons

in the long term after lesion.

Neonatal extraocular motoneurons die after axotomy

The present results have shown that enucleation at P1 in

neonatal rats led to a massive loss of motoneurons in the

oculomotor nucleus. In particular, near 80% of oculomotor

motoneurons died as quantified 2 months after injury. The

two procedures used, ChAT immunolabeling and Nissl

staining, produced similar percentages of cell death. In

contrast, the same type of insult performed in adult rats was

followed by the survival of the whole population of

oculomotor motoneurons, as assessed by ChAT immunos-

taining. Therefore, these data demonstrate that oculomotor

motoneurons are more vulnerable to axotomy during the

postnatal period than when they reach maturity. This finding

provides further support to previous studies in other

brainstem as well as spinal motoneurons in the rat showing

a massive cell death following axotomy during the neonatal

period (Lowrie et al., 1987; Snider and Thanedar, 1989; for

reviews, see Lowrie and Vrbova, 1992; Moran and Graeber,

2004). Moreover, time course experiments in which a motor

nerve has been transected at different developmental ages

until adulthood have revealed that motoneurons become

progressively resistant to cell death with increasing maturity

(Kou et al., 1995; Snider and Thanedar, 1989).

The differential response of neonatal and adult moto-

neurons to axotomy likely reflects a greater dependence of

developing motoneurons upon target muscle contact for

survival. In support of this, prevention of peripheral

reinnervation after section of the medial gastrocnemius

nerve in neonatal rats is followed by a marked increase in

the percentage of motoneuronal death (Kashihara et al.,

1987). The importance of target in regulating the survival of

motoneurons during development has been further rein-

forced by the finding that target-derived neurotrophic

molecules can attenuate the extent of motoneuron death

following neonatal axotomy when they are exogenously

administered. For instance, brain-derived neurotrophic

factor, neurotrophin-3, ciliary neurotrophic factor, and

glial-cell-line-derived neurotrophic factor have been

described to prevent the loss of spinal and facial motoneur-

ons induced by axotomy in newborn rats (Aszmann et al.,

2004; Clatterbuck et al., 1994; Koliatsos et al., 1993;

Sendtner et al., 1990, 1992a,b; Yan et al., 1992, 1993,

1995), although in some cases the rescue effects seem to be

transient (Schmalbruch and Rosenthal, 1995; Vejsada et al.,

1995, 1998). In addition, numerous other growth factors

have been found with survival-promoting effects on

motoneurons in vitro or in vivo, suggesting that target-

derived requirements for motoneurons likely comprise a

combination of multiple trophic molecules derived from

muscle (Houenou et al., 1994; Oorschot and McLennan,

1998; Oppenheim, 1996; Sendtner et al., 1996; Thoenen et

al., 1993).

Target dependence of abducens internuclear neurons

decreases with age

Abducens internuclear neurons constitute a group of

preoculomotor neurons projecting on the medial rectus

motoneurons of the oculomotor nucleus (Evinger, 1988;

Graybiel and Hartwieg, 1974; Highstein and Baker, 1978).

Thus, this projection represents a suitable model in which to

evaluate the consequences of selective target deprivation by

killing specifically the target motoneurons from the periph-

ery without affecting the integrity of afferent axons. For this

purpose, we induced the death of target motoneurons, in

both neonatal and adult rats, and quantified 2 months later

the number of abducens internuclear neurons surviving

target loss.

The procedure chosen to kill medial rectus motoneurons

in the newborn rat was the monocular enucleation

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256254

performed at P1. Enucleation induced the death of

approximately 80% of oculomotor motoneurons. Abducens

internuclear neurons were labeled 2 months after lesion by

using either retrograde HRP reaction or calretinin immu-

nostaining (de la Cruz et al., 1998). The possibility that

calretinin expression may be down-regulated by target loss,

thus altering the number of labeled abducens internuclear

neurons, is very unlikely. Calcium-binding proteins have

been previously shown to up-regulate in response to lesion,

such as in axotomized hypoglossal motoneurons of the rat.

This up-regulation is nevertheless transient since by

approximately 1 month the expression returns again to

normal values (Dassesse et al., 1998; Krebs et al., 1997). In

the case of feline abducens internuclear neurons, calretinin

immunolabeling assessed 1 month post-axotomy reveals a

similar number of labeled cells as in the control side,

indicating that at this time point calretinin expression is

normal (Pastor et al., 2000). Moreover, the two methods of

labeling used in the present experiments (HRP and

calretinin immunolabeling) yielded similar results, that is,

38.4% and 39.5% of cell death, respectively, in the abducens

internuclear population and were corroborated by Nissl

countings. Therefore, the present findings indicate that

nearly 40% of the abducens internuclear neurons died

following the loss of 80% of target motoneurons at early

postnatal development.

An interesting issue was that approximately 60% of

abducens neurons remained alive until adulthood following

the loss of target motoneurons. The possibility that the

survival of these neurons were due to the presence of

alternative targets is very unlikely. Previous works have

demonstrated by intra-axonal HRP labeling (Highstein et al.,

1982) or by anterograde biocytin staining (de la Cruz et al.,

1994a) that the projection from abducens to oculomotor

nucleus is highly specific. Rather, we argue in favor of some

other factors as likely explanations for the surviving cells.

First, some motoneurons remained alive in the oculomotor

nucleus (20%) after enucleation. These motoneurons could

support a proportion of the premotor abducens neurons.

Second, in addition to the motoneurons there are also

internuclear neurons within the oculomotor nucleus, which

are not normally contacted by the abducens internuclear

neurons (de la Cruz et al., 1994a; Nguyen et al., 1999).

However, in the adult cat we have shown that they are

innervated by abducens internuclear terminals following the

removal of medial rectus motoneurons (de la Cruz et al.,

1994a). In fact, since oculomotor internuclear neurons

receive axonal collaterals from oculomotor motoneurons

(Evinger et al., 1979, 1981; Spencer et al., 1982), they might

have membrane surface available for reinnervation follow-

ing motoneuronal ablation. Third, trophic support for

surviving abducens neurons can originate from sources

other than target cells, including glial cells, afferences, or

from neighboring cells in a paracrine or even autocrine

mode (Korsching, 1993). If so, neurons could remain alive

in the absence of target contact.

Our findings are in agreement with a previous work of

developing central neurons showing that peripheral nerve

transection in the neonatal rat leads to the death of

presumably interneurons in the spinal cord. In this case,

however, the simultaneous loss after injury of both target

cells (i.e., motoneurons) and afferences (i.e., dorsal root

ganglion cells) seems to contribute to the death of spinal

interneurons, revealing a double retrograde and anterograde

trophic dependence (Oliveira et al., 2002). Moreover, in

experiments using embryonic rat spinal cord explants, spinal

interneurons have been demonstrated to depend on the

presence of motoneurons for survival and more specifically

on neurotrophin-3 which is produced by motoneurons

(Bechade et al., 2002).

In contrast to the neonate, the loss of target motoneur-

ons in the adult (induced by ricin injection in the medial

rectus muscle) was followed by the survival of the entire

population of abducens internuclear neurons. Therefore,

similar to the oculomotor motoneurons, the susceptibility

of these central premotor neurons to target deprivation

diminished with maturation. In the case of the abducens

internuclear neurons, moreover, target ablation was

performed by a selective procedure that left these cells

intact without the possible side effects of the axotomy

itself. Other model in which target dependence has been

evaluated after selective target removal and at different

ages is the septo-hippocampal projection. The removal of

the hippocampal target by excitotoxic lesion in adult rats

does not produce retrograde cell death in the population

of afferent septal neurons (Sofroniew et al., 1990, 1993).

On the other hand, when the same type of injury is

performed during postnatal development, septal neurons

die in a high percentage after target loss (Cooper et al.,

1996).

In the adult cat, we have previously shown that abducens

internuclear neurons survive target loss up to 1 year but that

they exhibit alterations in their discharge pattern, as assessed

by extracellular single-unit recordings in the alert animal (de

la Cruz et al., 1994b). In particular, these neurons show an

overall reduction in firing rate and a loss of eye-related

signals, i.e., eye position and velocity sensitivities. Dis-

charge characteristics recover, however, in about 1 month,

in correlation with the reinnervation of oculomotor inter-

nuclear neurons as a novel target (de la Cruz et al., 1994a,b).

Taken together, all these findings indicate that target-derived

factors are essential for the survival of developing neurons,

and although are not required for adult neurons to survive,

they play an important role in regulating neuronal physio-

logical properties.

Acknowledgments

This work was supported by grants from MCYT

(I+D+I)-FEDER BFI2003-01024 and Fundacion Eugenio

Rodrıguez Pascual.

S. Morcuende et al. / Experimental Neurology 195 (2005) 244–256 255

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