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THE JOURNAL OF COMPARATIVE NEUROLOGY 427:391– 404 (2000)

Discharge Characteristics of Axotomized Abducens Internuclear Neurons in the Adult Cat ´ M. DELGADO-GARCIÁ, AND A ´ NGEL M. PASTOR ROSA R. DE LA CRUZ,* JOSE Laboratorio de Neurociencia, Facultad de Biologıá, Universidad de Sevilla, 41012-Sevilla, Spain

ABSTRACT The aim of the present work was to characterize the axotomy-induced changes in the discharge properties of central nervous system neurons recorded in the alert behaving animal. The abducens internuclear neurons of the adult cat were the chosen model. The axons of these neurons course through the contralateral medial longitudinal fascicle and contact the medial rectus motoneurons of the oculomotor nucleus. Axotomy was carried out by the unilateral transection of this fascicle (right side) and produced immediate oculomotor deficits, mainly the incapacity of the right eye to adduct across the midline. Extracellular single-unit recording of abducens neurons was carried out simultaneously with eye movements. The main alteration observed in the firing of these axotomized neurons was the overall decrease in firing rate. During eye fixations, the tonic signal was reduced, and, on occasion, a progressive decay in firing rate was observed. On-directed saccades were not accompanied by the high-frequency spike burst typical of controls; instead, there was a moderate increase in firing. Similarly, during the vestibular nystagmus, neurons hardly modulated during both the slow and the fast phases. Linear regression analysis between firing rate and eye movement parameters showed a significant reduction in eye position and velocity sensitivities with respect to controls, during both spontaneous and vestibularly induced eye movements. These firing alterations were observed during the 3 month period of study after lesion, with no sign of recovery. Conversely, abducens motoneurons showed no significant alteration in their firing pattern. Therefore, axotomy produced long-lasting changes in the discharge characteristics of abducens internuclear neurons that presumably reflected the loss of afferent oculomotor signals. These alterations might be due to the absence of trophic influences derived from the target. J. Comp. Neurol. 427:391– 404, 2000. © 2000 Wiley-Liss, Inc. Indexing terms: oculomotor system; axotomy; medial longitudinal fascicle; firing alterations; neuronal injury; eye movements

It is well established that axotomy causes changes in the electrophysiological properties of neurons. Most of this evidence has been obtained in axotomized motor and autonomic neurons, in which axotomy can modify passive membrane properties, neuronal excitability, site of generation of impulses, distribution and density of ionic channels, discharge pattern, and synaptic transmission (Eccles et al., 1958; Kuno and Llina´s, 1970a,b; Purves, 1975; Takata et al., 1980; Gustafsson and Pinter, 1984; Foehring et al., 1986; Delgado-Garcıá et al., 1988; Laiwand et al., 1988; see also review by Titmus and Faber, 1990). In contrast to the wealth of information regarding the physiological response of neurons of the peripheral nervous system—particularly motoneurons—to axonal injury, few © 2000 WILEY-LISS, INC.

data are available about the changes that axotomy may produce in neurons residing entirely within the central nervous system. There is, however, evidence that electrical membrane properties, such as input resistance, afterhyperpolarization, or relationship between spike frequency and current intensity, are modified in axotomized corticospinal neurons recorded in vitro (Tseng and Prince, 1996). In the present experiments we sought at character-

*Correspondence to: Dr. Rosa R. de la Cruz, Laboratorio de Neurociencia, Facultad de Biologıá, Avda. Reina Mercedes 6, 41012-Sevilla, Spain. E-mail: [email protected] Received 13 March 2000; Revised 13 July 2000; Accepted 2 August 2000

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izing the discharge modifications induced by axotomy in a group of well-defined premotor neurons recorded in vivo under alert physiological conditions, i.e., during the performance of spontaneous and reflex movements. We consider this is an important approach for a more complete understanding of the physiological response to injury, insofar as it allows evaluation of how changes in intrinsic membrane properties combine with synaptic alterations in producing a final output signal encoded in terms of frequency of action potentials. The central neurons chosen for the present study were the internuclear neurons of the abducens nucleus. These neurons project specifically onto the medial rectus motoneurons of the oculomotor nucleus, establishing excitatory synaptic connections (Highstein and Baker, 1978; Nakao and Sasaki, 1980). Their axons cross the midline at the level of the abducens nucleus and ascend in the contralateral medial longitudinal fascicle (MLF) up to the oculomotor nucleus (Bienfang, 1978; Highstein et al., 1982). The abducens nucleus additionally contains the motoneurons that innervate the ipsilateral lateral rectus muscle (Delgado-Garcıá et al., 1986a). The functional role of abducens internuclear neurons in mediating the conjugacy of horizontal gaze is well-accepted (Evinger et al., 1977; Highstein, 1977; Highstein and Baker, 1978; Delgado-Garcıá et al., 1986b). They provide the motoneurons of the (contralateral) medial rectus muscle with the same signal as the motoneurons innervating the (ipsilateral) lateral rectus muscle, thereby allowing the simultaneous contraction of the synergistic pair involved in the generation of conjugate horizontal eye movements. The discharge pattern and afferent synaptic organization of abducens internuclear neurons have already been characterized (Delgado-Garcıá et al., 1986b; Evinger, 1988). In the present work, we axotomized these neurons by transecting the MLF unilaterally at the pontomesencephalic junction, approximately 5– 6 mm rostral to the abducens nucleus. The firing activity of axotomized abducens neurons was then recorded simultaneously with eye movements in the alert cat preparation. In a previous set of experiments, we have evaluated the response of abducens internuclear neurons to the selective loss of their target medial rectus motoneurons. Motoneuronal death was induced following the injection of toxic ricin into the medial rectus muscle (de la Cruz et al., 1994a,b). In the present work, special attention was paid to compare, in the same neuronal type, the physiological changes induced by axotomy with those previously reported following the selective loss of target neurons (de la Cruz et al., 1994b). This comparison will help evaluating the retrograde influence of target cells on the expression and maintenance of the functional properties of neurons. Of interest for the interpretation of the present results were the ultrastructural findings of a reduced number of synapses contacting with the axotomized abducens internuclear neurons and the failure of the transected axons to reinnervate a new target in the lesioned MLF (Pastor et al., 2000). Preliminary accounts of this study have appeared in abstract form (de la Cruz et al., 1997).

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Fig. 1. Experimental design. The abducens nucleus (ABD) contains motoneurons that innervate the lateral rectus muscle (LR), and internuclear neurons whose axons cross the midline and contact the motoneurons of the oculomotor nucleus (OCM) innervating the medial rectus muscle (MR). Eye movements were recorded with coils implanted bilaterally. Bipolar stimulating electrodes (St) were implanted on the VIth cranial nerve and in the medial longitudinal fascicle (MLF) for the antidromic identification of ABD motoneurons and control internuclear neurons during recording sessions (Rec). The right MLF was transected 0.5–1 mm caudal to the trochlear nucleus level.

cellular recordings in the abducens nucleus. All experimental procedures were in accordance with the guidelines of the European Union Directive (86/609/EU) and current Spanish legislation for the use and care of laboratory animals in chronic experiments (BOE 67/8509-12, 1988). Two of these animals were used in the morphological study presented in the accompanying paper (Pastor et al., 2000).

Surgical procedures A detailed description of the preparation has been published previously (de la Cruz et al., 1994b). Briefly, after a protective injection of atropine sulfate (0.5 mg/kg, i.m.) to reduce vagal reflexes, the animal was anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotaxic frame. Surgery was then performed under sterile conditions to implant stimulating electrodes, scleral coils, and the recording chamber. Two silver bipolar electrodes were implanted (Fig. 1) to stimulate the left VIth nerve at its exit from the brainstem and the right MLF next to the oculomotor nucleus. In one animal, however, the VIth nerve was stimulated from the orbit, after the implantation of a pair of stainless-steel hook electrodes in the left lateral rectus muscle. Coils, made up of two turns of Teflon-insulated stainless-steel wire, were implanted in the sclera of both eyes. A square window (5 ⫻ 5 mm) was drilled in the occipital bone to allow transcerebellar access to the brainstem during the recording sessions. An acrylic resin recording chamber was constructed around the window, which was sealed between recording sessions. The chamber was part of a restraining system to immobilize the head during the recordings.

MATERIALS AND METHODS

Chronic recordings

Experiments were conducted on three female adult cats weighing 2.0 –2.5 kg. Animals were prepared for the chronic recording of eye movements and neuronal extra-

After 2 weeks of postoperative recovery, recording sessions started. The animal was restrained with elastic bandages and placed in a perspex box inside the scleral coil

FIRING OF CENTRAL NEURONS AFTER AXOTOMY frame and resting on top of a servocontrolled table. The recording system rotated around the vertical axis to induce the vestibuloocular reflex. Both the micromanipulator and the head-restraining device were located inside the coil frame. Eye movements were recorded by means of the magnetic field search-coil technique (Fuchs and Robinson, 1966). Eye coils were calibrated by rotating at known angles the magnetic field frame relative to the cat. Extracellular recordings were carried out with glass micropipettes beveled to a resistance of 1–3 M⍀ and filled with 2 M NaCl. Micropipettes were advanced with a threeaxis micromanipulator through the intact cerebellum to reach the brainstem. The (left) abducens nucleus was approached stereotaxically and located with the aid of the antidromic field potential produced by electrical stimulation of the VIth nerve. Abducens motoneurons were identified by their antidromic activation from the VIth nerve and by the collision test between the orthodromic and antidromic action potentials. Control abducens internuclear neurons were identified similarly but from the electrode implanted in the MLF. The extracellular neuronal activity was amplified and filtered at a bandwidth of 10 Hz to 10 kHz for display and digitalization purposes.

Data storage and analysis Head position, horizontal and vertical eye position of both eyes, and neuronal activity were digitally stored for off-line analysis by means of an eight-channel video tape system at a sampling frequency of 11 kHz. Computer programs were written to display the histogram of instantaneous firing frequency of the neuronal discharge (i.e., the reciprocal of the interval between two adjacent spikes) and the position of both eyes. Relationships between neuronal firing rate (FR, in spikes/second) and horizontal eye position (EP, in degrees) were obtained by linear regression analysis to calculate the slope, i.e., the neuronal sensitivity to eye position (ks, in spikes/second/degree), and the intercept (F0, in spikes/second), i.e., the neuronal firing rate at straight-ahead gaze. Firing rate during fixations responded to the equation FR ⫽ ks 䡠 EP ⫹ F0. Relationships between neuronal firing and eye velocity during spontaneous saccades were also obtained by linear regression analysis after subtraction of the position component (ks 䡠 EP) calculated from the previously known sensitivity to eye position. Thus, the equation used was FR – ks 䡠 EP ⫽ rs 䡠 EV ⫹ F0, where rs (in spikes/second/degree/ second) is the neuronal sensitivity to eye velocity (EV, in degrees/second). Responses during vestibular stimulation were subjected to multiple linear regression analysis, after selecting the slow phases of the vestibular nystagmus with cursors. The regression equation used was FR ⫽ F0 ⫹ kv 䡠 EP ⫹ rv 䡠 EV, where the two regression coefficients represented the neuronal sensitivities to position (kv, in spikes/second/degree) and velocity (rv, in spikes/second/ degree/second).

Transection of the MLF After several control recording sessions in the abducens nucleus, the right MLF was transected. The MLF was approached with a microblade of 1.3 mm width using the location of the abducens nucleus as the reference system and according to the coordinates of Berman’s atlas (1968). The microblade was stereotaxically aimed at an angle of 45° in the anterior direction and driven 1 mm deeper than the MLF to ensure complete transection of the fascicle.

393 The MLF was transected at the pontomesencephalic level, 0.5 to 1 mm caudal to the trochlear nucleus (Fig. 1). The lesion was assessed by the immediate impairment of the right eye’s moving past the midline. Other physiological criteria that served to assess the lesion were the incapacity to evoke abducens field potentials and to activate the abducens internuclear neurons antidromically following electrical stimulation of the electrode located in the MLF next to the oculomotor nucleus. In the three animals, histological inspection of the brainstem sections confirmed the complete unilateral transection of the right MLF.

RESULTS Eye movement deficits Immediately after the unilateral section of the right MLF, the eye ipsilateral to the lesion demonstrated the inability to cross past the primary position towards the left oculomotor hemifield. That is, the right eye was unable to adduct across the midline (Fig. 2B). This incapacity produced a gaze deviation of the right eye in the temporal direction. Consequently, there was a great reduction in the oculomotor range covered by the right eye; virtually, the left hemifield could not be explored. The MLF lesion also produced a marked increase in the occurrence of disjunctive horizontal eye movements, in contrast to the high degree of conjugacy observed in control recordings (Fig. 2A). Thus, horizontal eye movements after MLF lesion coursed with frequent divergences and convergences as well as monocular displacements (Fig. 2B; dashed and dotted vertical lines). All these symptoms were maximally expressed for the first 7–10 days postlesion. Thereafter, there was a progressive motor recovery, although this was never complete, at least up to 3 months, which was the longest time interval studied. The right eye gradually recovered a central position in the orbit. In parallel, there was a successive increase in the oculomotor range covered by this eye, with the left hemifield being progressively explored. As is shown in Figure 2C for a recording carried out 50 days postlesion, the right eye was able to cross to the left hemifield and gaze was centered again near zero. However, the high degree of conjugacy of controls was never regained, and disconjugated eye movements were commonly executed throughout the 3-month period of study (Fig. 2C; dotted and dashed vertical lines). The increased incidence of these disjunctive horizontal eye movements after MLF lesion was manifested by the low correlation coefficients of the linear regression analysis obtained between left and right horizontal position. These correlation coefficients were always lower than 0.7, in contrast to control coefficients higher than 0.8. No deficit was observed in the maintenance of eye position at any time postlesion. The range of eye movements was measured in controls and at different intervals postlesion to evaluate both the degree of oculomotor restriction produced by the interruption of the fascicle and the later motor recovery. Control horizontal eye movements ranged approximately ⫾40° (⫹ indicating movement to the left). For instance, for cat 3, right eye position moved from ⫹41.2° to –39.8° in the control situation. In this same animal, the oculomotor range of the right eye was limited to between ⫹5.1° and –29.6° by 4 days postlesion. By 35 days, a certain recovery

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Fig. 2. Spontaneous horizontal eye movements recorded before (A) and after (B,C) the transection of the right MLF. Upward deflections represent eye movements to the left. At short time intervals postlesion (B; 2 days), oculomotor deficits were evident on the right eye (RH, right horizontal eye position, in degrees). These deficits included the incapacity of this eye to adduct across the midline (horizontal dotted line), the presence of slow and small nasal eye movements, and a higher incidence of disconjugate eye movements, as indicated by the dashed (convergence) and dotted (divergence) vertical lines. At longer time intervals (C; 50 days), there was a partial recovery of these deficits, except for the disconjugate eye movements, which were frequent at all times after lesion. LH is left horizontal eye position.

had occurred; right horizontal eye position ranged from ⫹15.4° to –39.2°, and by 58 days the recovery was more evident, with the right eye position ranging from ⫹27.1° to –38.8°. During the third month postlesion, no further significant improvement was observed in eye movements performed by the right eye. The changes in oculomotor range are illustrated in Figure 3, which displays the plots of vertical vs. horizontal eye position during 5 minutes of spontaneous eye movements. The affected (right) eye showed a dramatic reduction in range and a shift of central eye position toward the right hemifield (Fig. 3B) compared to control (Fig. 3A) during the first 7–10 days after MLF lesion. Then, there was a gradual improvement in the performance of right eye movements, so that, as illustrated in Figure 3C for data obtained at 66 days postlesion, both the range and the central eye position of the

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Fig. 3. Graphs illustrating the motor field covered by each eye during 5 minutes of spontaneous eye movements before (A) and after (B,C) right MLF transection. For each eye, the vertical component is plotted vs. the horizontal component (RV vs. RH and LV vs. LH). Leftward and upward movements have a positive sign. Note in B, at 7 days postlesion, the restriction of the right eye movement (RH) to the right hemifield (vertical dotted line). The plots in C (66 days postlesion) show the partial recovery of the motor range that occurred at longer time intervals. In C, note a strong relationship between the vertical and horizontal components of right eye movements (correlation coefficients of the regression lines were 0.81 and 0.48 for the right and left eyes, respectively).

right eye were partially recovered. Although the left eye also showed a reduction in oculomotor range, this was of lower magnitude (Fig. 3B). Probably, left eye movements diminished in amplitude as a compensatory response to right eye restrictions, thereby minimizing double vision. Interestingly, a relationship appeared between the horizontal and the vertical components of the right eye movements at long postlesion intervals (Fig. 3C). In particular, right eye movements directed to the left (i.e., nasally) tended to course downward and those to the right (i.e., temporally) upward. This tendency became evident from the third week postlesion. To test whether the tendency was significant, we carried out linear regression analysis

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between the vertical and the horizontal position of the right eye. At all time points analyzed from day 28 postlesion to the end of recording sessions (3 months), the relationship coursed with correlation coefficients higher than 0.75 and negative slopes (i.e., the eye moved from downnasal to up-temporal). As expected, there was no correlation between the vertical and the horizontal components of eye movements in the control recordings. During the first 2 weeks after lesion, the correlation was either absent or weak, in that the correlation coefficients were lower than 0.5. With respect to the left eye, although a tendency similar to that described for the right eye was sometimes observed in the recordings carried out at long time intervals (Fig. 3C), the correlation coefficients were always lower than for the right eye and, in every case, lower than 0.55. This finding is of relevance in that it indicates that the recovery in the motor range of the affected (right) eye might be explained, at least in part, by the secondary actions of other extraocular muscles.

Abducens field potentials and single-unit recordings Electrical stimulation from the electrode located in the MLF near the oculomotor nucleus showed a complete postaxotomy abolition of the antidromic field potential recorded within the abducens nucleus. The control antidromic field potential of abducens internuclear neurons is composed of two negative waves at 0.3 and 0.9 msec (Fig. 4C, upper record), corresponding to the activation of two populations of internuclear neurons of different conduction velocity, as previously described (Delgado-Garcıá et al., 1986b). A control depth profile of the antidromic field potentials of internuclear neurons and motoneurons following the electrical stimulation to the MLF and VIth nerve, respectively, is illustrated in Figure 4A. However, immediately after MLF lesion, the antidromic field potential of the abducens internuclear neurons disappeared without alteration of the motoneuronal antidromic field (Fig. 4B and lower record of Fig. 4C). As expected, transection of the MLF resulted in the lack of antidromic identification of abducens internuclear neurons. To circumvent this problem, we implanted in one animal a second bipolar electrode caudal to the lesion site. From this second site, eight abducens internuclear neurons were positively identified from a total of 25 neurons recorded. An example of the collision test of an abducens internuclear neuron recorded 4 days postaxotomy is shown in Figure 4D. In those animals (n ⫽ 2) with no implanted electrode at the lesion site, identification of axotomized abducens internuclear neurons was carried out using several criteria, including that the recorded unit 1) was located within the limits of the antidromic field potential of the abducens nucleus, 2) failed to be antidromically stimulated from the MLF, 3) increased its firing frequency for ipsilateral eye movements, and 4) could, in some cases, be activated synaptically from the MLF. Synaptic activation from the oculomotor area in our sample of control internuclear neurons yielded a total of 7 of 24 recorded neurons (29.2% of the control sample), whereas in the axotomized population (n ⫽ 100), 17% were activated synaptically from the electrode located in the MLF near the oculomotor nucleus. The main source of synaptic activation of abducens internuclear neurons from the oculomotor area comes from the internuclear neurons located in the oculomotor and perioculomotor region. This

Fig. 4. Abducens field potentials and antidromic identification. A: Control depth profile of the antidromic field potentials induced in the abducens nucleus after MLF stimulation followed by stimulation of the abducens (ABD) nerve. Fields were recorded from ventral to dorsal at 400 ␮m steps through the abducens nucleus (indicated by numerals, in mm). Each record has three traces superimposed. B: Same as A but immediately after axotomy. Note the lack of antidromic field potential after MLF stimulation. Calibrations in B also apply to A. C: Control (Con) and postaxotomy (Ax) traces of the antidromic field potentials recorded in the center of the abducens nucleus following stimulation of the MLF and illustrated at higher temporal resolution. D: Collision test of an axotomized abducens internuclear neuron. The upper record shows a spontaneous orthodromic spike followed by the antidromic identification (arrow) after stimulation of the MLF caudal to the lesion site (arrowhead). The unit was recorded within the abducens nucleus, as illustrated by the antidromic field potential induced by stimulation of the abducens nerve (star). The lower record shows the collision (asterisk) of the orthodromic and the antidromic spike when the interval was shortened.

projection courses through the MLF and terminates bilaterally in the abducens nucleus (Maciewicz et al., 1975; Maciewicz and Spencer, 1977; de la Cruz et al., 1992). The unilateral transection of the right MLF would preserve part of the oculomotor descending pathway to the abducens nucleus, which could explain the synaptic activation of some abducens internuclear neurons after axotomy.

Firing alterations during spontaneous eye movements Qualitatively, control abducens internuclear neurons displayed a characteristic burst-tonic firing during spontaneous eye movements, as previously reported (DelgadoGarcıá et al., 1986b). These neurons exhibited a fairly regular firing rate that increased monotonically for successive ocular fixations directed towards the ipsilateral side of recording, namely, the on-direction. Conversely, firing rate decreased as the eye adopted eye positions more eccentric in the off-direction, and eventually neurons ceased firing (Fig. 5A). During on-directed saccades, control neurons discharged a high-frequency burst of spikes (Fig. 5A, dotted line), and, for saccades in the off-direction, the instantaneous firing frequency either decreased abruptly or ceased completely (Fig. 5A, dashed line). Some abducens internuclear neurons were recorded immediately after the transection of the MLF. Despite the

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Fig. 5. Firing activity of four abducens internuclear neurons recorded during spontaneous eye movements in the control situation (A) and during the first week postlesion (B–D). Horizontal position of both left (LH) and right (RH) eyes is illustrated, in degrees (deg). Upward deflections represent movements to the left. Firing rate (FR) is shown in spikes/second. The dotted line in A illustrates for the control neuron the burst of spikes present for on-directed saccades and the dashed line the decrease in FR for off-directed saccades. The neuron

shown in B was recorded immediately after transection of the MLF. Note that the firing activity looked normal, but oculomotor deficits appeared. The horizontal dotted line in B–D through the RH trace shows the central eye position in the orbit. Neurons illustrated in C (2 days postlesion) and D (5 days postlesion) showed a decreased modulation of FR. The asterisk in C indicates a strong postsaccadic slide in FR that finally silenced the neuron.

motor abnormalities of the right eye, the firing of these axotomized neurons was similar to that of controls; that is, they fired at high frequencies for both tonic and burst component (Fig. 5B). Therefore, axotomy did not produce any immediate qualitative alteration in the discharge properties of abducens internuclear neurons. Abducens internuclear neurons were recorded up to 3 months postaxotomy. As early as 2 days after lesion, some qualitative changes in the discharge pattern were noticed. The most obvious change was an overall decrease in the firing rate, so that axotomized neurons hardly discharged at rates higher than 100 –150 spikes/second during eye fixations (Fig. 5C,D), in contrast to control neurons that could reach tonic firing rates of up to 200 –250 spikes/ second (Fig. 5A). On occasion, axotomized neurons displayed a progressive decay in firing rate during fixations, demonstrating the inability to maintain a sustained rate during stable eye positions (Fig. 5C, asterisk). During saccades in the on-direction, axotomized neurons showed a moderate increase in firing or a reduced burst of spikes that was of low rate compared to controls (Fig. 5C,D, cf. A). Abducens internuclear neurons showed similar alterations in their discharge pattern throughout the 3 month study period. No evidence was found for a recovery in firing properties during this period.

velocity of the eye was used for obtaining the position and velocity sensitivities, respectively. Control neuronal sensitivities were calculated for both eyes, but for axotomized internuclear neurons we will refer them to the ipsilateral (left) eye, because the transection of the right MLF reduced the range of movements of the right eye, as described above, and this interfered with the neuronal firing analysis. Thus, as exemplified in Figure 6A,B, the same data set (i.e., firing rates) of a control abducens internuclear neuron plotted against the left (A) and right (B) eye positions yielded similar position sensitivities (ks), and the correlation coefficients (r) of the linear regression analysis in the two cases were high and close. Correlation coefficients in the control sample were always higher than 0.84. The mean ks obtained for the 24 control abducens internuclear neurons was 7.01 ⫾ 1.64 (mean ⫾ SD) spikes/ second/degree when calculated using the left eye. This value was not different from that calculated with the right eye (6.80 ⫾ 1.51; P ⬎ 0.3, paired t-test). Such results indicated that analysis could be performed using either eye for the control neurons, owing to the high degree of conjugacy of horizontal eye movements. Conversely, for axotomized neurons recorded at short intervals after lesion (Fig. 6C,D), the firing-position plots calculated using the right eye (D) produced abnormally high ks values, because of the low range of eye movements, but yielded lower sensitivity values when using the left eye (C). Moreover, the correlation coefficients of the linear regression analysis obtained with the left eye were higher than those obtained with the right eye. At longer time intervals after

Neuronal sensitivities during spontaneous eye movements As indicated in Materials and Methods, linear regression analysis between the firing rate and the position and


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Fig. 6. Analysis of the firing rate of abducens internuclear neurons during spontaneous eye fixations. Firing rate (FR, in spikes/second) was plotted against left (LH) and right (RH) eye position, in degrees, for a control (A,B) and two axotomized abducens internuclear neurons recorded on days 4 (C,D) and 41 (E,F) postlesion. Eye positions to the left are shown as positive values on the abscissa axis. The slope of the regression line between FR and eye position represents the neuronal eye position sensitivity during spontaneous eye movements (ks, in spikes/second/degree). The correlation coefficient (r) is also indicated.

Note the reduced ks and r values obtained in the FR vs. LH plots of the two axotomized neurons (C,E) compared to the control neuron (A). At short time intervals after lesion, the restriction of right eye movements to the temporal (rightward) oculomotor hemifield yielded a higher slope in the rate-position plots when FR was analyzed with the right eye (D) rather than with the left eye (C). At longer time periods, there was a partial recovery of right eye movements, but the high degree of disconjugacy explained the lower ks and r values obtained when FR was plotted vs. RH (F) compared to LH (E).

lesion, the range of horizontal right eye movements partially recovered (Fig. 6F), but, because this recovery was due, at least in part, to the contribution of other vertical or torsional components (as described above), neuronal firing was highly uncorrelated with the horizontal position of the right eye. Correlations calculated with the left eye were higher (correlation coefficients between 0.58 and 0.92), though still lower than controls. Therefore, for the purposes of comparing to control, firing parameters obtained with the left eye were used. Despite the fact that morphological examination of the lesion demonstrated a complete transection of the MLF in the three animals, we found a small proportion of internuclear neurons that had normal discharge characteristics. An example of an axotomized abducens internuclear neuron exhibiting a normal firing pattern recorded 44 days postlesion is shown in Figure 7A. This type of neuron modulated with horizontal eye movements showing high rates of discharge, a sustained tonic firing during eye fixations, and high-frequency spike bursts during ondirected saccades. Figure 7A also shows the recovery in range of right eye movements occurring at long time intervals after lesion. However, horizontal eye movements coursed with a high degree of disconjugacy, so that divergences and convergences (Fig. 7A; dotted and dashed lines) were frequent. In general, most of the axotomized neurons modulated more closely in relation with the movements of the left (ipsilateral) eye. In these apparently normal abducens internuclear neurons recorded after le-

sion, the analysis of firing rate with eye position also yielded better correlations when performed with the left than with the right eye (Fig. 7B,C), as happened for the rest of the axotomized neurons (Fig. 6). For the whole population of control and axotomized abducens internuclear neurons, Figure 8A shows the time course of changes in position sensitivity (ks) obtained with the left eye. Seventeen neurons of the postlesion sample behaved qualitatively as normal cells and showed position sensitivity values that fell within 1 SD of the control mean; these included three neurons recorded immediately after lesion (day 0). Therefore, we separated these two groups of cells in the subsequent analyses, i.e., cells showing a normal physiology (n ⫽ 17) vs. those showing firing alterations (n ⫽ 83) after MLF lesion. To determine whether there was any variation in ks with time, the postlesion sample of affected internuclear neurons was grouped into three different time intervals: the first week (short-term), the period from the second to the fourth weeks (medium-term), and that from the fifth to the twelfth weeks (long-term). The results are shown in Figure 8B, which displays the mean ks and SD for each neuronal group. A significant reduction in the mean position sensitivity was found at all time intervals postaxotomy compared to the control data (P ⬍ 0.05; ANOVA test). Moreover, no significant differences were obtained between the three groups of neurons recorded at short, medium, or long time intervals. Therefore, axotomy pro-

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Fig. 7. Example of an abducens internuclear neuron recorded 44 days after MLF transection showing a normal discharge pattern. In A, the firing rate (FR, in spikes/second) of the neuron is illustrated, along with the horizontal position of both the left (LH, in degrees) and right (RH) eyes. Vertical lines indicate disjunctive eye movements (dotted lines, divergences; dashed lines, convergences). B: Linear regression plot of FR vs. LH during spontaneous eye fixations for the cell illustrated in A. The slope of the regression line (ks, in spikes/ second/degree) represents the neuronal eye position sensitivity during spontaneous eye movements. The correlation coefficient (r) is also indicated. C: Same as B, but after correlating the same FR data with the other eye (RH). Note the higher scattering of data points and lower ks and r values compared to the plot shown in B.

Fig. 8. A: Scatterplot of eye position sensitivity for spontaneous eye movements (ks; calculated with the left eye) of 100 abducens internuclear neurons recorded after MLF transection. Control data (n ⫽ 24) are indicated by an arrow on the abscissa axis. The horizontal dashed line represents the mean ks of the control sample minus 1 SD. Neurons whose ks was higher than this value were considered normal postlesion cells. B: Histogram showing the mean and standard deviation of ks values after grouping the neurons at the time intervals indicated (c, control; w, week). The group of neurons recorded after lesion and showing normal firing parameters is shown separately (n). Asterisks indicate statistically significant differences with respect to the control group (P ⬍ 0.05; ANOVA test).

duced a rapid and long-lasting decrease in the neuronal sensitivity to eye position. The firing rate at zero eye position (F0) showed similar changes after axotomy. For control neurons, mean F0 was 57.9 ⫾ 22.7 spikes/second. Axotomized abducens internuclear neurons exhibited a lower firing rate for the central left eye position, although the tendency was significant (P ⬍ 0.05; ANOVA test) only for the groups of neurons recorded at medium- and long-term postlesion intervals. For example, neurons recorded between weeks 5 and 12 had a mean F0 of 33.6 ⫾ 16.5 spikes/second. The sensitivity of neurons to eye velocity during spontaneous saccades (rs) was also calculated as the slope of the regression line between the firing rate (after subtraction of the position component) and the saccadic velocity of the left eye. Qualitatively, it was observed that most axotomized neurons showed a weak-to-moderate increase in firing rate during saccades in the on-direction, in contrast to the high-frequency spike burst of control neurons. This observation was reflected quantitatively as a decrease in

the eye velocity sensitivity. Thus, control neurons had a mean rs value of 1.38 ⫾ 0.41 spikes/second/degree/second. During the first week postlesion, rs dropped to 0.62 ⫾ 0.21 spikes/second/degree/second. The decrease in rs was of similar magnitude in the groups of neurons recorded at medium and long time intervals. At all time intervals postlesion, the difference in mean rs vs. the control group reached the significance level (P ⬍ 0.05; ANOVA test). For the group of cells recorded after lesion and showing a normal firing pattern, mean rs was similar to the control value.

Behavior during vestibular stimulation Horizontal sinusoidal head rotation in the dark induced a vestibular nystagmus that consisted of a sinusoidal slow eye movement in the opposite direction to the head, interspersed with fast eye movements in the direction of head movement. Control abducens internuclear neurons modulated in relation to both the slow and the fast phases of the vestibular nystagmus (Fig. 9A). The lesion of the MLF


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Fig. 9. Discharge pattern of three abducens internuclear neurons recorded during vestibularly induced eye movements in the control situation (A) and 5 days (B,C) following MLF transection. From top to bottom: horizontal position of the left and right eyes (LH and RH, in degrees), horizontal eye velocity of both eyes (LH⬘ and RH⬘, in degrees/second), head position (H, in degrees), and firing rate (FR, in spikes/second). Note the reduced velocity of RH (arrowheads) result-

ing from the impairment in the performance of the vestibuloocular reflex (asterisks in RH) after lesion. The majority of axotomized internuclear neurons showed a decreased firing modulation during the vestibular nystagmus, as illustrated in B. Arrows indicate the reduced bursts in FR during the fast phases of the nystagmus. A few axotomized cells (such as the one illustrated in C) showed firing characteristics similar to those of the control neurons.

affected the performance of the vestibuloocular reflex in the right eye, which coursed with a slower velocity (Fig. 9B,C; asterisks and arrowheads), but left eye movements were preserved. Axotomy also altered the discharge of abducens internuclear neurons during vestibular eye movements. Most axotomized neurons fired at low rates, showing a reduced degree of modulation during both the slow and the fast phases of the nystagmus (Fig. 9B). However, as described for spontaneous eye movements, a few internuclear neurons exhibited an apparently normal firing during the vestibular nystagmus (Fig. 9C). Neuronal sensitivities to eye position and velocity during vestibular stimulation (kv and rv, respectively) were calculated using multiple regression analysis. Figure 10 illustrates the procedure for a neuron recorded 7 days postaxotomy and during one cycle of vestibular stimulation. The plot of firing rate vs. left eye position produced an ellipsoid figure (Fig. 10B, left) that collapsed into a line when the velocity component was removed (Fig. 10C, left). The slope of this line represented the position sensitivity. The scatterplot of firing rate vs. left eye velocity showed circulation according to the direction of movement (Fig. 10D, left). After subtraction of the position component, the scatterplot similarly collapsed into a line whose slope represented the velocity sensitivity (Fig. 10E, left). Thus, during vestibular stimulation, the firing rate was propor-

tional to both the position and the velocity of the eye. Because the lesion of the MLF altered the performance of the vestibuloocular reflex in the right eye, the analysis of firing rate with right eye movements produced abnormally high kv values because of the reduced range of eye position (Fig. 10B,C, right). The position sensitivity of control neurons obtained with the left eye, kv, was 9.9 ⫾ 3.26 spikes/second/degree. This parameter showed a significant decrease (P ⬍ 0.05; ANOVA test) at all time intervals postaxotomy, i.e., in the first week, from the second to the fourth weeks, and from the fifth to the twelfth weeks (Fig. 11A). No significant differences were found between the three postaxotomy groups. Therefore, no recovery occurred with time in this firing parameter. The analysis of eye velocity sensitivity during vestibularly induced eye movements, rv, showed similar results. Mean control rv was 1.94 ⫾ 1.06 spikes/ second/degree/second. Abducens internuclear neurons recorded during the first week postlesion showed a mean rv of 0.91 ⫾ 0.42 spikes/second/degree/second, which was significantly different from controls (P ⬍ 0.05; ANOVA test). A similar degree of reduction in rv was obtained in the neurons recorded at medium and long terms after lesion (Fig. 11B). As happened for spontaneous eye movements, the group of neurons whose firing appeared normal

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Fig. 10. Analysis of an abducens internuclear neuron recorded 7 days postaxotomy during the vestibuloocular reflex. In A, the firing rate (FR, in spikes/second) of the cell is shown simultaneously with the left and right horizontal eye positions (LH and RH, in degrees) and velocity (LH⬘ and RH⬘, in degrees/second) and head position (H, in degrees). B: Scatterplots of FR vs. LH (left) and RH (right). C: Partial regression plots of the data shown in B. After subtraction of the firing component from eye velocity, the scatterplots shown in B collapsed to a line (rl and rr are left and right eye velocity sensitivity, respectively). Note that, when analyzed for the right eye, data points were restricted to the right oculomotor hemifield, yielding a higher slope (C, right). The slope of the regression line (kv) corresponds to the neuronal

sensitivity to eye position during vestibular stimulation (4.17 and 11.9 spikes/second/degree for left and right eyes, respectively). D: Scatterplots of FR vs. LH⬘ (left) and RH⬘ (right) for the same data points plotted in B. E: Partial regression plots of the data shown in D. After subtraction of the firing component resulting from eye position (where kl and kr indicate left and right eye position sensitivities, respectively), the scatterplots shown in D collapsed closer to a line. The slope of the linear regression line represents the neuronal sensitivity to eye velocity during vestibular stimulation (rv, which was 0.86 and 0.41 spikes/second/degree/second for the left and right eyes, respectively). Correlation coefficients were 0.91 and 0.90 for regression lines with LH and LH⬘ and 0.90 and 0.38 for regression lines with RH and RH⬘.

exhibited mean kv and rv that did not differ significantly from those of the control group.

eye movements, no alteration was found in the firing activity of the postlesion group of motoneurons. Therefore, the unilateral transection of the MLF at a rostral level did not significantly alter the discharge characteristics of abducens motoneurons.

Firing of abducens motoneurons Control abducens motoneurons discharge with a pattern similar to that of internuclear neurons, as previously described (Delgado-Garcıá et al., 1986a,b). Thus, during spontaneous eye movements they show a tonic firing proportional to eye position, and for on-directed saccades they discharge a burst of spikes proportional to the eye velocity. During the vestibular nystagmus, motoneurons are also modulated in relation to both eye position and eye velocity. Abducens motoneurons recorded after the lesion of the MLF showed a normal firing pattern in relation to the movement of the ipsilateral (left) eye (not illustrated). For the control sample of motoneurons (n ⫽ 13), mean position and velocity sensitivities were ks ⫽ 6.3 ⫾ 1.26 spikes/ second/degree and rs ⫽ 0.98 ⫾ 0.31 spikes/second/degree/ second. These firing parameters were similar in the group of motoneurons recorded after MLF lesion (n ⫽ 33), with ks ⫽ 6.05 ⫾ 1.15 spikes/second/degree and rs ⫽ 1.05 ⫾ 0.35 spikes/second/degree/second. During vestibularly induced

DISCUSSION The present study has evaluated the physiological changes produced by axotomy in the discharge characteristics of abducens internuclear neurons of the adult cat. During spontaneous eye movements there was a general reduction in firing rate, affecting both the tonic and the burst components. During the vestibuloocular reflex, axotomized neurons showed low rates and a small degree of modulation. Using linear regression analysis, we quantified the sensitivities of axotomized neurons to eye position and velocity, and compared the data to those of control neurons. The results showed a significant reduction in eye position and velocity sensitivities during spontaneous fixations and saccades as well as during the vestibular nystagmus (ks, rs, kv, and rv firing parameters, respectively).


Fig. 11. A: Eye position sensitivity of abducens internuclear neurons during vestibularly induced eye movements (kv). Mean values and their SD are represented for controls (c) and at different time intervals in weeks (w) following axotomy. Asterisks indicate statistically significant differences with respect to control data (P ⬍ 0.05; ANOVA test). A group of axotomized abducens internuclear neurons showed normal kv values and are illustrated separately (n). B: Same as A, but for the eye velocity sensitivity (rv). The parameters kv and rv were obtained by multiple linear regression analysis with respect to the left eye.

Moreover, firing alterations were present throughout the whole period of study after lesion (3 months), without any sign of recovery. Abducens motoneurons showed no significant change in their discharge pattern, having normal firing parameters. Therefore, the discharge alterations of abducens internuclear neurons were due to the axotomy effects and reflected a loss of oculomotor-related signals in their firing activity.

Oculomotor deficits after MLF transection The unilateral transection of the (right) MLF affected the performance of spontaneous and reflex movements of the ipsilateral eye. The right eye showed an incapacity to adduct across the midline, so that eye movements were restricted to the right hemifield. Whereas control horizontal eye movements coursed with a high degree of conjugacy, after MLF lesion convergent and divergent eye movements were commonly observed. Similar oculomotor

401 deficits have been described in the monkey following the bilateral transection of the MLF (Evinger et al., 1977) and the reversible unilateral blockade of the MLF after lidocaine injection (Gamlin et al., 1989), except that the effects produced by bilateral lesions were more drastic, especially those concerning the vertical eye movements. These oculomotor deficits are identified clinically as anterior internuclear ophthalmoplegia (Christoff et al., 1960; Carpenter and McMasters, 1963; Pola and Robinson, 1976), a syndrome produced by lesions of the MLF rostral to the abducens nucleus and characterized by specific disturbances of conjugate eye movements, with paralysis of ocular adduction on attempted lateral gaze but preservation of convergence. Based on electrophysiological and lesion experiments, it has been claimed that the deficits observed in this form of ophthalmoplegia can be explained by the interruption of the internuclear neuron pathway arising in the abducens nucleus and terminating on the contralateral medial rectus motoneurons of the oculomotor nucleus (Evinger et al., 1977; Highstein, 1977; Highstein and Baker, 1978; Delgado-Garcıá et al., 1986b). The excitatory connection of abducens internuclear neurons with the contralateral medial rectus motoneurons and their firing activity similar to that of lateral rectus (abducens) motoneurons ensure the synergistic contraction of the medial and lateral rectus muscles of each eye for conjugate horizontal gaze. The present data further support a functional role for abducens internuclear neurons in mediating the conjugacy of horizontal eye movements. The interruption of this projection would, therefore, explain the high incidence of disjunctive eye movements observed in the present experiments. Moreover, this deficit did not revert with time, and horizontal eye movements remained highly disconjugate throughout the 3 month period of study. This indicated that conjugacy cannot be restored by compensatory mechanisms involving other intact oculomotor pathways lying outside the MLF, such as the ascending tract of Deiters. Despite of the lack of recovery in the conjugacy of horizontal gaze, other deficits improved notably with time. In particular, there was a gradual return of the right eye to the central position in the orbit, so that this eye could progressively explore the left hemifield. This was accompanied by the consequent increase in oculomotor range. Recovery was not complete, however; the most eccentric eye positions in the nasal direction were not reached, even after 3 months. Two possible mechanisms might contribute to the motor recovery observed after lesion. One could be based on collateral sprouting or increased synaptic efficacy of other inputs to medial rectus motoneurons that were preserved by the MLF lesion. In addition to abducens internuclear neurons, a major afferent source to medial rectus motoneurons arises in the ventral lateral vestibular nucleus. The axons of these excitatory vestibular neurons course ipsilaterally by means of the ascending tract of Deiters (Baker and Highstein, 1978; Nguyen et al., 1999). This tract, which is located laterally to the MLF, was preserved in our lesions (see accompanying paper; Pastor et al., 2000). Neurons in the ascending tract of Deiters encode head velocity and eye position signals (Reisine and Highstein, 1979; Reisine et al., 1981). Although the eye position signal of these vestibular neurons is not as strong as that of abducens internuclear neurons (Reisine et al., 1981), Deiters’ neurons might contribute to the recovery of the primary eye position and range of movements of the

402 affected (right) eye, particularly if the synaptic efficacy of this vestibular input on the motoneurons was increased. This could happen, for example, by axonal sprouting of vestibular fibers on the vacated postsynaptic space left over the motoneuronal membrane after the interruption of the abducens internuclear pathway, a phenomenon commonly observed in the injured central nervous system (see reviews by Cotman et al., 1981, and Seil, 1989). Therefore, compensatory mechanisms involving the intact ascending tract of Deiters neurons might participate in the oculomotor recovery observed after MLF lesion. A second possible explanation for this motor recovery is the contribution of other extraocular muscles. In support of this idea is the good correlation (r ⬎ 0.75) found between the horizontal and the vertical components of eye movements, especially at a long time after lesion. Thence, the motor range covered by the right eye could be restored by the secondary actions of other vertical or torsional extraocular muscles. In particular, in frontally eyed animals, the inferior rectus muscle produces adduction besides its primary depressor action, and the secondary actions of the inferior oblique muscle include elevation and abduction (Graf and Simpson, 1981). The contribution of these other extraocular muscles might, therefore, support the recovery of the motor range in the right eye and would be in accordance with the correlation found between the vertical and the horizontal components of right eye movements.

Firing alterations of axotomized abducens internuclear neurons The discharge characteristics of abducens internuclear neurons exhibited several changes after MLF lesion. Axotomized neurons fired at lower rates and were modulated to a smaller degree during the performance of different types of eye movement. Quantitatively, there was a significant decrease in the neuronal sensitivities to eye position and velocity, during both spontaneous and vestibularly induced eye movements. Two possible processes might explain these findings. First, alterations of the membrane electrical properties could contribute to the observed changes in the discharge pattern of axotomized neurons by reducing the efficacy in the input-to-output transformation of the preoculomotor synaptic drive. Axonal injury has been described as inducing changes in the electrophysiological properties of neurons, such as in excitability, input resistance, rheobase current, electrogenesis, afterhyperpolarization characteristics, and ionic channel expression and distribution (see review by Titmus and Faber, 1990). The relative contribution of electrical changes to the altered discharge of abducens internuclear neurons observed under alert physiological conditions remains to be determined. However, modifications of the electrical membrane properties may be subtle, in spite of striking changes occurring in the discharge activity, as happens for axotomized abducens motoneurons (Baker et al., 1981; Delgado-Garcıá et al., 1988). This case probably reflects the great influence of the synaptic inputs that are partially lost after axotomy and is, in fact, the second mechanism likely involved in the discharge alterations of axotomized abducens internuclear neurons, i.e., a depression in the synaptic transmission from afferent inputs. Synaptic depression is a common phenomenon occurring in axotomized neurons (Kuno and Llina´s, 1970b; Purves and Lichtman, 1978; Mendell, 1984). In many cases, the

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decrease in synaptic efficacy has been correlated ultrastructurally with a reduced number of synaptic endings contacting with the membrane surface of axotomized neurons (Matthews and Nelson, 1975; Purves, 1975; Sumner, 1975; Delgado-Garcıá et al., 1988). Indeed, we have ultrastructural evidence for synapse loss as one mechanism involved in the firing alterations of abducens internuclear neurons following their axotomy. As described in the accompanying paper (Pastor et al., 2000), as early as 6 days after axotomy, the percentage of the somatic perimeter of abducens internuclear neurons that appeared covered by synaptic boutons was significantly reduced compared to that of control neurons. This synaptic loss was maintained (and even accentuated) during the 3 month period of study. We consider that the reduction in the density of afferent synapses is one major factor responsible for the decreased firing rate and the low eye position and velocity sensitivities of abducens internuclear neurons following their axotomy.

Absence of changes in abducens motoneurons In contrast to the internuclear neurons, abducens motoneurons recorded postlesion showed normal discharge characteristics during the different types of eye movement. This finding indicated that the main inputs terminating on the abducens nucleus were not sectioned by the MLF lesion. Vestibular, prepositus hypoglossi, and reticular neurons constitute the major afferent sources to the abducens neurons (Hikosaka et al., 1978; Igusa et al., 1980; Bu¨ttner and Bu¨ttner-Ennever, 1988; Escudero and Delgado-Garcıá, 1988; Evinger, 1988; Escudero et al., 1992), and they are all located caudal to the lesion site in the MLF. Insofar as abducens motoneurons and internuclear neurons share common afferent inputs (Highstein et al., 1976; Ishizuka et al., 1980; Evinger, 1988), the presence of a normal discharge pattern in the motoneurons ruled out the possibility that the physiological alterations found in the internuclear neurons were the consequence of deafferentation and confirmed that they were due to the axotomy effects. However, the lesion to the MLF performed in the present experiments probably sectioned one descending input to the abducens nucleus arising in the oculomotor area. This projection corresponds to the oculomotor internuclear neurons, terminating bilaterally in the abducens nucleus (Maciewicz et al., 1975; Maciewicz and Spencer, 1977; de la Cruz et al., 1992). Little is known about the discharge characteristics and connectivity of the oculomotor internuclear neurons in the cat. For the monkey, although their involvement in the generation of vergence eye movements was suggested initially (Mays, 1984; Mays et al., 1986), recent experiments indicate that they code conjugate horizontal signals (Clendaniel and Mays, 1994). Our lesions were unilateral, so the projection from the oculomotor internuclear neurons was not completely transected, and a part was preserved. The fact that part of this pathway remained intact, along with the likely minor contribution in the processing of oculomotor signals at the motoneuronal level (compared to the vestibular, prepositus, and reticular neurons), might explain the absence of significant changes in the discharge of the motoneurons following the unilateral transection of the MLF.


Comparison of axotomy with selective target removal We have previously characterized the response of abducens internuclear neurons to the selective loss of their target motoneurons induced by the injection of toxic ricin into the medial rectus muscle (de la Cruz et al., 1994a,b). This type of lesion, in contrast to axotomy, leaves the axons and terminals of the internuclear neurons intact. However, both experimental procedures disconnect these neurons from their target. The firing alterations found in the present work share some similarities with those previously reported following selective target lesion. Thus, in both cases, the overall firing rate and the degree of firing modulation of abducens internuclear neurons are reduced during the different types of eye movement, yielding a significant decrease in neuronal eye position and velocity sensitivities. However, there is an important difference between the two types of lesion; after the selective target ablation, firing alterations are present only during an initial period of approximately 3 weeks. Thereafter, the firing of abducens internuclear neurons recovers a normal pattern (de la Cruz et al., 1994b). This functional recovery is coincident with the reinnervation of a new target within the terminal area: the internuclear neurons of the oculomotor nucleus (de la Cruz et al., 1994a). In contrast, after axotomy, abducens internuclear neurons did not restore a normal discharge pattern with time, and firing alterations remained throughout the 3 months of the postlesion study. Absence of functional recovery in axotomized neurons might be linked to the lack of reinnervation of a new target. We have shown that the proximal stumps of transected axons in the MLF remain devoid of postsynaptic target for these 3 months (Pastor et al., 2000). Thus, according to the present data and those obtained after selective target removal (de la Cruz et al., 1994a,b), it seems that alterations in the firing properties of abducens neurons could be explained by the absence of contact with a target. This conclusion is in agreement with previous studies in axotomized motoneurons showing that reinnervation is required for the full restoration of normal electrical properties and synaptic inputs (Sumner, 1976; Pinter and Vanden Noven, 1989; Vanden Noven and Pinter, 1989). However, following transection of the MLF, a small group of abducens internuclear neurons displayed normal discharge characteristics and firing parameters. In particular, 17 of 100 neurons recorded after lesion behaved as normal units. If we ignore the three neurons recorded on the day of axotomy, the group of normal cells represented 14% of the total population of neurons recorded postlesion. A similar proportion of abducens internuclear neurons maintains normal firing properties following the selective removal of their target motoneurons (de la Cruz et al., 1994b). Probably, these neurons send collaterals to other sites in addition to medial rectus motoneurons in the oculomotor nucleus. Such terminal areas of some abducens internuclear neurons could be the prepositus hypoglossi nucleus, according to the results of intracellular tracer injection and anterograde biocytin labeling (Highstein et al., 1982; de la Cruz et al., 1994a). The connection with prepositus hypoglossi neurons, located caudally to the abducens nucleus, is preserved after transecting the MLF at a rostral level. Therefore, this other target might sustain the expression of normal functional properties in

403 this subset of abducens internuclear neurons, even when its main axon, coursing rostrally in the fascicle, is cut. Considerable evidence has been obtained during the last decades that target cells regulate numerous properties of neurons. The agents of this retrograde influence are considered to be neurotrophic factors (Purves, 1990). Neurotrophins have been implicated in the maintenance of synapses (Njå and Purves, 1978; Miyata et al., 1986) as well as in the modulation of neuronal excitability (Gonzalez and Collins, 1997; Oyelese et al., 1997; Scharfman, 1997; see also review by McAllister et al., 1999). Altogether, these findings suggest that target-derived factors could be implicated in the expression and maintenance of the electrical and synaptic properties of abducens internuclear neurons. The lack of these neurotrophic factors might be responsible, at least in part, for the functional alterations observed following their axotomy (present results), as well as after selective target removal (de la Cruz et al., 1994b).

ACKNOWLEDGMENTS We thank Mr. R. Churchill for assistance in the preparation of the manuscript. This study was supported by grants from the Spanish CICYT (SAF96-0160 to A.M.P.), DGICYT (PB93-1175 to J.M.D.-G. and R.R.d.l.C.), and Fundacioń MAPFRE Medicina to R.R.d.l.C.

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