Abstract
Multiply-innervated muscle fibers (MIFs) are peculiar to the extraocular muscles as they are non-twitch but produce a slow build up in tension on repetitive stimulation. The motoneurons innervating MIFs establish en grappe terminals along the entire length of the fiber, instead of the typical en plaque terminals that singly-innervated muscle fibers (SIFs) motoneurons establish around the muscle belly. MIF motoneurons have been proposed to participate only in gaze holding and slow eye movements. We aimed to discern the function of MIF motoneurons by recording medial rectus motoneurons of the oculomotor nucleus. Single-unit recordings in awake cats demonstrated that electrophysiologically-identified medial rectus MIF motoneurons participated in different types of eye movements, including fixations, rapid eye movements or saccades, convergences, and the slow and fast phases of the vestibulo-ocular nystagmus, the same as SIF motoneurons did. However, MIF medial rectus motoneurons presented lower firing frequencies, were recruited earlier and showed lower eye position (EP) and eye velocity (EV) sensitivities than SIF motoneurons. MIF medial rectus motoneurons were also smaller, had longer antidromic latencies and a lower synaptic coverage than SIF motoneurons. Peristimulus time histograms (PSTHs) revealed that electrical stimulation to the myotendinous junction, where palisade endings are located, did not recurrently affect the firing probability of medial rectus motoneurons. Therefore, we conclude there is no division of labor between MIF and SIF motoneurons based on the type of eye movement they subserve.
SIGNIFICANCE STATEMENT In addition to the common singly-innervated muscle fiber (SIF), extraocular muscles also contain multiply-innervated muscle fibers (MIFs), which are non-twitch and slow in contraction. MIF motoneurons have been proposed to participate only in gaze holding and slow eye movements. In the present work, by single-unit extracellular recordings in awake cats, we demonstrate, however, that both SIF and MIF motoneurons, electrophysiologically-identified, participate in the different types of eye movements. However, MIF motoneurons showed lower firing rates (FRs), recruitment thresholds, and eye-related sensitivities, and could thus contribute to the fine adjustment of eye movements. Electrical stimulation of the myotendinous junction activates antidromically MIF motoneurons but neither MIF nor SIF motoneurons receive a synaptic reafferentation that modifies their discharge probability.
- eye movements
- motoneuron
- multiply innervated fiber
- oculomotor system
- palisade endings
- singly innervated fiber
Introduction
Multiply-innervated muscle fibers (MIFs) of mammals show remarkably different structural and functional characteristics as compared with the canonical singly-innervated muscle fibers (SIFs). MIFs are only found in extraocular, laryngeal, and middle inner ear muscles (Schiaffino and Reggiani, 2011). MIFs are small fibers with a poorly developed sarcoplasmic reticulum which do not respond on electrical stimulation with a twitch-like tension, rather a graded slow and small-amplitude contraction on repetitive stimulation, and are also known as slow or non-twitch fibers (Bach-y-Rita and Ito, 1966; Pilar, 1967; Shall and Goldberg, 1992). The motoneuronal innervation of MIFs is also unusual since they receive multiple en grappe synaptic terminals along their length arising from thin axons (Hess and Pilar, 1963; Namba et al., 1968; Mayr, 1971; Bondi and Chiarandini, 1983). In contrast, SIFs are large-size muscle fibers innervated by large-diameter motoneuronal axons forming the classical en plaque neuromuscular junction (Namba et al., 1968; Mayr, 1971), as in the skeletal musculature, and generating a twitch on stimulation.
The physiological role of MIF motoneurons is intriguing due, in part, to the peculiarities of the muscle fibers they innervate. It has been suggested that they could contribute differentially to the generation of eye movements, compared with SIF motoneurons (Büttner-Ennever and Horn, 2002; Wasicky et al., 2004; Eberhorn et al., 2006; Ugolini et al., 2006). Thus, MIF motoneurons might participate in gaze holding and slow eye movements, whereas SIF motoneurons would participate in all types of eye movements. Two types of experiments have led to this suggestion. First, morphologic studies have demonstrated that MIF motoneurons are mainly located at the periphery of the oculomotor nuclei, anatomically segregated from SIF motoneurons. This has been shown in humans (Horn et al., 2018), monkeys (Büttner-Ennever et al., 2001; Wasicky et al., 2004; Büttner-Ennever, 2006; Erichsen et al., 2014; Tang et al., 2015), rats (Eberhorn et al., 2006), and in the medial rectus subgroup of the oculomotor nucleus in cats (Bohlen et al., 2017b). Nevertheless, in the cat abducens nucleus, MIF and SIF motoneurons have been found intermingled (Hernández et al., 2019). Second, anatomic studies of the afferent inputs to MIF and SIF motoneurons have revealed that these two types of motoneuron receive, in general, different projections. Thus, in primates, the anterograde labeling of afferents to oculomotor MIF and SIF motoneurons (Wasicky et al., 2004) and the retrograde transneuronal tracing of rabies virus injected into the distal portion of the lateral rectus muscle (where en grappe terminals of MIF motoneurons are located) have shown a differential innervation pattern of MIF and SIF motoneurons, so that “tonic” inputs, those responsible for a sustained firing during fixations such as vestibular and prepositus hippoglossi neurons, terminate on MIF motoneurons, which virtually do not receive “fast” afferents (i.e., burst reticular neurons), whereas SIF motoneurons receive all known types of preoculomotor, “slow” and “fast,” afferents (Ugolini et al., 2006).
It is also important to highlight that MIF, but not SIF motoneurons, in addition to the en grappe terminals along the muscle fiber, give rise to the so-called palisade endings at the myotendinous junction, which are particularly abundant in the medial rectus muscle (Blumer et al., 2017). Palisade endings were described as sensory structures based on their morphology (Ruskell, 1978; Alvarado-Mallart and Pinçon-Raymond, 1979). Nonetheless, their function is under debate as connectional and molecular characteristics suggest a motor role, that is, palisade endings are cholinergic, are traced from the oculomotor nuclei, contain key proteins of the exocytotic machinery but lack, however, postsynaptic nicotinic acetylcholine receptors and have little, if any, extracellular acetylcholinesterase (Blumer et al., 2009, 2016, 2020; Zimmermann et al., 2013).
Given that oculomotor organization is more complex than initially devised, we aimed at describing the function of electrophysiologically-identified MIF motoneurons innervating the medial rectus muscle and its relative contribution to the different types of eye movements.
Materials and Methods
Ethics statement
All procedures were performed in accordance with the guidelines of the European Union (2010/63/EU) and the Spanish legislation (R.D. 53/2013, BOE 34/11370-421) for the use and care of laboratory animals and approved by the ethics committee (protocol #13/04/2018/047). All efforts were made to reduce the number of animals used.
Animals and surgical procedures
Experiments were performed on adult female cats weighing 2.0–2.5 kg obtained from authorized suppliers (Universidad de Córdoba, Spain). A total of 8 animals was used for the present study. Five of them were prepared for extracellular single-unit recordings of medial rectus motoneurons in the oculomotor nucleus (Fig. 1A), and the other three for the morphologic study. None of these three cats used for morphology derived from electrophysiological experiments.
Animals were prepared for chronic recordings as previously described (de la Cruz et al., 1989; Hernández et al., 2017). Briefly, atropine sulfate (0.5 mg/kg, i.m.) was injected to reduce vagal reflexes and ketamine hydrochloride (20 mg/kg, i.m.) mixed with xylazine (0.5 mg/kg, i.m.) to induce anesthesia. Thereafter, animals were placed in a stereotaxic frame. Surgery was performed under sterile conditions to implant stimulating electrodes, scleral coils, and the recording chamber (Fig. 1A). For the antidromic identification of the oculomotor nucleus, silver bipolar electrodes were implanted in the third nerve bilaterally (Fig. 1A, St. 1). In addition, a pair of hook electrodes, made of three-strand stainless steel Teflon insulated wire was inserted at the myotendinous junction of both medial rectus muscles to activate (Eladly et al., 2020) the axons of MIF motoneurons (Fig. 1A, St. 2). Hook electrodes were located ∼10 mm apart from the muscle's belly where the band of en plaque contacts of SIF motoneurons is located. It has been shown that, because of volume conduction attenuation, the current decays in muscle to 10% over a 10 mm distance (Enoka et al., 2020). The criterion for stimulating MIF axons was 1–1.1× the threshold to produce a maximal field potential from the tendon electrode using currents always <100 µA. Coils, made up of two turns of Teflon-isolated stainless-steel wire, were implanted in the sclera of both eyes for the recording of eye movements (Fig. 1A). A square window (6 × 6 mm) was drilled in the parietal bone to allow the lateral access to the midbrain oculomotor nucleus. A restraining system was also constructed to immobilize the head during the recordings. Postoperative care was provided daily, as needed, to ensure the healthy state of the animal.
Chronic extracellular recordings
After 10 d of postoperative recovery, recording sessions started. The animal was comfortably seated in a fabric bag, bandaged, and placed in a Perspex box, which was located inside the magnetic field for eye movement recordings (Fuchs and Robinson, 1966). Extracellular recordings were conducted with glass micropipettes, filled with 2 m sodium chloride, attached to a three-axis micromanipulator to advance through the intact brain to reach the medial rectus subdivision of the oculomotor nucleus. The oculomotor nucleus was located by its antidromic field potential after the electrical stimulation to the ipsilateral IIIrd nerve (50-µs pulses of < 0.1 mA). The medial rectus subdivision was located stereotaxically and medial rectus motoneurons were identified, as previously described (Hernández et al., 2017), by their increase in firing frequency selectively during nasally-directed horizontal eye movements. In addition, the collision test of the orthodromic and antidromic spike was systematically used to assure that the activated unit was indeed the recorded one.
Both MIF and SIF medial rectus motoneurons were antidromically activated from the electrode placed at the third nerve (collision tests in Fig. 1B,C, IIIn). This procedure indicated that the axons of both types of units coursed through the oculomotor nerve. MIF motoneurons were distinguished from SIF motoneurons because only the former were antidromically activated from the electrode located at the myotendinous junction (collision tests in Fig. 1B,C, MTJ). Once identified, the extracellular action potentials of the motoneuron were recorded along with eye movements under alert conditions. For some cells, it was also possible to record their firing during vestibularly-induced eye movements in the horizontal plane by means of a servo-controlled motor attached to the turntable oscillating at a frequency of 0.125 Hz and ±20° peak-to-peak amplitude.
Peristimulus time histograms (PSTHs) were conducted following the repetitive electrical stimulation to the electrode placed at the myotendinous junction simultaneously with the recording of an identified MIF or SIF motoneuron, to check whether the activation of palisade endings could change the probability of discharge of the recorded motoneuron. Parameters to generate the PSTHs were the same as we have used previously (Davis-López de Carrizosa et al., 2009).
Data storage and analysis
Horizontal eye position (EP) and associated neuronal activity were digitally stored for off-line analysis (Power 1401, Cambridge Electronic Design). Computer programs written in MATLAB 7.5 were used for selecting the data of instantaneous firing frequency along with the corresponding EP and eye velocity (EV). The correlations between firing rate (FR) and EP and EV were conducted according to the equation FR = F0 + k·EP + r·EV, where k and r are the neuronal sensitivities to EP (k, in spikes/s/°) and velocity (r, in spikes/s/°/s), and F0 is the FR at straight ahead gaze, that is, when EP = 0° (Robinson, 1970; de la Cruz et al., 1989; Davis-López de Carrizosa et al., 2011). During spontaneous eye fixations, EV = 0, and therefore this equation can be expressed as FR = F0 + k·EP. Accordingly, the correlation between FR and EP was fitted by a linear regression line where the slope of the line represents the neuronal EP sensitivity (k). The intercept of the line with the abscissa axis represents the estimated EP threshold for recruitment (Robinson, 1970; Delgado-Garcia et al., 1986). To obtain the neuronal eye sensitivity during spontaneous rapid eye movements, or saccades, we correlated FR, after the subtraction of the previously calculated EP component (k·EP – F0), with EV during saccades, according to the equation FR – k·EP – F0 = r·EV, by means of linear regression analysis.
During vestibularly-induced eye movements, neuronal EP and EV were obtained by multiple linear regression using the equation FR = F0 + k·EP + r·EV, selecting between cursors only the slow phases of the nystagmus. We named ks and rs to neuronal EP and EV sensitivities, respectively, obtained during spontaneous eye movements, and kv and rv to neuronal EP and EV sensitivities calculated during vestibular eye movements. Correlations between FR and EP and EV were made with the eye ipsilateral to the recorded nucleus.
Morphologic identification of MIF motoneurons
Retrograde labeling of rhodamine B isothiocyanate (Sigma-Aldrich) was used to identify MIF motoneurons. Animals were anesthetized as described above and placed in a stereotaxic frame. The medial rectus muscle was isolated under a dissecting microscope and with the help of a muscle hook, 1 µl of 20% rhodamine dissolved in 2% dimethylsulfoxide was injected at the myotendinous junction of the medial rectus muscle to selectively label only MIF motoneurons, according to previous reports (Büttner-Ennever et al., 2001; Eberhorn et al., 2006; Bohlen et al., 2017b; Hernández et al., 2019).
Two weeks after rhodamine injection, animals were transcardially perfused under deep anesthesia (sodium pentobarbital, 100 mg/kg, i.p.) with 500 ml of saline followed by fixative (4% paraformaldehyde prepared in 0.1 m phosphate buffer, pH 7.4). The brainstem was removed and cut on a vibratome in coronal sections of 50-µm thickness. Medial rectus muscles were dissected to reveal the extent of rhodamine injection.
Immunofluorescence for confocal microscopy
For the identification of motoneurons, choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, was used. Double immunofluorescence was used to combine ChAT with synaptic markers, either synaptophysin (a general marker of synaptic boutons) or vesicular GABA and glycine amino acid transporter (VGAT), to selectively label inhibitory boutons. MIF medial rectus motoneurons were, in addition, retrogradely labeled with rhodamine. Therefore, we focused within the medial rectus subdivision and distinguished MIF medial rectus motoneurons as those that were for rhodamine-positive and ChAT-immunoreactive (IR), whereas SIF medial rectus motoneurons were those ChAT-IR motoneurons that lacked the retrograde rhodamine labeling.
For immunofluorescence, we used as primary antibodies: (1) goat polyclonal antibody against ChAT (1:500; Millipore); (2) mouse monoclonal antibody against synaptophysin (1:1000; Millipore); and (3) rabbit polyclonal antibody against VGAT (1:500; Millipore). Secondary antibodies were: (1) donkey anti-goat IgG coupled to FITC (to detect ChAT); (2) donkey anti-mouse IgG coupled to Cy5 (to reveal synaptophysin); (3) and donkey anti-rabbit IgG coupled to Cy5 (for VGAT visualization). All secondary antibodies were used at a dilution of 1:50 (Jackson ImmunoResearch). Images were captured with a confocal microscope (Zeiss LSM 7 DUO) using different filters with the Zeiss microscope program ZEN.
Morphologic analysis
The following analyses were performed using the captured confocal images: (1) the topographic distribution of MIF medial rectus motoneurons within the oculomotor nucleus; (2) the somatic size of SIF and MIF motoneurons; and (3) the synaptic coverage around cell bodies with either synaptophysin or VGAT, in both SIF and MIF motoneurons. The distribution of MIF medial rectus motoneurons within the oculomotor nucleus was conducted using 4 × 4 tile confocal images at 10× magnification to visualize the entire nucleus. The topographical analysis was performed using the Adobe Illustrator program (Adobe Systems Incorporated). For the measurement of the somatic size of both motoneuronal types, three parameters were calculated: (1) the average of the large and small diameter; (2) the cell body area; and (3) the somatic perimeter. For these measurements, stacks of confocal planes of 1-µm virtual thickness captured at 63× magnification were used, selecting the plane at which the cell nucleus showed its maximum diameter. The program ImageJ (NIH) was used to calculate cell size parameters. Synaptic coverage around SIF and MIF motoneurons was measured as the percentage of the somatic perimeter that appeared surrounded by immunoreactive boutons, either synaptophysin-IR or VGAT-IR.
Statistics
To compare morphologic and physiological measurements between SIF and MIF motoneurons the Student's t test, the two-way ANOVA, the Wilcoxon signed-rank test or the Mann–Whitney rank-sum test were used at a level of significance of p < 0.05 (SigmaPlot 11 program; Systat Software). All regression analyses were significant (p < 0.05). The effect size was measured as Cohen's d (d) and determined with online calculators (https://www.psychometrica.de/effect_size.html). Data are shown as mean ± SEM.
Results
Electrophysiological identification of SIF and MIF medial rectus motoneurons
When a unit was isolated within the limits of the medial rectus subdivision of the oculomotor nucleus, we systematically proceeded to characterize the unit as either MIF or SIF motoneuron by the antidromic activation and collision test performed from the electrodes placed at the IIIrd nerve (Fig. 1A, St.1) and in the myotendinous junction of the medial rectus muscle (Fig. 1A, St. 2). The unit was considered to be a SIF motoneuron if it was antidromically activated and collided after the electrical stimulation to the IIIrd nerve, but activation and collision failed from the electrode placed at the myotendinous junction (Fig. 1B, see collision from the IIIrd nerve at the left of the panel, and failure of collision after electrical stimulation to the myotendinous junction, at the right of the panel). On the other hand, MIF motoneurons were identified because they were antidromically activated and collided from both electrodes, that is, from the IIIrd nerve and from the myotendinous junction (Fig. 1C).
To evaluate whether there was a difference in the antidromic latencies from the nerve between MIF and SIF motoneurons, the time interval between the onset of the stimulus artifact and the negative peak of the spike was measured. Figure 1D illustrates the distribution of antidromic latencies for MIF and SIF medial rectus motoneurons. As can be observed, the distribution of latencies for MIF motoneurons was displaced toward the right, thus toward higher latency values. The arrows in Figure 1D point to the mean antidromic latency for both motoneuronal types, illustrating a higher latency for the MIF population of motoneurons. When MIF and SIF motoneurons were pooled in two groups, the statistical comparison of antidromic latencies between both populations revealed that MIF motoneurons showed a significantly higher antidromic activation latency when compared with SIF motoneurons (0.723 ± 0.009 and 0.644 ± 0.007 ms, respectively; t test, t(113) = 6.750, p < 0.001, d = 1.29; n = 45 MIF and n = 70 SIF motoneurons; Fig. 1E).
Peristimulus time intervals (PSTHs) were performed after repetitive stimulation (>150 pulses, 1 Hz) to the electrode placed at the myotendinous junction while recording the extracellular potentials of an identified MIF or SIF motoneuron to determine whether palisade ending stimulation could exert any synaptic influence on the motoneuron discharge probability. We constructed binned histograms (bin width 0.5 ms) from 20 ms before to 50 ms after the stimulation, with the stimulus artifact pooled at time 0 bin, but excluded from computations (Davis-López de Carrizosa et al., 2009). The stimulus intensity applied was the minimum to obtain the maximum antidromic field potential from the muscle-tendon electrode. An example of the PSTH for a SIF and for a MIF motoneuron is illustrated in Figure 1F. In both motoneuronal types, it can be appreciated that the discharge before and after the stimulation (indicated by the red arrowhead and vertical dashed line) shows similar rates. To quantify PSTHs, we obtained the number of counts (in spikes) during the initial 20 ms (before stimulation) and compared this value with the 20-ms period after stimulation. The results indicated absence of significant differences in the PSTHs (Fig. 1G) for both types of motoneuron (Wilcoxon signed-rank test, Z = 1.260, p = 0.250, n = 8 for MIF motoneurons and Z = 1.753, p = 0.125, n = 5 for SIF motoneurons).
Firing pattern of MIF and SIF medial rectus motoneurons during spontaneous, convergent, and vestibularly-induced eye movements
Medial rectus motoneurons showed a tonic-phasic discharge pattern in correlation with eye movements, as previously described (de la Cruz et al., 1989). During fixations, medial rectus motoneurons fired tonically, with higher rates for nasal-directed EPs, that is, movements contralateral to the recording side (on direction). During rapid eye movements or saccades, these motoneurons discharged a high-frequency burst of spikes for on-directed saccades, and a pause or an abrupt firing decay for off-directed saccades. As shown in Figure 2A,B, the discharge of MIF and SIF motoneurons during spontaneous eye movements revealed that both motoneuronal types discharged in a tonic-phasic way. However, MIF motoneurons displayed overall lower FRs compared with SIF motoneurons (Fig. 2A,B).
The behavior of MIF and SIF medial rectus motoneurons during spontaneous convergent eye movements was also evaluated. The great majority of MIF and SIF motoneurons increased their firing during convergence, as expected (for SIF motoneurons: 154 epochs of convergence, in 45 cells, with 83.8% of appropriate responses; for MIF: 87 epochs of convergence, in 27 cells, with 86.2% of appropriate response). It is important to emphasize that in any case neither MIF nor SIF medial rectus motoneurons displayed an inappropriate response (i.e., a decrease in FR) for convergent eye movements, in contrast with the inappropriate firing we reported previously for a small percentage of MIF and SIF abducens motoneurons during divergences (Hernández et al., 2019).
During vestibularly-induced eye movements, medial rectus motoneurons discharged in proportion to EP and EV, increasing their FR for eye movements in the nasal direction, during both the slow and the fast phases of the nystagmus, and decreasing their FR in the opposite direction. As occurred during spontaneous eye movements, MIF motoneurons discharged at lower firing frequencies as compared with SIF motoneurons during vestibularly-induced eye movements (Fig. 2E,F).
Quantitative comparison of eye-related parameters between MIF and SIF medial rectus motoneurons during spontaneous and vestibular eye movements
For spontaneous eye movements, we calculated neuronal EP sensitivity (ks) as the slope of the rate-position plot during eye fixations, when the velocity component was zero. The regression lines between FR and EP (Fig. 2C) for the motoneurons shown in Figure 2A,B illustrated the method of analysis to calculate ks (the slope of the regression line), F0 (the FR at straight ahead gaze, when EP is zero), and the recruitment threshold (Th; the EP at which the motoneuron is recruited into activity). To calculate neuronal eye EP and EV during saccades, FR (after subtracting the EP component) was plotted versus EV (Fig. 2D). The slope of the regression line thus obtained corresponded to neuronal eye velocity sensitivity (rs). Note in Figure 2D that for the MIF motoneuron illustrated in Figure 2B, its rs value was lower than that of the SIF motoneuron shown in Figure 2A.
Therefore, we proceeded to compare these four parameters, obtained during spontaneous eye movements (ks, F0, Th, and rs), between the whole population of SIF (n = 70) and MIF (n = 45) motoneurons. For ks, SIF motoneurons showed a mean of 5.59 ± 0.19 and MIF motoneurons 2.17 ± 0.10 spikes/s/°. The statistical analysis demonstrated that MIF motoneurons showed a significantly lower mean ks as compared with SIF motoneurons (t test, t(113) = −13.125, p < 0.001, d = −2.508; Fig. 2I). F0 was also significantly lower for MIF motoneurons (35.56 ± 1.25 spikes/s) than for SIF motoneurons (53.02 ± 1.48 spikes/s), as can be appreciated in Figure 2J (t test, t(113) = −8.294, p < 0.001, d = −1.585). MIF motoneurons were recruited at more eccentric EPs in the off-direction than SIF motoneurons, thereby presenting lower thresholds (−18.12 ± 1.04° vs −10.30 ± 0.45°, respectively; Fig. 2K). The difference in thresholds between both motoneuronal types was also significant (t test, t(113) = −7.794, p < 0.001, d = −1.489). During saccades, EV sensitivity (rs) reached lower values for MIF than for SIF motoneurons (0.49 ± 0.02 vs 0.89 ± 0.03 spikes/s/°/s, respectively) yielding a significant difference (t test, t(113) = −9.289, p < 0.001, d = −1.775; Fig. 2L).
There were also differences between the firing of MIF and SIF motoneurons during on-directed saccades. Thus, the latency between the saccade onset and the peak FR occurred ∼9 ms later in MIF motoneurons than in SIF motoneurons (64.2 ± 1.35 vs 55.3 ± 1.28 ms, respectively), a difference that was significant (t test; t(413) = 4.797, p < 0.001, d = 0.451). Peak FR preceded peak saccadic velocity for both motoneuron types. However, the peak firing of MIF motoneurons was closer to peak saccadic velocity (6.5 ms; paired t test, t(206) = 10.251, p < 0.001, d = 0.349), whereas SIF motoneurons fired maximally with a longer latency preceding peak EV (13.4 ms; paired t test, t(207) = 23.810, p < 0.001, d = 0.763). These findings suggest that MIF motoneurons contributed mainly during the last part of the saccade, which requires a fine adjustment to set the eye still on visual target while relaxing the viscoelastic forces implied in the saccade. On the other hand, SIF motoneurons were more influential for starting the saccade and reaching its peak velocity, when more force is needed to overcome the viscoelastic properties of the oculomotor plant. Similar differences were also found between MIF and SIF motoneurons of the abducens nucleus (Hernández et al., 2019).
During vestibularly-induced eye movements, neuronal EP and EV sensitivities (kv and rv, respectively) were calculated by multiple regression analysis and compared between MIF (n = 31) and SIF (n = 54) motoneurons. Partial regression plots of FR versus EP (Fig. 2G) or EV (Fig. 2H) for the SIF and MIF motoneurons illustrated in Figure 2E,F, respectively, clearly showed that both sensitivities (kv and rv) were higher for the SIF than for the MIF motoneurons. As a pool, MIF motoneurons showed lower kv values (3.07 ± 0.11 spikes/s/°) as compared with SIF (5.99 ± 0.19 spikes/s/°) motoneurons (Fig. 2M), reaching statistical significance (t test, t(83) = −11.100, p < 0.001, d = −2.501). The same happened with rv, which was significantly lower for MIF motoneurons (0.43 ± 0.02 spikes/s/°/s) with respect to SIF motoneurons (0.99 ± 0.05 spikes/s/°/s), as can be appreciated in Figure 2N (t test, t(83) = −8.956, p < 0.001, d = 2.018).
Recruitment order and correlations between parameters in MIF and SIF medial rectus motoneurons
According to the analyses described above, MIF medial rectus motoneurons discharged at lower FRs and with lower sensitivities, and were recruited first, as compared with SIF medial rectus motoneurons. These differences can be clearly appreciated in the plots of Figure 3A,B, in which the entire samples of 70 SIF and 45 MIF motoneurons were represented. Thus, MIF motoneurons showed lower slopes in their rate-position (Fig. 3A) and rate-velocity (Fig. 3B) plots, reaching for a given EP or EV lower values of firing frequency, as compared with the SIF motoneuron population. In addition, MIF motoneurons started their discharge at more negative EPs, i.e., with the eye located more laterally in the orbit (the off direction for medial rectus motoneurons), indicating that they had lower recruitment thresholds (Fig. 3A). Since MIF motoneurons had longer antidromic latencies and smaller cell size (see below), the present data are in congruence with the size principle (Henneman et al., 1965), so that smaller motoneurons are recruited first and contribute generating less force than larger motoneurons.
Correlations between these parameters (ks, Th, rs) have been described previously for extraocular motoneurons considered as a homogeneous pool (Delgado-Garcia et al., 1986; Fuchs et al., 1988; Pastor and González-Forero, 2003; Davis-López de Carrizosa et al., 2011; Hoh, 2021). We aimed at investigating whether similar relationships could be present in MIF and SIF medial rectus motoneurons treated as separate populations. Results are shown in Figure 3C–E. First, when correlating ks with threshold, a significant exponential fit was found for both motoneuronal types (Fig. 3C), so that the higher the threshold (less negative EPs), the higher the EP sensitivity (ks), for both MIF (blue dots and line, r = 0.719, p < 0.001) and SIF motoneurons (orange dots and line, r = 0.801, p < 0.001). It was also observed that lower ks and thresholds were present in MIF motoneurons as compared with SIF motoneurons. Figure 3C, black line, represents the exponential fit of the whole population of MIF + SIF motoneurons (r = 0.798, p < 0.001). Second, the correlation between ks and rs also fitted to an exponential equation (Fig. 3D), for both MIF (r = 0.542, p < 0.001) and SIF (r = 0.794, p < 0.001) motoneurons, meaning that motoneurons with higher ks values also presented, in general, higher rs values. Figure 3D, black line, represents the correlation ks versus rs for the entire population of MIF and SIF motoneurons, which also fitted to an exponential equation (r = 0.834, p < 0.001). Third, the correlation between rs and threshold was significant for SIF motoneurons (r = 0.652, p < 0.001; Fig. 3E, orange dots and line), but not for MIF motoneurons (Fig. 3E, blue dots). However, when both motoneuronal types were grouped as a single population, an exponential equation resulted from the relationship between rs and threshold (black line, r = 0.647, p < 0.001).
When ks, Th and rs values were represented in a 3D plot (Fig. 3F) for MIF and SIF motoneurons, the two motoneuronal types distributed, in general, as distinct populations. Thus, it can be clearly appreciated that MIF motoneurons (blue dots) grouped toward lower Th, rs and ks values as compared with SIF motoneurons (orange dots).
Distribution of MIF medial rectus motoneurons within the oculomotor nucleus
The retrograde tracer rhodamine, injected at the myotendinous junction of the medial rectus muscle (Fig. 4A, top) was used to anatomically identify MIF medial rectus motoneurons in the oculomotor nucleus. We obtained similar results for the three animals processed for this purpose. MIF motoneurons appeared located within the medial rectus subdivision of the oculomotor complex, that is, in the rostral two thirds of this nucleus, both at the dorsal and ventrolateral groups, with more MIF motoneurons found in the dorsal group (Fig. 4A), the main subgroup of the medial rectus motoneuron pool in cats (Akagi, 1978; Miyazaki, 1985; de la Cruz et al., 1994; Büttner-Ennever, 2006). However, as illustrated in Figure 4B–D, some MIF motoneurons appeared in the ventrolateral group, some of them interspersed among the bundles of the medial longitudinal fascicle. A previous study in cats has described MIF medial rectus motoneurons mainly in the ventrolateral subgroup, in accordance with our work, although we also found them in the dorsal pool (Bohlen et al., 2017b).
To determine the percentage of MIF motoneurons with respect to the total population of medial rectus motoneurons, rhodamine-positive motoneurons (MIF) were counted and compared with the total number of ChAT-IR motoneurons (SIF+MIF) located at the medial rectus motoneuron subdivision of the oculomotor nucleus (Akagi, 1978; Miyazaki, 1985; de la Cruz et al., 1994; Büttner-Ennever, 2006). The results indicated that the number of MIF motoneurons in relation to the population of medial rectus motoneurons was, on average, 23.77%. The proportion found of MIF motoneurons is in agreement with previous reports in monkeys (Büttner-Ennever et al., 2001), rats (Eberhorn et al., 2006), and in the cat abducens nucleus (Hernández et al., 2019).
Differences between MIF and SIF medial rectus motoneurons in cell size and synaptic coverage
To compare the somatic cell size between MIF and SIF motoneurons, three parameters were measured (average diameter, area, and perimeter) in ChAT-IR motoneurons. We obtained significant differences between both motoneuronal groups, with MIF motoneurons showing significantly lower average diameter (28.87 ± 0.35 µm; t test, t(528) = −11.155, p < 0.001, d = −1.138), area [651.21 ± 14.94 µm2 (Fig. 4E); t test, t(528) = −8.762, p < 0.001, d = −0.894 (Fig. 4F)], and perimeter (95.82 ± 1.06 µm; t test, t(528) = −9.917, p < 0.001, d = −1.012; Fig. 4G) in comparison with SIF motoneurons (34.05 ± 0.24 µm, 866.75 ± 12.92 µm2, 110.50 ± 0.76 µm, for average diameter, area, and perimeter, respectively). The number of motoneurons analyzed was n = 126 MIF and n = 404 SIF motoneurons for the three measurements.
The overall lower FR of MIF motoneurons led us to study the density of synaptic terminals that MIF motoneurons receive on their cell body and to compare this value with that of SIF motoneurons. We did this study using immunocytochemistry against synaptophysin, as a general marker for both excitatory and inhibitory boutons, as well as against VGAT, a selective marker of inhibitory boutons. By confocal microscopy, the percentage of the somatic perimeter that was contacted by either synaptophysin-IR or VGAT-IR (i.e., linear density, in %) was calculated.
Regarding synaptophysin, it was observed that MIF motoneurons were covered by less synaptic boutons as compared with SIF motoneurons. This can be appreciated in the SIF and MIF motoneurons illustrated in Figure 5B,C, respectively, whose location in the oculomotor nucleus is indicated by white arrows in Figure 5A. When all measured motoneurons were grouped, the statistical analysis of synaptophysin coverage (an index of synaptic linear density) yielded a significant difference, with MIF (n = 65) motoneurons showing a significantly lower synaptic coverage than SIF (n = 206) motoneurons (31.64 ± 0.96% and 51.84 ± 0.60%, respectively; t test, t(269) = −16.933, p < 0.001, d = −2.409; Fig. 5D).
Similar results were obtained with the inhibitory synaptic marker VGAT. As can be appreciated in Figure 5F,G, SIF motoneurons showed a higher density of VGAT-IR boutons contacting their soma than MIF motoneurons. The location within the oculomotor nucleus of the SIF and MIF motoneurons illustrated in Figure 5F,G, respectively, is indicated by white arrows in Figure 5E. Our measurements showed that MIF (n = 61) motoneurons presented a significantly lower VGAT-IR coverage when compared with SIF (n = 198) motoneurons (17.17 ± 0.63% and 29.53 ± 0.41%, respectively; t test, t(257) = −15.076, p < 0.001, d = −2.208; Fig. 5H).
The balance inhibitory-to-total synaptic boutons was estimated as the linear density of VGAT-IR terminals (i.e., inhibitory) with respect to the total linear density of synaptophysin-IR boutons (i.e., excitatory and inhibitory), for both SIF and MIF motoneurons in each animal (n = 3). The statistical comparison between MIF and SIF motoneurons revealed no significant difference in the inhibitory-to-total synaptic coverage (Mann–Whitney rank-sum test, U = 2.000, p = 0.400, d = 0.995).
Discussion
We demonstrate for the first time that electrophysiologically-identified MIF and SIF medial rectus motoneurons discharge during slow and fast eye movements. However, MIF motoneurons fired at lower rates, had lower recruitment thresholds, and lower EP and EV sensitivities during spontaneous and vestibular eye movements. A size-principle-based recruitment order was present in both motoneuronal populations. PSTHs obtained following stimulation to the myotendinous junction, where palisade endings are located, revealed no change in the discharge probability of MIF and SIF motoneurons. Finally, MIF motoneurons were also smaller in size, presenting longer antidromic latencies, and lower synaptic coverage.
MIF medial rectus motoneurons discharge during different types of eye movements but with a distinct contribution
The present results show that MIF (and SIF) motoneurons fired during eye fixations, saccades, the slow and fast phases of the vestibulo-ocular reflex and convergent eye movements. These data indicated there are no motoneurons (SIF or MIF) exclusive of certain eye movement type as previously suggested (Wasicky et al., 2004; Ugolini et al., 2006; Bohlen et al., 2017a,b). In line with our findings, all reports on extraocular motoneurons recorded in different vertebrate species, including monkeys, cats, rabbits and fish, have shown that all motoneurons display a tonic-phasic discharge pattern and participate in all types of eye movements (Robinson, 1970; Schiller, 1970; Fuchs and Luschei, 1971; Delgado-Garcia et al., 1986; de la Cruz et al., 1989; Evinger and Baker, 1991; Pastor et al., 1991; Stahl and Simpson, 1995; Davis-López de Carrizosa et al., 2011; Hernández et al., 2017). However, a full exploration of frequencies and amplitudes of stimulation is required to further ascertain the differences in the dynamics of MIF and SIF motoneurons. The vestibulo-ocular pathways manifest differential adaptation properties based on their frequency selectivity (Lisberger et al., 1983), thus it is possible that these separate channels (i.e., low vs high frequency) extend to the motoneurons (Dietrich et al., 2017) and that intrinsic membrane properties along vestibular neurons and motoneurons are tuned to determine the firing and discharge properties of MIF and SIF motoneurons (Serafin et al., 1991a,b; Nieto-Gonzalez et al., 2007; Mayadali et al., 2021). Altogether, it can be suggested that individual extraocular motoneurons do not specialize in producing selectively any of the different types of eye movements (see, however, Henn and Cohen, 1972).
Nonetheless, the present data demonstrated significant differences in several physiological parameters between MIF and SIF medial rectus motoneurons. In general, MIF motoneurons fired at lower frequencies and showed lower EP and EV sensitivities during spontaneous and vestibularly-induced eye movements. Another important finding was that MIF motoneurons were recruited first, as they presented lower thresholds, i.e., EPs more in the off-direction, where small muscle tension is required. As the eye moved toward positions attained in the on-direction they continued firing at increasingly higher rates but still at lower frequencies as compared with SIF motoneurons. This indicates that SIF motoneurons would contribute with higher frequencies at EPs where more tension is required, and therefore, the contribution of MIF and SIF motoneurons throughout the oculomotor range would be different. MIF motoneurons would contribute with low discharge generating small muscle forces and activating first in the off-oculomotor field, whereas SIF motoneurons would contribute with higher FRs to impulse more force for those EPs more in the on-direction.
MIF and SIF medial rectus motoneurons display convergent signals
The majority of MIF and SIF medial rectus motoneurons had appropriate signals during spontaneous convergent eye movements, i.e., a small increase in FR. These findings are quite different to those previously reported for cat abducens motoneurons, where most MIF and SIF motoneurons do not exhibit an appropriate response during divergences, but instead a variety of discharge patterns. Thus, a high percentage of MIF and SIF abducens motoneurons either discharged inappropriately, or did not respond, or responded in a variable way to divergences (Hernández et al., 2019).
This discrepancy can be explained considering previous findings of neurons located dorsal and lateral to the oculomotor nucleus that increase their FR with increases in the angle of ocular convergence and that project on medial rectus motoneurons. The output of these so-called midbrain near response cells that might provide the appropriate vergence command needed by the medial rectus motoneurons (Zhang et al., 1992). In contrast, no divergent gaze signal has been reported to drive abducens motoneurons during disjunctive eye movements so far (Clendaniel and Mays, 1994).
MIF motoneurons have smaller size and receive fewer synaptic boutons
MIF medial rectus motoneurons showed significantly smaller size than SIF medial rectus motoneurons, in agreement with previous reports in monkeys and rats (Eberhorn et al., 2005, 2006). In analogy, previous reports describe that MIF axons are smaller than SIF axons (Mayr, 1971; Browne, 1976; Nelson et al.,1986). The percentage of MIF motoneurons with respect to the total population constituted ∼20%, which is also in congruence with previous studies (Büttner-Ennever et al., 2001; Wasicky et al., 2004; Spencer and Porter, 2006; Erichsen et al., 2014; Bohlen et al., 2017b). The smaller size of MIF motoneurons could explain their longer antidromic latencies, and consequently, their expected lower conduction velocity. Similarly, MIF motoneurons of the cat abducens nucleus are smaller and have higher antidromic latencies as compared with SIF abducens motoneurons (Hernández et al., 2019). The finding that MIF motoneurons had smaller size could also correlate with their lower EP threshold, since a small cell responds to a given amount of current with higher excitability, because of its higher input resistance, and therefore, it is recruited first according to the size principle (Henneman et al., 1965).
As demonstrated by synaptophysin immunoreactivity, MIF motoneurons received less synaptic boutons than SIF motoneurons, in agreement with a previous study in monkey medial rectus motoneurons (Erichsen et al., 2014). The smaller density of afferent synaptic boutons driving MIF motoneurons could correlate with their lower FR and sensitivities. The ratio between inhibitory terminals (VGAT-positive) versus the total number of boutons (synaptophysin-positive) was the same for MIF and SIF motoneurons, as happens in the cat abducens nucleus (Hernández et al., 2019).
Relationship between MIF motoneurons and palisade endings
Palisade endings are terminal varicosities at the myotendinous junction around single muscle fiber tips. The muscle fibers associated with palisade endings are the MIFs of the global layer of extraocular muscles, which are characterized by the presence of en grappe nerve endings along their length (Ruskell, 1978; Alvarado-Mallart and Pinçon-Raymond, 1979). We have previously shown that palisade endings are in continuity with MIF motoneuronal axons (Zimmermann et al., 2013; Blumer et al., 2020), thereby en grappe terminals and palisade endings arise both from the same MIF motoneuron. Other authors, however, have hypothesized that palisade endings arise from sensory neurons located in the oculomotor nucleus, which in turn would synapse with MIF motoneurons, suggesting in this way a proprioceptive reflex (Lienbacher and Horn, 2012).
To evaluate this hypothesis, we conducted PSTHs after electrical stimulation to the myotendinous junction while recording the spontaneous discharge of a MIF (or a SIF) motoneurons. It is important to mention that medial rectus muscle in the cat, and in all species studied so far, is the extraocular rectus muscle containing the highest number of palisade endings (Blumer et al., 2016). PSTH analysis revealed no change in the probability of discharge of either MIF or SIF motoneurons, which indicated that the stimulation of palisade endings did not produce any increase or decrease in motoneuronal firing. Therefore, our data indicate that it is unlikely that any signal arrives to motoneurons arising from palisade endings through sensory oculomotor neurons.
An interesting possibility on the functional role of palisade endings and their associated MIF motoneurons (Lienbacher and Horn, 2012; Hoh, 2021) would be that palisade endings, which are in a good location to sense muscle tension, might be activated by muscle stretching during an eye movement and the generated signal would electrically reach their associated en grappe terminals, which in turn would act on the non-twitch muscle fibers, thereby generating a local reflex (Lienbacher and Horn, 2012; Hoh, 2021). Since palisade endings have been described displaying motor (Konakci et al., 2005; Blumer et al., 2009; Rungaldier et al., 2009; Zimmermann et al., 2013) and sensory (Ruskell, 1978; Alvarado-Mallart and Pinçon-Raymond, 1979; Billig et al., 1997; Büttner-Ennever et al., 2003; Lienbacher et al., 2011) characteristics, the hypothesis of an intrinsic sensory-motor reflex seems to reconcile all data (Hoh, 2021).
New perspectives arise in motor control with the tandem of MIF and palisade endings. If palisade endings were proprioceptive organs originating from MIF motoneurons, the possibility of an intrinsic local reflex between palisade endings and en passant boutons of MIF in the muscle is an attractive hypothesis to be studied in the near future. It is clear that proprioception in extraocular muscles would open a new vision beyond the classic proprioception in skeletal muscle. Moreover, the significance of heterogeneity of motoneuron pools, as shown in here, is a crucial question for motor control.
Footnotes
↵*G.C.-R. and R.G.H are co-first authors.
↵#R.R.d.l.C. and A.M.P. are co-senior authors.
This paper is part of the I+D+i project PGC2018-094654-B-100 supported by MCIN/ AEI/10.13039/501100011033, FEDER “A way of making Europe”. Also supported by Austrian Science Fund, Grant P32463-B and FIUS project PRJ202104162. RGH was a Postdoctoral fellow funded by Junta de Andaluía and European Social Fund. Confocal microscopy was performed at the Central Research Services of the Universidad de Sevilla (CITIUS).
The authors declare no competing financial interests.
- Correspondence should be addressed to Angel M. Pastor at ampastor{at}us.es