A Single Intraventricular Injection of VEGF Leads to Long-Term Neurotrophic Effects in Axotomized Motoneurons

Disorders of the Nervous System A Single Intraventricular Injection of VEGF Leads to Long-Term Neurotrophic Effects in Axotomized Motoneurons Paula M. Calvo, Rosa R. de la Cruz, and Angel M. Pastor https://doi.org/10.1523/ENEURO.0467-19.2020 Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla 41012, Spain Abstract Vascular endothelial growth factor (VEGF) has been recently demonstrated to induce neuroprotective and synaptotrophic effects on lesioned neurons. Hitherto, the administration of VEGF in different animal models of lesion or disease has been conducted following a chronic protocol of administration. We questioned whether a single dose of VEGF, administered intraventricularly, could induce long-term neurotrophic effects on injured motoneurons. For this purpose, we performed in cats the axotomy of abducens motoneurons and the injection of VEGF into the fourth ventricle in the same surgical session and investigated the discharge characteristics of axotomized and treated motoneurons by single-unit extracellular recordings in the chronic alert preparation. We found that injured motoneurons treated with a single VEGF application discharged with normal characteristics, showing neuronal eye position (EP) and velocity sensitivities similar to control, thereby preventing the axotomy-induced alterations. These effects were present for a prolonged period of time (50 d) after VEGF administration. By confocal immunofluorescence we also showed that the synaptic stripping that ensues lesion was not present, rather motoneurons showed a normal synaptic coverage. Moreover, we demonstrated that VEGF did not lead to any angiogenic response pointing to a direct action of the factor on neurons. In summary, a single dose of VEFG administered just after motoneuron axotomy is able to prevent for a long time the axotomy-induced firing and synaptic alterations without any associated vascular sprouting. We consider that these data are of great relevance due to the potentiality of VEGF as a therapeutic agent in neuronal lesions and diseases. Key words: angiogenesis; injured motoneurons; neurotrophic factors; oculomotor system; synaptic stripping Significance Statement Vascular endothelial growth factor (VEGF) is a novel neurotrophic factor whose chronic administration protects motoneurons from cell death in animal models of lesion or disease. Because of the promising therapeutic perspectives of this factor, it would be interesting to know whether a single dose could produce long-lasting effects on injured motoneurons. We pursued this issue by using a behaving chronic preparation showing that single intraventricular administration of VEGF prevented the firing and synaptic alterations induced by axotomy in abducens motoneurons without provoking an angiogenic response. These data demonstrate that VEGF administration does not require a continuous supply to obtain physiological improvements, data of great value due to its potential therapeutic use. Received November 11, 2019; accepted March 23, 2020; First published May 5, 2020. The authors declare no competing financial interests. Author contributions: P.M.C., R.d.l.C., and A.M.P. performed research; P.M. C. analyzed data; R.d.l.C. and A.M.P. designed research; R.d.l.C. and A.M.P. wrote the paper. This work was supported by Ministerio de Economía y Competitividad- FEDER (Grant reference: BFU2015-64515-P) in Spain and Ministerio de Ciencia, Innovación y Universidades (Grant reference: PGC2018-094654- B-100). Confocal images were performed in the Central Research Services of University of Sevilla (CITIUS). P.M.C. was a scholar of Ministerio de Economía, Industria y Competitividad (BES-2016-077912) in Spain. Acknowledgements: Confocal images were performed in the Central Research Services of University of Sevilla (CITIUS). May/June 2020, 7(3) ENEURO.0467-19.2020 1–15 Research Article: Confirmation Introduction Vascular endothelial growth factor (VEGF) was initially characterized by its effects on the vascular system, promoting vasculogenesis and angiogenesis (Senger et al., 1983;Ferrara and Henzel, 1989;Yancopoulos et al., 2000). Interestingly, it has also been proven to be neuroprotective. Numerous works have demonstrated its trophic effects after different types of neuronal lesions (Pan et al., 2013;Beecher et al., 2018;Calvo et al., 2018; Zeng et al., 2018;Chi et al., 2019), excitotoxic injury (Matsuzaki et al., 2001;Tovar-y-Romo et al., 2007;Tovar- y-Romo and Tapia, 2010), ischemia (Sun et al., 2003;Guo et al., 2016;Geiseler and Morland, 2018;Wang et al., 2018), epilepsy (Nicoletti et al., 2008,2010), and neurologic diseases such as motoneuronal degeneration in amyotrophic lateral sclerosis (ALS) and spinal-bulbar muscular atrophy (Kennedýs disease), peripheral neuropathies, Alzheimer¨s disease, Parkinson¨s disease, demyelinating diseases, and also in traumatic spinal injury (enhancing nerve repair) and in neovascular ocular diseases (Storkebaum et al., 2005;Zachary, 2005;Wang et al., 2007,2016;Lange et al., 2016;Guo et al., 2019). Although VEGF can act on different neuronal types (Storkebaum et al., 2004;Cabezas et al., 2019), a clear link has been established between VEGF and motoneurons (Lladó et al., 2013), mainly due to the appearance of symptoms resembling ALS when the levels of VEGF are low, as is the case for the VEGF d / d mutant mice. These mice lack the hypoxia response element in the promotor region of the VEGF gene, so that as the animal matures the levels of VEGF decrease, leading to adult-onset motoneuron degeneration reminiscent of ALS (Oosthuyse et al., 2001). VEGF delivery to SOD G93A transgenic mice (a classical model of ALS) by different gene therapies yields to a significant delay in the onset of motor deficits, slows motoneuron degeneration and prolongs survival (Azzouz et al., 2004;Wang et al., 2007,2016). In human postmortem studies of ALS patients, the levels of VEGF and its receptors in the anterior horn cells of the spinal cord have been shown to be decreased (Brockington et al., 2006). Altogether, these works point to VEGF as a potent neurotrophic factor for motoneurons. Previous studies supplying VEGF have delivered the factor chronically through different methods, such as osmotic pumps, viral vectors, genetic crossings with mice overexpressing VEGF, or repeated injections. Our recent work showed the effects of maintained supply of VEGF retrogradely via the proximal stump of the recessed abducens nerve (Calvo et al., 2018). Chronic VEGF retrograde supply produces the recovery of motoneuronal discharge activity and synaptic inputs altered by axotomy. In the present work, we aimed at determining the time span of the effects of VEGF after a single intraventricular application as a potential treatment route for neurologic diseases. We chose the abducens nucleus as a good model for studies of neuronal plasticity since its motoneurons can be recorded readily in the chronic alert animal preparation, and their normal discharge activity and synaptic inputs are well characterized (Delgado-Garcia et al., 1986; Escudero and Delgado-García, 1988;Escudero et al., 1992;Büttner-Ennever, 2006;Davis-López de Carrizosa et al., 2011). We sectioned the VIth nerve at the orbital level in the same surgical session as the (single) administration of VEGF into the fourth ventricle, and thereafter, we performed a neurophysiological and synaptic study of the axotomized motoneurons. We have also evaluated whether the findings obtained after VEGF intraventricular administration can be explained by its well-known angiogenic activity, or rather indicate a direct neurotrophic action on neurons. Interestingly, we have found that a single administration of VEGF intraventricularly prevents the alterations induced by axotomy on the structure/function of abducens motoneurons for a prolonged period of time, and that these effects reflect a direct neurotrophic, nonangiogenic, activity of VEGF on motoneurons. Materials and Methods 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). 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 34/11 370–421) for the use and care of laboratory animals and approved by the ethics committee. A total of 11 animals was used in the present study. Three of these animals were prepared for extracellular single-unit recordings of abducens motoneurons. Motoneurons were recorded in the control situation as well as after axotomy plus VEGF treatment. The electrophysiological axotomy data were taken from our previous work by Calvo et al. (2018). In addition, in the present work we prepared eight additional animals for the morphologic study, which were not used for recordings to preserve an intact morphology: three control animals for the vascularization study (see below), two axotomized animals which were prepared exclusively for the morphologic study (one of them exclusively for the vascular study), and three animals which were axotomized and received VEGF intraventricularly. Data from two additional axotomized animals were taken from our previous study by Calvo et al. (2018). A detailed description of the preparation for chronic recordings has been published previously (Davis-López de Carrizosa et al., 2009,2010). Briefly, animals received an injection of atropine sulfate (0.5 mg/kg, i.m.) to reduce vagal reflexes, then they were anesthetized with ketamine hydrochloride (20 mg/kg, i.m.) mixed with xylazine (0.5 mg/ kg, i.m.) and placed in a stereotaxic frame. Two bipolar stimulating electrodes were implanted intracranially at the exit of both abducens (VIth) nerves from the brainstem (Fig. 1A, only left electrode illustrated) to allow the antidromic Correspondence should be addressed to Rosa R. de la Cruz at rmrcruz@ us.es. https://doi.org/10.1523/ENEURO.0467-19.2020 Copyright © 2020 Castrén et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Research Article: Confirmation 2 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org identification of abducens motoneurons. Recordings were performed in the left abducens nucleus. Coils, made up of two turns of Teflon-isolated stainless-steel wire, were implanted in the sclera of both eyes to record eye movements, and a pedestal was constructed to facilitate head-fixed extracellular recordings. A square window (5 5 mm) was drilled in the occipital bone to allow transcerebellar access to the brainstem for recordings (Fig. 1A). Postoperative care was provided daily, as needed. Chronic extracellular recordings Control recording sessions started ;10 d after surgery. The animal was gently restrained inside a fabric bag, wrapped with elastic bandages, and placed in a Perspex box (with its head immobilized), which was located inside the magnetic field for eye movement recordings (Fuchs and Robinson, 1966). Single-unit extracellular recordings were conducted with glass micropipettes filled with 2 M NaCl, attached to a three-axis micromanipulator and advanced through the intact cerebellum to reach the brainstem (Fig. 1A). The (left) abducens nucleus was located by recording the antidromic field potential induced by electrical stimulation of the ipsilateral VIth nerve (Delgado-Garcia et al., 1986). All recorded abducens motoneurons of the present work were identified by their antidromic activation from the VIth nerve followed by the collision test between the orthodromic and the antidromic spikes (Fig. 1B–D; examples for a control, an axotomized, and an axotomized 1VEGF-treated motoneuron, respectively). After motoneuron identification, single-unit extracellular spikes were recorded simultaneously with eye movements under alert conditions. In some cells, the discharge activity during vestibularly-induced eye movements in the horizontal plane was also conducted. For this, the recording system rotated around the vertical axis by a servo-controlled motor attached to the turntable. Data storage and analysis The horizontal eye position (EP) of both eyes and simultaneous neuronal activity were digitally stored for off-line analysis (Power 1401, Cambridge Electronic Design). For data selection, computer programs written in MATLAB 7.5 displayed instantaneous firing frequency and the position of both eyes. As previously described (Robinson 1970;Davis-López de Carrizosa et al., 2011), the firing rate (FR) of abducens motoneurons correlates with both EP and eye velocity (EV) according to the equation FR = F 0 1k·EP 1r·EV, where k and r are the position and velocity neuronal sensitivities, respectively, and F 0 is the FR at straight ahead gaze, i.e., when EP = 0 degrees. FR during ocular fixations was fitted to the equation FR = F 0 1k s EP, since during eye fixations, EV is zero. The slope of the linear regression line obtained between FR and EP corresponded, therefore, to k s (neuronal EP sensitivity during spontaneous eye movements, in spikes/ second per degree). Relationships between neuronal FR and EV during spontaneous rapid eye movements, or saccades, were obtained by linear regression analysis, after the subtraction of the position component calculated from the previously known sensitivity to EP. Thus, the equation used was FR –k s ·EP –F 0 =r s ·EV, where r s represents the neuronal EV sensitivity during saccades (in spikes/second per degree/second) and represents the slope of the rate-velocity regression line. During vestibular stimulation, the equation used was FR = F 0 1k v EP 1r v EV. In this case, we used multiple linear regression analysis to calculate the neuronal sensitivities k v (in spikes/second per degree) and r v (in spikes/ second per degree/second) during vestibular eye movements. Only the slow phases of the nystagmus were selected between cursors for the analysis. Because of the motor impairment of the eye ipsilateral to the axotomy, we used the eye movements of the contralateral (right) eye for computations. Previously, we demonstrated in control motoneurons that neuronal EP Figure 1. Experimental design and motoneuron identification. A, Diagram illustrating the recording (Rec.) of abducens (Abd.) motoneurons identified by their antidromic activation from the electrode (St.) placed at the VIth nerve. Abd. motoneurons of the left side were axotomized at the orbital level, the lateral rectus (LR) muscle removed and the nerve proximal stump inserted into the IOD to prevent reinnervation. In the same surgical session, VEGF was delivered by pressure injection into the VIth ventricle. Recordings and VEGF infusion were conducted through the cerebellum (CB). B–D, Collision tests were used for the identification of Abd. motoneurons (Mn). Three examples are shown: in B, a control Mn, in C, an axotomized Mn (10 d after axotomy), and in D, a Mn recorded 10 d after lesion and VEGF administration. The orthodromic spontaneous spikes are indicated by dots and the arrows point to the stimulus artifact. Research Article: Confirmation 3 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org and velocity sensitivities during spontaneous and vestibularly-induced eye movements were similar when the analysis was performed with either the left or the right eye. This similarity in motoneuronal firing properties with either eye can be explained by the high degree of conjugacy in horizontal eye movements in the cat (de la Cruz et al., 2000;Davis-López de Carrizosa et al., 2009,2010;Calvo et al., 2018). Axotomy and intraventricular administration of VEGF After the postoperative recovery, we performed several control recording sessions for approximately two weeks before lesion and VEGF administration. The animal was anesthetized (ketamine hydrochloride, 20 mg/kg, i.m., mixed with xylazine, 0.5 mg/kg, i.m.) and placed in a stereotaxic frame to perform the axotomy of the VIth nerve, as previously described in detail (Davis-López de Carrizosa et al., 2008,2009;Calvo et al., 2018). Briefly, the left VIth nerve was sectioned at its entrance into the lateral rectus muscle, which was removed, and an intraorbital device (IOD) implanted in the nerve stump. The proximal stump of the nerve was tightly introduced into the IOD, consisting in a custom-made conical chamber that had two pieces of tubing inserted for fluid replacement of the chamber with 0.01 Mphosphate buffer, pH 7.4, with 0.9% saline (PBS) every 48 h. The IOD implantation also prevented the reinnervation of the severed nerve to any adjacent tissue, therefore allowing chronic axotomy. VEGF was delivered in the fourth ventricle immediately after axotomy. We located the ventricle by lowering the recording micropipette through the cerebellum until an electrical activity silence in the recording was noticed just at the exit of the cerebellum (i.e., the IVth ventricle). Moreover, the entrance into the brainstem was also discernible by the mechanical pressure of the micropipette touching the brainstem. It is important to indicate that the abducens nucleus is located dorsally in the pons, at a distance of ;1 mm below the floor of the fourth ventricle. For VEGF injection, we used a micropipette whose tip was beveled to a diameter of ;20 mm. The micropipette was filled with the VEGF solution (recombinant rat VEGF 164 ; R&D Systems) containing PBS and 0.1% bovine serum albumin. We delivered at this position a single application of 1 mg of VEGF in a total volume of 10 ml by means of a pressure injector. Recording sessions of about 2 h each on alternate days restarted 2–4 d after axotomy plus VEGF administration and lasted for a maximum of 50 d. Immunocytochemical procedures We conducted immunofluorescence at the confocal microscopy level to evaluate the synaptic coverage in axotomized motoneurons treated with VEGF, as well as the possible angiogenic response to VEGF exogenous supply. In the three situations (control, axotomy, and axotomy plus intraventricular administration of VEGF), animals did not derive from the electrophysiological experiments. Animals were deeply anesthetized (sodium pentobarbital, 100 mg/kg, i.p.) 21 d after injury (with or without VEGF administration) for their transcardial perfusion with physiological saline followed by fixative (4% paraformaldehyde in 0.1 Msodium phosphate buffer, pH 7.4). Following 2 h of postfixation, the brainstem at the level of abducens nucleus was cut at 50- mm-thick coronal sections on a vibratome and processed for immunocytochemistry. Double immunofluorescence was conducted sequentially,thatis,eachprimaryantibodyfollowedbyitscorresponding secondary antibody. Sections were first washed in PBS, pH 7.4, with 0.1% Triton X-100 (PBS/ TX) and then blocked with normal donkey serum (1:10 in PBS/TX). In all sections through the abducens nucleus, motoneurons were identified by their immunoreactivity against choline acetyltransferase (ChAT), obtained from goat (1:750; Millipore). Double immunofluorescence was conducted to combine ChAT immunostaining with other primary antibodies: either mouse monoclonal antibody against synaptophysin as a general marker of synaptic boutons (1:1000; Millipore), or mouse monoclonal antibody against GFAP to label astrocytes and their processes (1:400; Sigma-Aldrich). In order to check the possibility that VEFG administration could mediate its neuronal effects, at least in part, through its well-known angiogenic activity, we characterized by immunocytochemistry the density of the vascular network in the abducens nucleus using an antibody against the glucose transporter 1 (GLUT-1), expressed selectively by endothelial cells of the blood-brain barrier (Krum et al., 2002; mouse monoclonal antibody, 1:1000; Millipore). Secondary antibodies were obtained from Jackson ImmunoResearch, used at a dilution 1:50 in PBS/TX, and were the following: donkey anti-goat IgG coupled to TRITC (for ChAT detection) and donkey anti-mouse IgG coupled to FITC (for synaptophysin, GFAP, or GLUT-1) Finally, sections were rinsed in PBS, mounted on glass slides, and coverslipped with Dako fluorescence mounting medium (Dako North America). Images were captured with a confocal laser scanning microscope (Zeiss LSM 7 DUO) at the same focal plane using different filters and later merged by using the Zeiss microscope software ZEN. Gray scales were adjusted to expand the maximum dynamic range of the image. Confocal images were analyzed by using the program ImageJ (NIH). For the quantification of the intensity of synaptophysin or GFAP immunostaining, two types of measurements were conducted by means of the program ImageJ, as previously described (Davis-López de Carrizosa et al., 2009,2010; Morado-Díaz et al., 2014;Calvo et al., 2018). First, we measured the linear density of synaptophysin-immunore- active (IR) boutons or GFAP-IR profiles surrounding the somata of ChAT-IR abducens motoneurons by using images captured at high magnification (63) in confocal planes containing the cell nucleus. The percentage of the total perimeter of the ChAT-IR cell body occupied by synaptophysin-IR or GFAP-IR profiles was measured as an index of the linear density of the immunolabeling. Second, the intensity of synaptophysin or GFAP immunolabeling was measured also in the neuropil of the abducens nucleus using 4 4 tile images captured at 20. Mean gray value Research Article: Confirmation 4 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org (optical density) was considered as an index of the intensity of immunolabeling in the abducens neuropil (the area surrounding abducens motoneurons) and was analyzed as the average gray value of 20 square boxes of 42-mmside per image, after background subtraction. Data obtained in the neuropil were then normalized with respect to control. To evaluate the possibility of an angiogenic response to VEGF treatment, we measured, in the three situations (control, axotomy, axotomy 1VEGF), three different vascular parameters (blood vessels identified as GLUT-1-IR) in the abducens nucleus (delimited by the ChAT labeling), in all cases by stereology using a grid pattern to measure only those vessels interacting with the grid to avoid subjective bias (Croll et al., 2004;Nicoletti et al., 2008), by means of the ImageJ program. First, we measured vascular density as the area of vessels relative to the total area of the abducens nucleus (in %). Moreover, we also compared vessel lumen size between the three situations by measuring the diameter of the blood vessels, as described in detail previously (Croll et al., 2004;Nicoletti et al., 2008) and also used by others (Oosthuyse et al., 2001; Azzouz et al., 2004). A third measurement of vasculature consisted in counting the number of blood vessels as has been used previously (Oosthuyse et al., 2001), and expressed relative to unit area (2500 m m 2 ) by a stereology procedure using regions of interest (ROIs) of 2500 mm 2 . The number of animals for the vasculature analysis was the following: n= 3 for control, n= 4 for axotomy, and n=3 for axotomy 1VEGF. All quantification of fluorescent images, including synaptic, GFAP, and vascular measurements, were conducted by experimenters blind to the different groups of animals (i.e., control, axotomy, and axotomy 1VEGF). Statistics Results between groups were statistically contrasted by using one-way or two-way ANOVA tests, in all cases at an overall level of significance of p,0.05. ANOVA tests were followed by post hoc pairwise multiple comparisons using the Holm–Sidak method. The statistical program used was SigmaPlot version 11 (Systat Software). Data were expressed as mean 6SEM. Results VEGF prevents axotomy-induced alterations on motoneuron discharge during spontaneous eye movements The discharge activity of abducens motoneurons highly correlates with EP and velocity, as previously reported (Delgado-Garcia et al., 1986;Davis-López de Carrizosa et al., 2011). Thus, a tonic firing is present during eye fixations, which increases as the eye moves toward the ipsilateral side (i.e., the on direction; in our case leftwards), and decreases for fixations in the off direction (Fig. 2A). Similarly, when the eye performs a rapid eye movement, or saccade, in the on-direction, motoneurons discharge a high-frequency burst of action potentials, whereas they stop firing or exhibit an abrupt drop in FR for off-directed saccades (Fig. 2A). Therefore, abducens motoneurons display a tonic-phasic discharge pattern during spontaneous eye movements. The discharge of axotomized abducens motoneurons changed noticeably. Injured motoneurons discharged at lower firing frequencies than in control, during both the static and the dynamic components of the discharge (Fig. 2B). The absence of high-frequency bursts during on-di- rected saccades was very conspicuous, instead a weak increase in firing was observed during this type of eye movements (Fig. 2B). VEGF administration prevented all these lesion-induced alterations in axotomized motoneurons. Figure 2C,Dillustrates the discharge activity of two motoneurons recorded 18 d (Fig. 2C)or38d(Fig. 2D) after axotomy 1VEGF delivery. As can be observed, their discharge pattern resembled that of control motoneurons (compare Fig. 2C,Dand A). It is important to emphasize that: (1) all motoneurons recorded after VEGF delivery exhibited, from a qualitative perspective, a firing pattern similar to control throughout the entire period of experimental recordings (50 d), and (2) VEGF administration was acute, that is, it was supplied only once and just at the time of axotomy. Motoneuronal EP sensitivity (k s , in spikes/second per degree) was obtained as the slope of the rate-position plot during eye fixations. Linear regression lines of the rate-position plot for the four motoneurons illustrated in Figure 2A–Dare shown in Figure 2E. Except for the untreated axotomized motoneuron, whose k s value was low (4.5 spikes/s per degree), the motoneurons that received the single dose of VEGF, either at 18 (k s = 9.6 spikes/s per degree) or 38 d post-VEGF (k s = 7.48 spikes/s per degree), showed rate-position slopes and, therefore, k s values, similar to the control motoneuron (k s = 7.85 spikes/s per degree; Fig. 2E). The same happened for the motoneuronal EV sensitivity (r s , in spikes/second per degree/second) during saccades, the slope of the regression line obtained between FR, previous subtraction of the EP component, and EV (Fig. 2F). For the motoneurons illustrated in Figure 2A–D, we obtained that after VEGF administration, r s values (1.4 and 1.36 spikes/s per degree/s, for the motoneurons recorded at 18 and 38 d post-VEGF, respectively) were similar to control (0.93 spikes/s per degree/s), whereas the r s of the axotomized motoneuron differed clearly showing a lower value (r s = 0.30 spikes/s per degree/s; Fig. 2F). VEGF neurotrophic effects on axotomized motoneurons are long-lasting The time course of changes in k s and r s in motoneurons was evaluated in the two experimental conditions: axotomy only and axotomy plus a single dose of intraventricular VEGF. As a first approximation, we elaborated a graph displaying over time the values of k s (Fig. 3A) and r s (Fig. 3B) of the motoneurons recorded in each condition. It can be observed that the great majority of k s and r s data in the VEGF-treated motoneurons appeared distributed within the mean 6SD of the control group of motoneurons (Fig. 3A,B). In contrast, axotomized motoneurons presented lower k s and r s values, which in many cases appeared below the mean minus 1 SD of control data. Although our Research Article: Confirmation 5 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org data of axotomy were obtained only during 21 d after lesion (Calvo et al., 2018), it has been previously described that chronic axotomy produces long-lasting changes as long as reinnervation is not allowed (Delgado-Garcia et al., 1988). Therefore, as a first approximation, these data showed similar k s (Fig. 3A) and r s (Fig. 3B) values in the VEGF-treated motoneurons as compared with the control cells and, in addition, this similarity remained throughout the experiment. To further evaluate whether the single administration of VEGF led to a long-time recovery, motoneurons were grouped in week intervals beginning from the day of surgery (i.e., the day when axotomy plus VEGF intraventricular injection was performed) until the last week of recordings (seventh week), and mean k s and r s values were calculated per week and compared with control (Fig. 3C,D). The overall comparison of the k s values obtained per week after treatment along with the control data revealed similarity between groups (one-way ANOVA, F (7,253) = 1.251, p= 0.275; nranged between 12 and 40 motoneurons per week; for control n= 64), therefore indicating the absence of any change in k s over time, and similarity of all groups in relation to control (Fig. 3C). In contrast, axotomy data grouped per week were significantly lower than the VEGF data for the same week [for k s : two-way ANOVA, F (5,141) = 69.465, p,0.001 (Fig. 3C); for r s : two-way ANOVA, F (5,141) = 28.732, p,0.001 (Fig. 3D)]. Of especial relevance was the fact that k s did not fall toward lower values at long periods after VEGF application, and therefore motoneuronal EP sensitivity did not return to the axotomy situation. For r s values, we obtained similar results. Thus, the data grouped per week plus the control group were compared with each other, and the results of this comparison revealed absence of any statistically significant difference (one-way ANOVA, F (7,253) = 1.341, p= 0.231; same nas for k s data, see above). Therefore, as can be appreciated in Figure 3D, there was no tendency in r s values to decrease with time after treatment. Since no differences were found in k s and r s over time, we grouped all motoneurons recorded during each situation: control (n= 64), axotomy alone (n= 57), or axotomy plus VEGF delivery (n= 199), and calculated their respective k s and r s mean values. The results obtained indicated Figure 2. Discharge pattern of motoneurons during spontaneous eye movements. A, Example of a control motoneuron showing its FR (in spikes/second) recorded simultaneously with EP (in degrees). A typical tonic-phasic discharge activity can be appreciated. Leftwards (L) and rightwards (R) eye movements are indicated by the double arrow. B, Axotomized motoneurons discharged at lower rates showing the partial loss of the static and dynamic components of their firing. The illustrated cell corresponds to a motoneuron recorded 18 d after axotomy. C, Example of a motoneuron recorded 18 d after axotomy 1VEGF administration showing a firing pattern similar to control. D, Same as Cbut for a motoneuron recorded 38 d after axotomy and VEGF. E, Plots showing firing rate versus EP during fixations for the motoneurons shown in A–D. The slope of each regression line corresponds to the EP sensitivity for that motoneuron (k s , in spikes/s per degree). F, Regression lines obtained between FR (minus the EP component) and EV (in degrees/second) during saccades for the same four motoneurons in A–D. The slope of these regression lines corresponds to the EV sensitivity (r s , in spikes/second per degree/second). Research Article: Confirmation 6 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org statistically significant differences [k s : one-way ANOVA, F (2,317) = 35.674, p,0.001 (Fig. 3E); r s : one-way ANOVA, F (2,317) = 26.196, p,0.001 (Fig. 3F)] so that k s and r s of axotomized motoneurons were significantly (p,0.001) lower than control, whereas the values obtained after the administration of VEGF were similar to control (p= 0.437 for k s and p= 0.072 for r s ). Altogether, these results indicated that a single dose of VEGF prevented for a long time the changes induced by axotomy in the EP and velocity sensitivities of motoneurons during spontaneous eye movements. Vestibular signals remain normal in axotomized motoneurons after a single dose of VEGF During vestibularly-induced eye movements produced by horizontal rotation of the table, abducens motoneurons discharged in correlation with EP and EV as previously Figure 3. Time course of changes in EP and velocity sensitivities during spontaneous eye movements after axotomy and VEGF administration. A, EP sensitivities (k s ) of motoneurons recorded at the indicated days after axotomy (red dots; lesion corresponds to time 0) and after axotomy followed by VEGF administration (green dots). The mean control value (solid dot, in blue) and its SD (blue bars) are shown at the left of the graph. B, Same as A, but for EV sensitivity (r s ). C, Mean 6SEM of k s values for the control population (c) and at different weeks (w) after either axotomy (in red) or axotomy plus the single VEGF delivery (in green). Horizontal dashed lines represent the a alone (lower line). There were significant differences between axotomy and VEGF groups comparing per week (two-way ANOVA, p,0.001). D, Same as C, but for r s . No significant differences were found between the mean values obtained per week after the supply of VEGF for k s (C; one-way ANOVA, p= 0.275) or r s (D; one-way ANOVA, p= 0.201). For C,D, the number of motoneurons recorded at the different weeks after treatment ranged between 12 and 40, and for axotomy between 8 and 28; n=64 control motoneurons. E, Bar chart illustrating k s data and its mean value for in the population of motoneurons recorded in the control situation, after only axotomy and after axotomy plus VEGF administration. The asterisk indicates significant difference between the axotomy and control groups (one-way ANOVA, p,0.001). On the contrary, the VEGF-treated group showed no significant difference with control (p= 0.437). F, Same as E, but for r s values. The axotomy group showed a significant difference in r s with respect to control (asterisk, one-way ANOVA, p,0.001), whereas the VEGF-treated group was similar to control (p= 0.064). For E,F, the number of motoneurons analyzed in each situation was n= 64, 57, and 199 for the control, axotomy, and VEGF-treated groups, respectively. Research Article: Confirmation 7 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org described (Delgado-Garcia et al., 1986;Fuchs et al., 1988;Davis-López de Carrizosa et al., 2009,2010). Control motoneurons increased their discharge for ipsilateral eye movements during both the slow and the fast phases of the vestibulo-ocular reflex, and reduced their FR for eye movements in the opposite direction (Fig. 4A). In contrast, axotomy led to a significant reduction in motoneuronal discharge during vestibular eye movements (Fig. 4B). When VEGF was applied in the fourth ventricle immediately after axotomy, motoneurons recorded thereafter showed a normal discharge pattern during vestibular eye movements that resembled the pattern observed in control. Axotomized VEGF-treated motoneurons behaved normally throughout the whole period of recordings after VEGF administration (50 d), as illustrated for the two examples of motoneurons recorded 4 and 36 d after VEGF administration (Fig. 4C,D, respectively). We analyzed EP and EV sensitivities (k v and r v , respectively) during the slow phases of the nystagmus in control, axotomized and VEGF-treated motoneurons. As shown in Figure 4. Behavior of abducens motoneurons during vestibularly-induced eye movements. A–D, Examples of motoneurons recorded during vestibular eye movements in control (A), axotomy (B; 4 d after lesion), and after VEGF administration (C; 4 d and D; 36 d). Traces correspond to EP (in degrees) and FR (in spikes/second). Eye movements to the left (L) are upwards in the EP trace (double arrow). E,F, Time course of changes in EP (k v ;E) and EV (r v ;F) during vestibular eye movements. Control data (mean 6SD) are indicated by a blue dot and bars. k v and r v values for the axotomized population are indicated by red dots in E,F, respectively, as a function of time following the day of lesion (day 0). For VEGF-treated motoneurons, k v data are illustrated in E(green dots) and those of r v in F(green dots) throughout time after the day of surgery (lesion plus VEGF administration, day 0). G,H, All the population of control (n= 25), axotomized (n= 13) or VEGF-treated motoneurons (n= 64) recorded during vestibular eye movements were grouped in three pools, and their EP (k v ;G) and EV (r v ;H) sensitivities were compared. For k v , axotomized motoneurons showed a significantly lower sensitivity as compared with control (G; asterisk; one-way ANOVA, p= 0.021), whereas similar k v values were found between control and VEGF-treated motoneurons (G;p= 0.470). The same happened for r v (in H): axotomized motoneurons presented a significantly lower value than control (asterisk; one-way ANOVA, p= 0.021), but motoneurons of the VEGF group showed r v values similar to control (p= 0.657). Research Article: Confirmation 8 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org Figure 4E,F,k v and r v values of axotomized cells were low in comparison with control (mean 6SD), whereas those of injured motoneurons that received VEGF appeared similar to control. In addition, it can be observed that these parameters were maintained within a normal range in the VEGF-treated motoneurons throughout the whole period of recordings. Pooling the motoneurons per week after VEGF delivery, we found no significant difference between the seven VEGF groups (from the first to the seventh week) and control (k v : one-way ANOVA, F (7,81) = 1.442, p= 0.2; r v : one-way ANOVA, F (7,81) = 1.535, p= 0.167; nranged between 5 and 13 treated motoneurons per week, and for the control group n= 25; not illustrated). Therefore, we clustered the motoneurons in three groups: control, axotomy alone, and axotomy plus VEGF delivery. Comparison between groups revealed significant differences between groups [for k v : one-way ANOVA, F (2,99) = 5.121, p= 0.008 (Fig. 4G); for r v : one-way ANOVA, F (2,99) = 4.490, p= 0.014; n= 25, 13, and 64 motoneurons for control, axotomy, and VEGF groups, respectively (Fig. 4H)]. In particular, axotomized motoneurons presented significantly lower k v (p= 0.021; Fig. 4G) and r v (p= 0.021; Fig. 4H) values than control, whereas injured motoneurons treated with VEGF had k v (p= 0.470; Fig. 4G) and r v (p= 0.657; Fig. 4H) values similar to control cells. Therefore, as happened for spontaneous eye movements, a single dose of VEGF applied to axotomized motoneurons was able to prevent the alterations in motoneuronal sensitivities to EP and EV during vestibularly-in- duced eye movements for a prolonged period of time. Synaptic density is preserved in the long-term after a single pulse of VEGF to axotomized motoneurons Synaptophysin immunoreactivity was used as a general marker of synaptic boutons. We quantified the percentage of the somatic perimeter of abducens motoneurons that appeared contacted by synaptophysin-positive boutons, an index of linear density (in %) 21 d after lesion (with or without VEGF). A simple inspection of synaptophysin-IR boutons in the three situations revealed that control motoneurons presented a high density of perisomatic boutons (Fig. 5A) that contrasted with the low synaptophysin-IR boutons contacting axotomized motoneurons (Fig. 5B). In contrast, VEGF-treated motoneurons showed a normal density of synaptophysin-positive boutons contacting their somatic perimeter that resembled that found in control (Fig. 5C). Quantification of the linear density of synaptophysin-immunostained terminals (Fig. 5D) revealed significant differences between groups (one-way ANOVA, F (2,191) = 144.762, p,0.001). Thus, control motoneurons (n= 69) presented a somatic synaptic coverage of 41.44 60.84%, whereas in axotomized cells (n= 54), synaptic coverage fell Figure 5. VEGF prevents the synaptic loss induced by axotomy. Confocal images obtained after double immunofluorescence against ChAT, to label the motoneurons, and synaptophysin, to label synaptic boutons, around cell bodies (A–C) and in the neuropil (E–G). Images are shown for control (A,E), axotomy (B,F), and after axotomy 1VEGF supply (C,G). D, Bar chart illustrating the percentage of the somatic perimeter of motoneurons covered by synaptophysin-positive boutons (i.e., linear density) in the three situations. The statistical analysis revealed a significant (p,0.001) decrease in synaptic coverage after axotomy as compared with control (asterisk), whereas data obtained after VEGF delivery were similar (p= 0.703) to control (one-way ANOVA). H, Same as Dbut for measurements of synaptophysin optical density in the neuropil of the abducens nucleus. Axotomy induced also a significant (p,0.001) decrease (asterisk) in synaptic boutons at the neuropil level, but VEGF administration prevented this reduction producing values similar (p= 0.503) to control (one-way ANOVA). For D,H, mean 6SEM are illustrated. Scale bars: 20 mminA(for A–C) and 50 mminE(for E–G). Research Article: Confirmation 9 of 15 May/June 2020, 7(3) ENEURO.0467-19.2020 eNeuro.org