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Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats
Neuroscience Letters 494 (2011) 196–201
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats Yulin Luo, Xiaoying Wu ∗ , Shuangzhen Liu, Kuanshu Li Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China
a r t i c l e
i n f o
Article history: Received 21 July 2010 Received in revised form 4 February 2011 Accepted 4 March 2011 Keywords: Visual cortex Plasticity Dendritic spine Ultrastructure Visual evoked potentials (VEP)
a b s t r a c t Amblyopia is difficult to cure in adult due to the declination of visual cortical plasticity with age. However, the mechanisms limiting adult cortical plasticity are still unclear. Inhibition factors associated with myelin are suggested to be crucial for the ocular dominance plasticity in the visual cortex. We hypothesize that blocking Nogo-NgR system with NEP1–40 in adult visual cortex will reactivate the structural and functional plasticity. To back up this hypothesis, we subjected postnatal day 21 (P21) rats to monocular deprivation (MD) model until P45. Then the deprived eyes of MD model rats were reopened and followed by NEP1–40 or PBS administration for 7 days. Dendritic spine densities, ultrastructral modifications of synaptic junctions and objective visual function were examined at P52 to determine the therapeutic effects of NEP1–40. Our findings suggest a new curative role for NEP1–40 in structural and functional recovery from the deficits of adult MD rats, and offer a potential therapeutic tool for curing amblyopia and other cortically based visual disorders. © 2011 Elsevier Ireland Ltd. All rights reserved.
In the primary visual cortex, connection of neurons is dramatically influenced by visual experience from each eye during an early postnatal period called the critical period [15]. For instance, depriving one eye of pattern vision during the critical period, as is the case following unilateral congenital cataract, anisometropia or strabismus, changes the structure of geniculocortical afferents [17], shifts the ocular dominance distribution of cortical neurons [32] and leads to a severe impairment of visual function. The anatomical and physiological effects as well as defective visual function can be totally recovered only if the therapy is performed in early infancy [26], which has been attributed to a decline of plasticity in visual cortex with age. Although this theory has been extensively studied over several years, the underlying molecular mechanisms are still partially known. There is some evidence that the reduced plasticity and the termination of the critical period in adult mammalian visual cortex are partly due to the existence of growth-inhibitory molecules associated with myelin. Nogo-A [3], myelin-associated glycoprotein (MAG) [24], and oligodendrocyte myelin glycoprotein (OMgp) [30] appear to be responsible for this inhibition of axonal growth. A receptor molecule, Nogo-66 receptor (NgR) [10], mediates the action of one of two Nogo-A inhibitory domains, as well as the
∗ Corresponding author at: Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. Tel.: +86 731 84327121; fax: +86 731 84327121. E-mail address: [email protected] (X. Wu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.011
action of MAG and OMgp on axons. All three myelin molecules bind to NgR in a complex with p75 neurotrophin receptor and a leucine-rich repeat transmembrane protein LINGO-1, which acts to transduce the inhibitory signal across the cell membrane [25]. Then Rho/Rho kinase pathway is activated, resulting in rearrangement of the cytoskeleton and inhibition of axonal growth [33]. These pathways have been studied most intensively in ischemic stroke and traumatic spinal cord injury. Several previous studies have shown that Nogo-NgR system is implicated in plasticity in the hippocampal and cortical microcircuitry as well as spine cord [2,12], yet few investigation has examined their expression and role in the visual cortex. Are they also involved in plasticity in the visual cortex? DNA microarray analysis of transcription in mouse primary visual cortex surrounding the critical period suggested that myelin-associated genes increased developmentally, but neither nogo-A nor NgR was significant [18]. Furthermore, McGee and colleagues [23] have provided the firsthand persuasive evidence that the critical period window for ocular dominance plasticity is substantially extended in NgR null mice. As the result of that, we speculate the Nogo-NgR system, which inhibits axon regeneration and regulates cortical plasticity, presents a promising target for therapeutic intervention. A NgR competitive antagonist, NEP1–40 (Nogo-66 residue 1–40), binds to the leucine-rich region of the receptor and also blocks the inhibitory effect of Nogo. Intrathecal administration of NEP1–40 induced regenerative growth of corticospinal axons and partial recovery of function after dorsal hemisection injury in adult rats [13].
Y. Luo et al. / Neuroscience Letters 494 (2011) 196–201
In the present study, we used NgR antagonist peptide NEP1–40 to block the inhibition action of Nogo-NgR system, which results in the recovery of synaptic structure and function from early monocular deprivation (MD) in adult rats. Our data demonstrates the therapeutic potential of NEP1–40 for amblyopia and other cortically base visual disorders in the clinic. Female Sprague-Dawley rats with litters were obtained from Laboratory Animal Department of Central South University. Experiments followed the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Chinese Government Committee for Animal Experimentation Ethics. The mother was raised in a temperature and light controlled facility until the pups were 21 days old. Pups were randomly allocated into 6 groups: normal animals (Nor), MD animals without any treatment (MD), normal animals treated with PBS (Nor + PBS) or NEP1–40 (Nor + NEP), MD animals treated with PBS (MD + PBS) or NEP1–40 (MD + NEP). For the MD model, we used a method previously described by Maffei [19] and Prusky [28] at P21. Suture was checked daily until the deprived eye was reopened, and even minimal eye opening was excluded. At P45, the deprived eyes were reopened and received antibiotic and cortisonic drops. Meanwhile, lateral ventricles microinjections of drug or vehicle were done as described previously by Tsushima [29]. NEP1–40 was dissolved in vehicle (83% PBS plus 17% DMSO) at a concentration of 0.02 mg/l and rats received 0.2 mg of the solution daily from P45 to P51. For the control groups, equal-volume PBS was administrated per day. When the experiments were finished, the microinjection sites were histologically verified under a microscope. The Golgi method was employed as already reported [4]. All rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde and 1.5% picric acid in 0.1 M PBS. After postfixing in the same solution, brains were sectioned using a vibratome (Leica, Germany). Coronal sections were received in a 3% potassium dichromate, and then mounted on glass coverslips and impregnated in 1.5% silver nitrate. After removing coverslips, sections were rinsed in distilled water, dehydrated in an ascending series of alcohols, cleared with xylene, mounted on slides and covered with non-acidic synthetic balsam and coverslips. The neurons in binocular zone of primary visual cortex (Rat Brain Atlas [27]) contralateral to the deprived eye were observed. For each rat, ten well-stained Golgi impregnated neurons were selected, resulting in 40 neurons being analyzed from each group (n = 4). Apical dendrites of layer II–III pyramidal neurons were examined by counting spine density in their first 120 m of dendritic length. The specimens of binocular zone in primary visual cortex contralateral to the deprived eye for electron microscopy observation were removed, post-fixed in 2.5% glutaraldehyde in 0.1 M PBS, and then put into 1% osmium tetroxide. After several washes, tissue blocks were dehydrated, treated in a 1:1 mixture of epen812 epoxy resin and 100% acetone, and finally embedded in epen812 epoxy resin. Ultra-thin sections were cut and stained in saturated Uranyl acetate and lead nitrate. After repeated washes, ultra-thin sections were dried and examined with a JEOL-1230 electron microscope at 100 kV. In the present study, synapses in layer II–III pyramidal neurons were examined for synaptic measurement. According to methods by Jones [16] and Güldner [14], the width of synaptic cleft, the thickness of postsynaptic density (PSD) at the thickest part, the length of the active zones and the curvature of the synaptic interface (Fig. 2) were examined using Scion Image Beta 4.02 (Scion Corporation). Fifty synapses were selected for each rat, resulting in 200 synapses being analyzed from each group (n = 4). Visual evoked potentials [8] were performed on rats to assess the visual function objectively. Before examination, the head of rat was placed approximately 10 cm from the video monitor. Electrodes were placed according to the Sharon’s method [11]. Flashes
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stimuli were generated at 1 Hz and the evoked potentials were read, averaged from 256 flashes. P2 peaks were selected to characterized peak latencies, and amplitudes were measured between the N2 and P2 peaks. Deprived eye of each rat was examined at P52 (n = 8 each group). All statistical analysis was performed using SPSS11.5 statistical software. Data of all index are described as mean ± standard error of mean (SEM). One-way ANOVA followed by the LSD-t-test were used to evaluate significant differences among three groups. Statistical differences between two groups were analyzed by unpaired
Fig. 1. NEP1–40 causes a significant recovery of dendritic spine density in adult MD rats. (A) Apical dendrite segments showing dendritic spine densities of layer II–III pyramidal cells from binocular zone in primary visual cortex contralateral to the deprived eye. The spine densities in MD group and MD + PBS group decrease dramatically in comparison to Nor group, while MD + NEP group shows little difference. Scale bar = 10 m (B) Mean spine density in rats from all groups (n = 4 rats in each group, 40 dendritic spines studied per group, mean data per animal used for comparisons). In both MD group and MD + PBS group, spine density in the visual cortex is smaller than that of in Nor group (unpaired Student’s t-test, p < 0.05). In contrast, spine density in MD + NEP group does not differ from Nor group (unpaired Student’s t-test, p < 0.05). There is no statistical difference in dendritic spine density among Nor group, Nor + PBS group and Nor + NEP group (One-way ANOVA, p > 0.05).
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Fig. 2. Electromicrographs show the ultrastructural features of synapses in binocular zone of primary visual cortex contralateral to the deprived eye in different groups. (A) The typical synapse from each group. The limits of the length of synaptic active zone are indicated by two arrowheads. The thickness of PSD is at the thickest part of PSD and limited by two long arrows. Po and Pn show the synaptic curvature measurement points. SC, SV represent the synaptic cleft and synaptic vesicles, respectively. Scale bar = 0.2 m. (B) Effects of NEP1–40 on the structural modification of synaptic interface in binocular zone of primary visual cortex contralateral to the deprived eye. The results are expressed as mean ± SEM. (n = 4 rats in each group, 200 synapses studied per group, mean data per animal used for comparisons.).
Student’s t-test. A value of p < 0.05 was considered statistically significant. To assess whether the effects of NEP1–40 could be mediated by the activation of structural plasticity of dendritic spines, we measured the density of apical dendrites on layer II–III pyramidal neurons from binocular zone in primary visual cortex contralateral to the deprived eye using Golgi staining. As is shown in Fig. 1, monocular deprivation in early postnatal period decreased dendritic spine density in the visual cortex contralateral to the
deprived eye (Nor vs. MD, unpaired Student’s t-test, p < 0.05). After 7 days treatment, the average dendritic spine density of visual cortical neuron in MD + NEP group increased significantly and was on the verge of that of in Nor group, which were much more than those of in MD + PBS group (p < 0.05) or MD group (p < 0.05). However, there was no statistical difference in dendritic spine density among Nor group, Nor + PBS group and Nor + NEP group (p > 0.05), indicating that NEP1–40 had no effect on normal animals without monocular deprivation.
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Fig. 3. NEP1–40 treatment allows well recovery of objective visual function in adult monocular deprivation rats examined by VEP. (A) Typical traces of the flash-elicited visual evoked potentials in different groups of rats at P52. P2 peaks were selected to characterized peak latencies, and amplitudes were measured between the N2 and P2 peaks. (B) In MD group rats, VEP showed prolonged latency and low amplitude, which represented impaired visual function; NEP1–40 recovered the latency and amplitude of VEP in MD + NEP group rats, which approximated to that of the Nor group; while VEP in MD + PBS group remained abolished visual function, which did not recover with treatment of PBS. The results are expressed as mean ± SEM (n = 8 each group).
To investigate the effects of NEP1–40 on synaptic plasticity of primary visual cortex in rats following monocular deprivation, we measured the structural modifications of synaptic junctions by electromicrographs. As were shown in Fig. 2, all of the structural parameters of the synaptic junction in visual cortex were altered by monocular deprivation in MD group compared with the Nor group, displaying a increased width of synaptic clefts, shortened synaptic active zone, decreased curvature of synaptic interface and decreased thickness of PSD. However, synaptic ultrastructural analysis showed that NEP1–40 treatment could recover all of the structural index in monocular deprivation rats (MD vs. MD + PBS vs. MD + NEP, One-way ANOVA, p < 0.05) but not normal rats (Nor vs. Nor + PBS vs. Nor + NEP, One-way ANOVA, p > 0.05). It was noteworthy that, although the width of synaptic clefts in MD + NEP group decreased remarkably in comparison with that of in MD group, it still had not reach the normal level (Nor vs. MD + NEP, unpaired Student’s t-test, p > 0.05). The latencies and amplitudes of deprived eyes examined by VEP at P52 are presented in Fig. 3. We could see that monocular deprivation prolonged the latency and reduced the amplitude of VEP in deprived eyes (Nor vs. MD, unpaired Student’s t-test, p < 0.05). There was no change in VEP examination among Nor, Nor + PBS and Nor + NEP groups (One-way ANOVA, p > 0.05). While comparing with the results in MD group and MD + PBS group, the latencies of VEP in MD + NEP group were shortened and the amplitudes were increased (One-way ANOVA, p < 0.05), which were similar with
that of in Nor group (Nor vs. MD + NEP, unpaired Student’s t-test, p > 0.05). We have used three independent indexes to explore the role of the Nogo-NgR system in reactivation of visual cortical plasticity in adult MD rats. In each case, blocking Nogo-NgR system function by NEP1–40 improves visual cortical plasticity, in some cases to normal levels. Dendritic spines are highly motile structures, and it is believed that their plasticity reflects adaptive alterations in synaptic strength as a result of altered neural activity [9,37]. In typically developing rats, dendritic spine densities increase steadily after eye opening and reach a plateau level into adulthood. However, robust pruning of protrusions is observed through competitive mechanisms after brief monocular deprivation during the physiological critical period, as reported previously [20]. Several specific findings are evident regarding dendritic spine density reduction on pyramidal neurons in visual cortex contralateral to the deprived eye follow MD [21]. It has been proposed that the decreased spine densities in rats with MD attribute to inadequate environmental inputs. In previous study, NgR has been proposed to regulate RhoA activity levels in dendritic spines, thereby causing changes in spine structure [35,36]. Sensory deafferentation of somatosensory cortex leads to a downregulation of neuronal NgR expression in the cortex and correlates with increased cortical plasticity [5]. Consistent with the idea that NgR limits neuronal plasticity, ocular dominance plasticity in NgR mutants is prolonged and continues to adulthood [23]. The underlying structural change of synapse for plasticity, however, has not yet been defined. For this study, in
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comparison with MD + PBS group, we found that NEP1–40 administration to adult MD rats can increase the dendritic spine density of neuron in binocular zone of primary visual cortex contralateral to the deprived eye, which appears no difference with that of normal rats. It is notably that NEP1–40 has no significant effect on normal rats without eye closing. NgR is a receptor for multiple myelin inhibitors, all of which signal neuronal growth inhibition through activating the Rho/Rho kinase pathway [22] and regulating anatomical rearrangements of cytoskeleton [7]. We speculate that, after breaking the balance of neuronal circuitry by monocular deprivation, NEP1–40 can block the Nogo-NgR system to regulate RhoA activity levels and mediate rearrangement of the cytoskeleton, thereby, influence spine dynamic changes and reactivate the cortical plasticity. Alternatively, the increase in dendritic spine density observed in MD + NEP group rats may be primarily a reflection of altered synaptic strength, which is associated with the recovery of ocular dominance plasticity. Along with alteration of dendritic spine density, ultrastructural modifications in the synaptic junctions are also considered to a key aspect of synaptic plasticity. Ultrastructural changes, such as tightened synaptic cleft, thickened PSD, lengthened synaptic active zone and increased curvature of synaptic interface, were basis for enhanced synaptic transmissive efficiency. In previous study, NgR had been detected locating at synapses in CNS, which raised the possibility that the Nogo-NgR system might contribute to ultrastructural plasticity at synapses [31]. In consistent with the alteration of dendritic spine density, NEP1–40 regulated the structural modification of synaptic interface in binocular zone of primary visual cortex contralateral to the deprived eye, both of which could improve the synaptic efficiency and reactivate synaptic plasticity. The increased thickness of PSD has been known as the most important component in synaptic plasticity. PSD is a protein complex that lays postsynaptic membranes at the synaptic contact zone. In addition to postsynaptic receptors, PSD also contains key elements involved in variable postsynaptic activity and plasticity, such as signal- and scaffolding-proteins that organize the functional postsynaptic structure [34]. In aggregate, these findings point out that NEP1–40 could also reactivate synaptic plasticity in adult MD rats by modifying ultrastructural features in synaptic junctions. Growing evidence suggests that altered spine structure impacts synaptic physiology, and reciprocally, changes in synaptic strength lead to altered spine morphology [6]. Electrophysiological studies were conducted to assess the synaptic strength which can reveal the objective visual function. In present study, it has been identified that NEP1–40 could shorten the latency and increase the amplitude of VEP on adult deprived eye, which appeared a well recovery from the poor visual function. The changes of VEP were accompanied by significant recovery of dendritic spine density and synaptic plasticity. As we all known, adult visual cortex displayed a remarkable level of structural stability which might be caused by the presence of inhibitory factors [1]. NgR supported high affinity binding of myelin inhibitors and had been implicated in exerting an inhibitory control over structural plasticity. NEP1–40 administration could competitive block inhibitory effects of Nogo-NgR system, thus removed an obstacle for experience-dependent structural modifications in adult animals, at least at the spine level. Therefore, re-opening the formerly deprived eye combined with NEP1–40 treatment promoted formation of synaptic contacts on the newly formed spines, which might be the mechanism underlying the visual functional recovery observed in NEP1–40 treated animals. In summary, our results indicate a new role for NgR antagonist peptide NEP1–40 in structural and functional recovery from the deficits of adult MD rats, and offer the theoretical foundation for curing amblyopia and other cortically based visual disorders in the clinic for the first time. However, further experiments are necessary
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats Yulin Luo, Xiaoying Wu ∗ , Shuangzhen Liu, Kuanshu Li Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China
a r t i c l e
i n f o
Article history: Received 21 July 2010 Received in revised form 4 February 2011 Accepted 4 March 2011 Keywords: Visual cortex Plasticity Dendritic spine Ultrastructure Visual evoked potentials (VEP)
a b s t r a c t Amblyopia is difficult to cure in adult due to the declination of visual cortical plasticity with age. However, the mechanisms limiting adult cortical plasticity are still unclear. Inhibition factors associated with myelin are suggested to be crucial for the ocular dominance plasticity in the visual cortex. We hypothesize that blocking Nogo-NgR system with NEP1–40 in adult visual cortex will reactivate the structural and functional plasticity. To back up this hypothesis, we subjected postnatal day 21 (P21) rats to monocular deprivation (MD) model until P45. Then the deprived eyes of MD model rats were reopened and followed by NEP1–40 or PBS administration for 7 days. Dendritic spine densities, ultrastructral modifications of synaptic junctions and objective visual function were examined at P52 to determine the therapeutic effects of NEP1–40. Our findings suggest a new curative role for NEP1–40 in structural and functional recovery from the deficits of adult MD rats, and offer a potential therapeutic tool for curing amblyopia and other cortically based visual disorders. © 2011 Elsevier Ireland Ltd. All rights reserved.
In the primary visual cortex, connection of neurons is dramatically influenced by visual experience from each eye during an early postnatal period called the critical period [15]. For instance, depriving one eye of pattern vision during the critical period, as is the case following unilateral congenital cataract, anisometropia or strabismus, changes the structure of geniculocortical afferents [17], shifts the ocular dominance distribution of cortical neurons [32] and leads to a severe impairment of visual function. The anatomical and physiological effects as well as defective visual function can be totally recovered only if the therapy is performed in early infancy [26], which has been attributed to a decline of plasticity in visual cortex with age. Although this theory has been extensively studied over several years, the underlying molecular mechanisms are still partially known. There is some evidence that the reduced plasticity and the termination of the critical period in adult mammalian visual cortex are partly due to the existence of growth-inhibitory molecules associated with myelin. Nogo-A [3], myelin-associated glycoprotein (MAG) [24], and oligodendrocyte myelin glycoprotein (OMgp) [30] appear to be responsible for this inhibition of axonal growth. A receptor molecule, Nogo-66 receptor (NgR) [10], mediates the action of one of two Nogo-A inhibitory domains, as well as the
∗ Corresponding author at: Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. Tel.: +86 731 84327121; fax: +86 731 84327121. E-mail address: [email protected] (X. Wu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.011
action of MAG and OMgp on axons. All three myelin molecules bind to NgR in a complex with p75 neurotrophin receptor and a leucine-rich repeat transmembrane protein LINGO-1, which acts to transduce the inhibitory signal across the cell membrane [25]. Then Rho/Rho kinase pathway is activated, resulting in rearrangement of the cytoskeleton and inhibition of axonal growth [33]. These pathways have been studied most intensively in ischemic stroke and traumatic spinal cord injury. Several previous studies have shown that Nogo-NgR system is implicated in plasticity in the hippocampal and cortical microcircuitry as well as spine cord [2,12], yet few investigation has examined their expression and role in the visual cortex. Are they also involved in plasticity in the visual cortex? DNA microarray analysis of transcription in mouse primary visual cortex surrounding the critical period suggested that myelin-associated genes increased developmentally, but neither nogo-A nor NgR was significant [18]. Furthermore, McGee and colleagues [23] have provided the firsthand persuasive evidence that the critical period window for ocular dominance plasticity is substantially extended in NgR null mice. As the result of that, we speculate the Nogo-NgR system, which inhibits axon regeneration and regulates cortical plasticity, presents a promising target for therapeutic intervention. A NgR competitive antagonist, NEP1–40 (Nogo-66 residue 1–40), binds to the leucine-rich region of the receptor and also blocks the inhibitory effect of Nogo. Intrathecal administration of NEP1–40 induced regenerative growth of corticospinal axons and partial recovery of function after dorsal hemisection injury in adult rats [13].
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In the present study, we used NgR antagonist peptide NEP1–40 to block the inhibition action of Nogo-NgR system, which results in the recovery of synaptic structure and function from early monocular deprivation (MD) in adult rats. Our data demonstrates the therapeutic potential of NEP1–40 for amblyopia and other cortically base visual disorders in the clinic. Female Sprague-Dawley rats with litters were obtained from Laboratory Animal Department of Central South University. Experiments followed the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Chinese Government Committee for Animal Experimentation Ethics. The mother was raised in a temperature and light controlled facility until the pups were 21 days old. Pups were randomly allocated into 6 groups: normal animals (Nor), MD animals without any treatment (MD), normal animals treated with PBS (Nor + PBS) or NEP1–40 (Nor + NEP), MD animals treated with PBS (MD + PBS) or NEP1–40 (MD + NEP). For the MD model, we used a method previously described by Maffei [19] and Prusky [28] at P21. Suture was checked daily until the deprived eye was reopened, and even minimal eye opening was excluded. At P45, the deprived eyes were reopened and received antibiotic and cortisonic drops. Meanwhile, lateral ventricles microinjections of drug or vehicle were done as described previously by Tsushima [29]. NEP1–40 was dissolved in vehicle (83% PBS plus 17% DMSO) at a concentration of 0.02 mg/l and rats received 0.2 mg of the solution daily from P45 to P51. For the control groups, equal-volume PBS was administrated per day. When the experiments were finished, the microinjection sites were histologically verified under a microscope. The Golgi method was employed as already reported [4]. All rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde and 1.5% picric acid in 0.1 M PBS. After postfixing in the same solution, brains were sectioned using a vibratome (Leica, Germany). Coronal sections were received in a 3% potassium dichromate, and then mounted on glass coverslips and impregnated in 1.5% silver nitrate. After removing coverslips, sections were rinsed in distilled water, dehydrated in an ascending series of alcohols, cleared with xylene, mounted on slides and covered with non-acidic synthetic balsam and coverslips. The neurons in binocular zone of primary visual cortex (Rat Brain Atlas [27]) contralateral to the deprived eye were observed. For each rat, ten well-stained Golgi impregnated neurons were selected, resulting in 40 neurons being analyzed from each group (n = 4). Apical dendrites of layer II–III pyramidal neurons were examined by counting spine density in their first 120 m of dendritic length. The specimens of binocular zone in primary visual cortex contralateral to the deprived eye for electron microscopy observation were removed, post-fixed in 2.5% glutaraldehyde in 0.1 M PBS, and then put into 1% osmium tetroxide. After several washes, tissue blocks were dehydrated, treated in a 1:1 mixture of epen812 epoxy resin and 100% acetone, and finally embedded in epen812 epoxy resin. Ultra-thin sections were cut and stained in saturated Uranyl acetate and lead nitrate. After repeated washes, ultra-thin sections were dried and examined with a JEOL-1230 electron microscope at 100 kV. In the present study, synapses in layer II–III pyramidal neurons were examined for synaptic measurement. According to methods by Jones [16] and Güldner [14], the width of synaptic cleft, the thickness of postsynaptic density (PSD) at the thickest part, the length of the active zones and the curvature of the synaptic interface (Fig. 2) were examined using Scion Image Beta 4.02 (Scion Corporation). Fifty synapses were selected for each rat, resulting in 200 synapses being analyzed from each group (n = 4). Visual evoked potentials [8] were performed on rats to assess the visual function objectively. Before examination, the head of rat was placed approximately 10 cm from the video monitor. Electrodes were placed according to the Sharon’s method [11]. Flashes
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stimuli were generated at 1 Hz and the evoked potentials were read, averaged from 256 flashes. P2 peaks were selected to characterized peak latencies, and amplitudes were measured between the N2 and P2 peaks. Deprived eye of each rat was examined at P52 (n = 8 each group). All statistical analysis was performed using SPSS11.5 statistical software. Data of all index are described as mean ± standard error of mean (SEM). One-way ANOVA followed by the LSD-t-test were used to evaluate significant differences among three groups. Statistical differences between two groups were analyzed by unpaired
Fig. 1. NEP1–40 causes a significant recovery of dendritic spine density in adult MD rats. (A) Apical dendrite segments showing dendritic spine densities of layer II–III pyramidal cells from binocular zone in primary visual cortex contralateral to the deprived eye. The spine densities in MD group and MD + PBS group decrease dramatically in comparison to Nor group, while MD + NEP group shows little difference. Scale bar = 10 m (B) Mean spine density in rats from all groups (n = 4 rats in each group, 40 dendritic spines studied per group, mean data per animal used for comparisons). In both MD group and MD + PBS group, spine density in the visual cortex is smaller than that of in Nor group (unpaired Student’s t-test, p < 0.05). In contrast, spine density in MD + NEP group does not differ from Nor group (unpaired Student’s t-test, p < 0.05). There is no statistical difference in dendritic spine density among Nor group, Nor + PBS group and Nor + NEP group (One-way ANOVA, p > 0.05).
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Fig. 2. Electromicrographs show the ultrastructural features of synapses in binocular zone of primary visual cortex contralateral to the deprived eye in different groups. (A) The typical synapse from each group. The limits of the length of synaptic active zone are indicated by two arrowheads. The thickness of PSD is at the thickest part of PSD and limited by two long arrows. Po and Pn show the synaptic curvature measurement points. SC, SV represent the synaptic cleft and synaptic vesicles, respectively. Scale bar = 0.2 m. (B) Effects of NEP1–40 on the structural modification of synaptic interface in binocular zone of primary visual cortex contralateral to the deprived eye. The results are expressed as mean ± SEM. (n = 4 rats in each group, 200 synapses studied per group, mean data per animal used for comparisons.).
Student’s t-test. A value of p < 0.05 was considered statistically significant. To assess whether the effects of NEP1–40 could be mediated by the activation of structural plasticity of dendritic spines, we measured the density of apical dendrites on layer II–III pyramidal neurons from binocular zone in primary visual cortex contralateral to the deprived eye using Golgi staining. As is shown in Fig. 1, monocular deprivation in early postnatal period decreased dendritic spine density in the visual cortex contralateral to the
deprived eye (Nor vs. MD, unpaired Student’s t-test, p < 0.05). After 7 days treatment, the average dendritic spine density of visual cortical neuron in MD + NEP group increased significantly and was on the verge of that of in Nor group, which were much more than those of in MD + PBS group (p < 0.05) or MD group (p < 0.05). However, there was no statistical difference in dendritic spine density among Nor group, Nor + PBS group and Nor + NEP group (p > 0.05), indicating that NEP1–40 had no effect on normal animals without monocular deprivation.
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Fig. 3. NEP1–40 treatment allows well recovery of objective visual function in adult monocular deprivation rats examined by VEP. (A) Typical traces of the flash-elicited visual evoked potentials in different groups of rats at P52. P2 peaks were selected to characterized peak latencies, and amplitudes were measured between the N2 and P2 peaks. (B) In MD group rats, VEP showed prolonged latency and low amplitude, which represented impaired visual function; NEP1–40 recovered the latency and amplitude of VEP in MD + NEP group rats, which approximated to that of the Nor group; while VEP in MD + PBS group remained abolished visual function, which did not recover with treatment of PBS. The results are expressed as mean ± SEM (n = 8 each group).
To investigate the effects of NEP1–40 on synaptic plasticity of primary visual cortex in rats following monocular deprivation, we measured the structural modifications of synaptic junctions by electromicrographs. As were shown in Fig. 2, all of the structural parameters of the synaptic junction in visual cortex were altered by monocular deprivation in MD group compared with the Nor group, displaying a increased width of synaptic clefts, shortened synaptic active zone, decreased curvature of synaptic interface and decreased thickness of PSD. However, synaptic ultrastructural analysis showed that NEP1–40 treatment could recover all of the structural index in monocular deprivation rats (MD vs. MD + PBS vs. MD + NEP, One-way ANOVA, p < 0.05) but not normal rats (Nor vs. Nor + PBS vs. Nor + NEP, One-way ANOVA, p > 0.05). It was noteworthy that, although the width of synaptic clefts in MD + NEP group decreased remarkably in comparison with that of in MD group, it still had not reach the normal level (Nor vs. MD + NEP, unpaired Student’s t-test, p > 0.05). The latencies and amplitudes of deprived eyes examined by VEP at P52 are presented in Fig. 3. We could see that monocular deprivation prolonged the latency and reduced the amplitude of VEP in deprived eyes (Nor vs. MD, unpaired Student’s t-test, p < 0.05). There was no change in VEP examination among Nor, Nor + PBS and Nor + NEP groups (One-way ANOVA, p > 0.05). While comparing with the results in MD group and MD + PBS group, the latencies of VEP in MD + NEP group were shortened and the amplitudes were increased (One-way ANOVA, p < 0.05), which were similar with
that of in Nor group (Nor vs. MD + NEP, unpaired Student’s t-test, p > 0.05). We have used three independent indexes to explore the role of the Nogo-NgR system in reactivation of visual cortical plasticity in adult MD rats. In each case, blocking Nogo-NgR system function by NEP1–40 improves visual cortical plasticity, in some cases to normal levels. Dendritic spines are highly motile structures, and it is believed that their plasticity reflects adaptive alterations in synaptic strength as a result of altered neural activity [9,37]. In typically developing rats, dendritic spine densities increase steadily after eye opening and reach a plateau level into adulthood. However, robust pruning of protrusions is observed through competitive mechanisms after brief monocular deprivation during the physiological critical period, as reported previously [20]. Several specific findings are evident regarding dendritic spine density reduction on pyramidal neurons in visual cortex contralateral to the deprived eye follow MD [21]. It has been proposed that the decreased spine densities in rats with MD attribute to inadequate environmental inputs. In previous study, NgR has been proposed to regulate RhoA activity levels in dendritic spines, thereby causing changes in spine structure [35,36]. Sensory deafferentation of somatosensory cortex leads to a downregulation of neuronal NgR expression in the cortex and correlates with increased cortical plasticity [5]. Consistent with the idea that NgR limits neuronal plasticity, ocular dominance plasticity in NgR mutants is prolonged and continues to adulthood [23]. The underlying structural change of synapse for plasticity, however, has not yet been defined. For this study, in
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comparison with MD + PBS group, we found that NEP1–40 administration to adult MD rats can increase the dendritic spine density of neuron in binocular zone of primary visual cortex contralateral to the deprived eye, which appears no difference with that of normal rats. It is notably that NEP1–40 has no significant effect on normal rats without eye closing. NgR is a receptor for multiple myelin inhibitors, all of which signal neuronal growth inhibition through activating the Rho/Rho kinase pathway [22] and regulating anatomical rearrangements of cytoskeleton [7]. We speculate that, after breaking the balance of neuronal circuitry by monocular deprivation, NEP1–40 can block the Nogo-NgR system to regulate RhoA activity levels and mediate rearrangement of the cytoskeleton, thereby, influence spine dynamic changes and reactivate the cortical plasticity. Alternatively, the increase in dendritic spine density observed in MD + NEP group rats may be primarily a reflection of altered synaptic strength, which is associated with the recovery of ocular dominance plasticity. Along with alteration of dendritic spine density, ultrastructural modifications in the synaptic junctions are also considered to a key aspect of synaptic plasticity. Ultrastructural changes, such as tightened synaptic cleft, thickened PSD, lengthened synaptic active zone and increased curvature of synaptic interface, were basis for enhanced synaptic transmissive efficiency. In previous study, NgR had been detected locating at synapses in CNS, which raised the possibility that the Nogo-NgR system might contribute to ultrastructural plasticity at synapses [31]. In consistent with the alteration of dendritic spine density, NEP1–40 regulated the structural modification of synaptic interface in binocular zone of primary visual cortex contralateral to the deprived eye, both of which could improve the synaptic efficiency and reactivate synaptic plasticity. The increased thickness of PSD has been known as the most important component in synaptic plasticity. PSD is a protein complex that lays postsynaptic membranes at the synaptic contact zone. In addition to postsynaptic receptors, PSD also contains key elements involved in variable postsynaptic activity and plasticity, such as signal- and scaffolding-proteins that organize the functional postsynaptic structure [34]. In aggregate, these findings point out that NEP1–40 could also reactivate synaptic plasticity in adult MD rats by modifying ultrastructural features in synaptic junctions. Growing evidence suggests that altered spine structure impacts synaptic physiology, and reciprocally, changes in synaptic strength lead to altered spine morphology [6]. Electrophysiological studies were conducted to assess the synaptic strength which can reveal the objective visual function. In present study, it has been identified that NEP1–40 could shorten the latency and increase the amplitude of VEP on adult deprived eye, which appeared a well recovery from the poor visual function. The changes of VEP were accompanied by significant recovery of dendritic spine density and synaptic plasticity. As we all known, adult visual cortex displayed a remarkable level of structural stability which might be caused by the presence of inhibitory factors [1]. NgR supported high affinity binding of myelin inhibitors and had been implicated in exerting an inhibitory control over structural plasticity. NEP1–40 administration could competitive block inhibitory effects of Nogo-NgR system, thus removed an obstacle for experience-dependent structural modifications in adult animals, at least at the spine level. Therefore, re-opening the formerly deprived eye combined with NEP1–40 treatment promoted formation of synaptic contacts on the newly formed spines, which might be the mechanism underlying the visual functional recovery observed in NEP1–40 treated animals. In summary, our results indicate a new role for NgR antagonist peptide NEP1–40 in structural and functional recovery from the deficits of adult MD rats, and offer the theoretical foundation for curing amblyopia and other cortically based visual disorders in the clinic for the first time. However, further experiments are necessary
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