A total of 26 cats (aged 1–3 years and weighing 2.5–3.2 kg) were used as subjects in this study. All cats were purchased from Nanjing Qing-Long-Shan Animal Breeding Farm (Jiangning District of Nanjing, Certificate No. SX1207) and all of them were disease-free, healthy subjects with no optical or retinal abnormality. All animals were reared in rooms separated by transparent glass walls. Each room had comfortably organized living, feeding, and playing areas, and the room temperature was maintained at 25°C. The cats could get clean food and water freely. Each animal was fasted for 12 h before the experiment. Eight cats were randomly selected for electrophysiological experiments to examine, respectively, the effects of HFS (4 cats) and LFS (4 cats) on visually-evoked field potentials (VEPs) in the primary visual cortex (V1: area 17), and 18 cats were used for Western blot experiments to assess the membrane protein content of glutamatergic AMPA and NMDA receptors in the V1 cortex after HFS (6 cats) or LFS (6 cats) versus that of control cats (6 cats).

All experiments in this study were performed strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and conformed to the principles and regulations as described in the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experiments and animal treatments were approved by the Ethics Committee of Anhui Normal University (approval No: AHNU-ET 2023015).

Brain tissues containing V1 cortex (area 17) were collected 30 min after the end of repeated HFS or LFS. The membrane protein content of the key subunit GluA1 of AMPA receptors and the main subunit NR1 of NMDA receptors in the V1 cortex after repeated HFS or LFS versus controls was measured using Western blot techniques.

Western blot assays were conducted using methods similar to those in our previous studies (Yang et al., 2016; Ding et al., 2017; Zhao et al., 2020; Zhang et al., 2022). The frozen tissues of V1 cortex were cut, weighed, thawed, homogenized in 10 volumes of an ice-cold buffer [25-mM Tris–HCl (pH: 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS] and a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China), and spun down at 12,000 rpm for 15 min at 4°C. The supernatant was saved and its protein concentration was assessed using BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China, #P0010S). The plasma membrane proteins (the expression of glutamatergic receptors’ subunits in the plasma membrane) were prepared using a Membrane Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China, #P0033) containing protease inhibitor cocktail and phosphatase inhibitor cocktail, and homogenized with 20 full strokes in glass homogenizers. The lysates were centrifuged for 700 g at 4°C for 10 min. The supernatant was centrifuged again at 14,000 g at 4°C for 30 min. The pellet was resuspended in the lysis buffer and was centrifuged for 14,000 g at 4°C for 5 min. The protein concentration was assessed using BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China, #P0010S). We fractionated the proteins (30 μg) from each sample using 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred them onto polyvinylidene fluoride membranes (Beyotime Biotechnology). The membranes were blocked with 5% non-fat dry milk in TBS-Tween 20 for 1 h and incubated overnight at 4°C in TBS-Tween 20 containing primary antibodies, including rabbit anti-GluA1 (1: 1000, Cell Signaling Technology, #13185), rabbit anti-NR1 (1: 1,000, Abcam, #ab17345), and rabbit anti-β-Tubulin (1:2000, Affinity Biosciences, #AF7011). They were then washed three times for 10 min in TBS-Tween 20, incubated with peroxidase conjugated affinipure goat anti-rabbit IgG (1:8,000, Sangon Biotechnology, China, D10058) diluted in TBS-Tween 20 for 2 h at 25°C, and washed again in TBS-Tween 20.

Images of Western blot bands were captured by Tanon 5,200 Multi chemiluminescent imaging system (Tanon, Shanghai, China) (Li et al., 2019; Xu et al., 2020; Zhang et al., 2022). The optical density (OD) of western blot bands was measured using Image J software (National Institutes of Health, Montgomery, Bethesda, MA, USA). The OD value of GluA1 and NR1 bands were normalized against the OD values of the corresponding β- Tubulin bands in each sample, respectively.

All value were shown as individual measurement value and mean ± SD. Statistical comparison of VEPs amplitude before and after repeated HFS or LFS as well as the normalized OD of GluA1 and NR1 in the V1 cortex between groups was done using ANOVA and LSD (least significance difference) Post hoc pairwise tests (Zhao et al., 2020; Ding et al., 2022).

To explore whether repeated HFS and LFS can induce a long-term potentiation of VEPs in the V1 cortex, this study recorded the VEPs of V1 cortex in response to test visual stimuli (see Methods 2.2.2) before and at different time point (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180 min) after the end of repeated HFS or LFS. To examine if the effects induced by repeated HFS and LFS were specific to the stimulated orientation, we recorded the VEPs of V1 cortex in response to test visual stimuli at the stimulated orientation (SO: 90°) and at non-stimulated orientation (NSO: 180°) orthogonal to the SO both before and after HFS or LFS. The voltage-traces of VEPs in response to test visual stimuli at SO and NSO recorded both before and after repeated HFS and LFS were similar in components, containing wave N1, P1 and N2 (Figure 1). The latencies in the peak wave N1, P1 and N2 of VEPs showed no evident alteration after repeated HFS and LFS (Figures 1A–D). However, the amplitude of VEPs was increased after the end of HFS at first, and then returned to the level before HFS (Figures 1A,B). The amplitude of VEPs had no evident change before and after repeated LFS (Figures 1C,D).

Several previous studies show that repeated sensory stimulation can increase the expression of extrasynaptic glutamatergic receptors (Eckert et al., 2013) or induce a neural response potentiation which is blocked by antagonists of NMDA and AMPA receptors (Clapp et al., 2006; Kirk et al., 2010; Clapp et al., 2012). These evidences suggest that HFS-evoked LTP-like potentiation of VEPs in the V1 cortex may involve changes of AMPA and NMDA receptors. Therefore, we assessed the membrane content of the key subunit GluA1 of AMPA receptors and the main subunit NR1 of NMDA receptors in the V1 cortex after repeated HFS or LFS relative to control cats.

One-way ANOVA analysis showed that the mean normalized OD value of NR1 against that of β-Tubulin in the V1 cortex exhibited a significant difference among groups after repeated HFS, LFS and controls [F(2, 18) = 14.630, p < 0.0001]. Post-hoc test indicated that the mean normalized OD of NR1 in the V1 cortex after repeated HFS was significantly higher than that after LFS (p = 0.0002) and of controls (p = 0.0003) whereas the mean normalized OD of NR1 in the V1 cortex after LFS displayed no significant variation from that of controls (p = 0.633) (Figure 5A). Similarly, the mean relative OD of GluA1 against β-Tubulin in the V1 cortex exhibited a significant variation among groups after repeated HFS, LFS and controls [F(2, 18) = 30.114, p < 0.001]. Post-hoc test indicated that the mean OD value of GluA1 relative to β-Tubulin in the V1 cortex after HFS was significant larger than that after LFS (p < 0.001) and of controls (p < 0.001) whereas the relative OD of GluA1 in the V1 cortex after LFS was not significantly different from that of controls (p = 0.461) (Figure 5B).

The analysis above indicated that repeated HFS but not LFS could significantly increase the membrane content of glutamatergic AMPA and NMDA receptors in the V1 cortex, which might mediate the LTP-like amplitude increase of VEPs in the V1 cortex after repeated HFS.

The classic long-term potentiation (LTP) refers to an input-specific and long-lasting increase of postsynaptic potentials evoked by high-frequency electrical stimulation at the presynaptic fibers in in vitro studies (Sarvey et al., 1989; Bear et al., 1992; Colbert and Levy, 1993; Kudoh and Shibuki, 1994; Murphy et al., 1997; Volianskis and Jensen, 2003; Miyamoto, 2006). Recent in vivo studies report that noninvasive transcranial magnetic stimulation (TMS) (Naro et al., 2015; Chung et al., 2016), direct current stimulation (tDCS) (Ding et al., 2016; Agboada et al., 2020; Zhao et al., 2020; Frase et al., 2021) and even rapidly repeated visual or auditory stimulation (Clapp et al., 2005; Teyler et al., 2005; Ross et al., 2008; Kirk et al., 2010; Lengali et al., 2021) can also induce a LTP-like increase in hemodynamic response or visualy-evoked field potentials (VEPs). However, whether this LTP-like neural response improvement shows a property identical to the classic LTP remains in debate. Some studies show that sensory induced LTP-like improvement of neural response is similar in stimulus-specificity and longevity to the classic LTP (McNair et al., 2006; Ross et al., 2008; Aberg and Herzog, 2012) whereas others report inconsistent plasticity, including an increase in the amplitude of different VEP components, no change or decrease in neural activity as well as lack of stimulus-specific potentiation (Teyler et al., 2005; Lahr et al., 2014; Klöppel et al., 2015; Sumner et al., 2018; Dias et al., 2022; Kleeva et al., 2022). This study examined the effect of repeated HFS versus LFS on VEPs in the V1 cortex, and observed alterations of VEPs in response to test visual stimuli with stimulated orientation (SO) and non-stimulated orientation (NSO) before and at different time (0–180 min) after HFS or LFS. Statistical comparisons show that repeated LFS had no significant effect on VEPs in the V1 cortex, whereas repeated HFS could significantly increase N1P1 and P1N2 amplitude of VEPs in response to visual stimuli with both SO and NSO, but the effect was faster, stronger and lasted longer at SO than at NSO. Our results suggest that repeated HFS but not LFS can induce a long-lasting increase of VEPs in V1 cortex, which is similar to the classic LTP (Bliss and Lomo, 1973; Sarvey et al., 1989; Kirkwood and Bear, 1994; Volianskis and Jensen, 2003) and sensory stimulation-evoked LTP-like response improvement reported in human subjects (Sanders et al., 2018; Valstad et al., 2020; Rygvold et al., 2021, 2022). Nevertheless, our results indicate that HFS-induced VEP-amplitude improvement shows a less specificity to stimulus orientation and can partially generalize to visual stimuli at the other orientations, which differs from LTP-like neural response potentiation observed in human studies (McNair et al., 2006; Ross et al., 2008; Kirk et al., 2010; Clapp et al., 2012; Valstad et al., 2021) and is also unlike the perceptual learning effect with high specificity to trained stimulus parameters (Hua et al., 2010; Lu et al., 2011; Seitz, 2011; Li, 2016; Yan et al., 2018; Yang et al., 2020; Jing et al., 2021; Astorga et al., 2022). Thus, repeated HFS is likely an alternative noninvasive paradigm that can be used to enhance visual cortical excitability and help improve visual ability for patients with impaired vision (Zhang et al., 2014; Yan et al., 2015; Lu et al., 2016).

Reasons leading to the diverse reports about sensory (visual/auditory) induced long-term plasticity of neural activity are poorly understood. At least two factors may contribute. First, different authors have used various stimuli paradigms during LTP induction, such as stimulus type, temporal/spatial frequency and duration (McNair et al., 2006; Ross et al., 2008; Clapp et al., 2012; Eckert et al., 2013; Lahr et al., 2014; Klöppel et al., 2015; Sanders et al., 2018; Rygvold et al., 2021; Dias et al., 2022), which may potentiate the activity of different neuronal populations. Second, methods for measurement of neural response after sensory tetanization varied among different studies (Clapp et al., 2005; Ross et al., 2008; Clapp et al., 2012; Lahr et al., 2014; Valstad et al., 2020; Lengali et al., 2021), which could have measured neural activity at different temporal and spatial scales. Subsequent researches are needed to clarify these possibilities (Dias et al., 2022).

The cellular mechanisms underlying the classic LTP induced by in vitro electrical stimulation at presynaptic fibers have been extensively investigated over several decades (Bliss et al., 1986; Jay et al., 1995; Murphy et al., 1997; Malenka and Nicoll, 1999; Lynch et al., 2000; Liu et al., 2004; Zhong et al., 2006; Peng et al., 2010). It is known that trains of high-frequency presynaptic stimulation can trigger releasing of more neurotransmitter glutamate, which will open more AMPA receptors at postsynaptic membrane. Subsequent influx of more sodium ions through AMPA receptors cause a large membrane depolarization and lead to the opening of NMDA receptors, and influx of calcium ions through NMDA channels will trigger a series of cascades that cause an increased expression and assembling of AMPA receptors at postsynaptic membrane and thus mediate LTP process (Miyamoto, 2006; Peng et al., 2010).

How rapidly repeated sensory stimulation induce LTP-like increase of neural response in local field potentials or hemodynamic response remains poorly understood. For the long-lasting improvement of neural response at field potential level after repeated sensory stimulation is totally different from the firing rate reduction of single-unit response after sensory stimulus repetition (Peter et al., 2021; Stauch et al., 2021), this LTP-like neural response potentiation could not be mediated by the mechanism of adaptation. Recent studies reports that rapidly presented visual stimulation can enhance the subunit expression of both AMPA and NMDA receptors in the extrasynaptic regions of the membrane although no significant potentiation of VEPs is observed in the primary visual cortex (Eckert et al., 2013), and tDCS can also improve the membrane content of AMPA and NMDA receptors although the total cellular content has no significant change (Zhang et al., 2022). In addition, other studies have found that the LTP-like neural response potentiation is cancelled by blockers of NMDA receptors (Clapp et al., 2006, 2012). These studies suggest that sensory-induced LTP-like neural response improvement could likely be mediated by a mechanism similar to that in classic LTP process (Miyamoto, 2006; Zhong et al., 2006; Peng et al., 2010). However, no study has provided a direct evidence.

The current study comparatively examined the membrane protein content of the key subunit GluA1 of AMPA receptors and the main subunit NR1 of NMDA receptors in the V1 cortex after repeated HFS relative to that after LFS and in controls. The results showed that the membrane protein content of GluA1 and NR1 in V1 cortex was significantly increased after HFS but not LFS when compared with that in controls. This result provide a direct evidence that cellular mechanism underlying visual stimulation-induced LTP-like neural response potentiation is similar to the classic LTP (Miyamoto, 2006; Peng et al., 2010).

In summary, the results in this study indicate that repeated high- but not low-frequency visual stimulation can evoke a LTP-like improvement of VEPs amplitude in the V1 cortex of cats. Similar to the classic LTP (Sarvey et al., 1989; Bliss and Collingridge, 1993; Miyamoto, 2006; Peng et al., 2010), this LTP-like VEPs potentiation is long-lasting and involve membrane trafficking of AMPA and NMDA receptors. Considering that VEPs measure the membrane potentials from a large population of neurons (Lashgari et al., 2012; Haider et al., 2016; Krishna et al., 2021), LTP-like improvement of field potentials evoked by noninvasive sensory stimulation (Clapp et al., 2006, 2012; Eckert et al., 2013), TMS (Aydin-Abidin et al., 2006; Naro et al., 2015), tDCS (Agboada et al., 2020; Frase et al., 2021; Zhang et al., 2022) and perceptual learning (Sale et al., 2011; Lengali et al., 2021) could be a pooled response of postsynaptic potentiation (classic LTP) across neural network. However, the LTP-like VEPs improvement observed in this study shows a less stimulus-input specificity than the classic LTP (Kelso et al., 1986; Kirkwood and Bear, 1994), suggesting other neuronal plasticity, such as changes in intracortical inhibition (Bear et al., 1992; Field et al., 2021), may engage in the effect generalization process. Further studies are needed to elucidate the underlying mechanisms.