5-HTR2A and 5-HTR3A but not 5-HTR1A antagonism impairs the cross-modal reactivation of deprived visual cortex in adulthood

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR1A and 5-HTR3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR1A, 5-HTR2A and 5-HTR3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR1A and 5-HTR3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR2A and 5-HTR3A but not 5-HTR1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.


Introduction
Even though the mammalian brain is most susceptible to changes in sensory inputs during so-called critical periods early in life [1][2][3][4], it retains the intrinsic capacity to recover from sensory deprivation well into adulthood [1,[5][6][7]. In human patients, for example, late-onset vision loss triggers two types of cross-modal brain plasticity [8]: 'compensatory plasticity' is the result of experience-dependent refinement of the spared senses, whereas 'cross-modal recruitment' involves the take-over of the deprived visual cortical territory for the active processing of non-visual information, leading to enhanced sound localization abilities or improved tactile acuity as with Braille reading [9][10][11][12][13].
In an animal model of one-eyed vision, due to monocular enucleation (ME) in adult (P120) mice, we could previously establish extensive neuronal reactivation of both the binocular zone (Bz) and the medial monocular zone (Mmz) of the deprived contralateral visual cortex within a 7 week recovery period (7wME) (Fig. 1a, b). This functional recovery not only relied on the expected unimodal potentiation of spared-eye inputs occurring in the Bz during the first 3 weeks post-ME, but also on an ensuing tactile whisker-related reactivation of especially the Mmz from week four onwards [14][15][16]. This cross-modal effect was most pronounced in the infragranular layers of the Mmz [15]. The level of neuronal activity reached in the Mmz 7 weeks post-ME could be experimentally manipulated by reducing or intensifying whisker inputs, respectively by means of whisker removal or via natural whisker stimulation during exploration of a new, enriched cage environment in complete darkness. These observations confirmed the manifestation of the cross-modal recruitment of the medial monocular cortical territory by the whiskers [15]. The post-ME recovery period is thus characterized by the recruitment of the deprived visual areas by the spared senses in adult mice just as in human patients.
Despite the current understanding of molecular and cellular aspects of visual cortex plasticity [12,[17][18][19][20][21][22] and the well-described role of neuromodulators in brain plasticity [19,[23][24][25][26][27], only little is known about sensory deprivation-induced alterations in serotonin (5-HT) signaling across sensory areas or, in concreto, about how 5-HT is involved in the different types of cortical plasticity in the context of acquired blindness. A wide range of axonal projections, originating from the serotonergic Fig. 1 Schematic overview of the different cortical plasticity phases occurring in the adult ME-mouse model and illustration of the experimental setup. a Timeline indicating the early unimodal plasticity phase (week 1-3 post-ME) and the subsequent cross-modal plasticity phase (week 4-7 post-ME). b Overview of the ISH zif268-based reactivation pattern of the uni-and cross-modal plasticity phase in adult mice upon ME. Whisker deprivation and 5-HTR antagonism specifically suppress reactivation of the Mmz. c Illustration of the anatomical delineation of the visual cortex regions on a Nissl-stained coronal section which are then overlaid on the matching ISH zif268 section. The delineated visual cortical areas are indicated between large arrowheads: V2L, V1, V2M and RM with the distinction between monocular (m) and binocular (b) segments. The binocular zone (Bz) comprises V2Lb-V1b while the medial monocular zone (Mmz) includes V1m-V2M. The different cortical layers are indicated with Roman numbers: I-VI. d All animals had normal vision up to the age of P120 or in case of the non-deprived age-matched control mice (AMC), up to P169 (white bars). Mice that underwent monocular enucleation (ME, light gray bars) at P120 recovered under standard housing conditions during 1 week (1wME, n = 5; representing ongoing open-eye potentiation), 3 weeks (3wME, n = 5, representing the end of the open-eye potentiation phase), 5 weeks (5wME, n = 5; representing ongoing cross-modal plasticity phase) or 7 weeks (7wME, n = 11; representing the end of the cross-modal plasticity phase). All drug-treated ME mice recovered for 7 weeks and received daily intraperitoneal (i.p.) injections of either saline (n = 6), 5-HTR 1A antagonist (WAY-100635, n = 3), 5-HTR 2A antagonist (ketanserin, n = 3) or 5-HTR 3A antagonist (ondansetron, n = 3) during the last 3 weeks of the 7 weeks post-ME period (dark gray bar, representing the time window of whisker-driven cross-modal plasticity). The number of animals (n) is represented in the bars Raphe Nucleus in the brain stem [28], reach the different sensory cortices, where 5-HT and several of the 14 identified mammalian 5-HT receptor subtypes are involved in the integration of neuronal signals and in the processing of sensory information [29][30][31][32][33][34][35].
Of particular interest to the field of cortical plasticity are the serotonergic G-protein coupled receptors 5-HTR 1A and 5-HTR 2A , and ion channel 5-HTR 3A . These receptors are most abundantly expressed in the mammalian neocortex, predominantly on excitatory, excitatory and inhibitory, and exclusively on inhibitory neurons respectively. In addition, 5-HTR 1A and 5-HTR 2A have already been implicated in either unimodal or cross-modal plasticity [18,27,32,[36][37][38][39][40][41]. On the one hand, administration of the selective serotonin reuptake inhibitor (SSRI) fluoxetine during a period of visual deprivation via eyelid suture reinstated juvenile-like unimodal ocular dominance plasticity in adulthood. This extraordinary phenomenon was found to be mediated through, amongst others, changes in 5-HTR 1A receptor function leading to an experiencedependent shift in the cortical excitation/inhibition balance (E/I) [26,27,36,42,43]. On the other hand, 2 days of visual deprivation in young rats mediated a 5-HTR 2A/ 2C -dependent delivery of AMPAR1 specifically at layer IV-II/III synapses of the primary somatosensory barrel cortex (S1BF), ultimately leading to compensatory plasticity in the form of a sharpened whisker-barrel map and more fine-tuned barrel neuron responses to primary whisker stimulation [32].
Adult cortical plasticity in response to sensory deprivation was further found to involve the activation of dis-inhibitory cortical circuits including the vasoactive intestinal polypeptide (VIP)-positive interneurons [44][45][46][47][48][49][50][51]. VIP cells are a type of 5-HTR 3A expressing interneurons, which account for approximately 1/3 rd of the entire interneuron population [52]. We discovered before that GABA A R α1 -mediated intracortical inhibition elicits a pivotal role in the different response of the Bz and the Mmz to ME [53]. Since 5-HT and the 5-HT receptors are considered important regulators of the cortical E/I balance [37,54,55], they may well exert a major impact on the excitability of the specific brain circuits involved in different types of cortical plasticity.
All these findings indicate that 5-HT may indeed elicit a very important role in the cortical response to sensory loss. We therefore initiated a set of experiments to elucidate if and how exactly 5-HT or one of the three above described 5-HT receptor subtypes, take part in ME-induced plasticity in adult mice. HPLC analysis was performed on whole-tissue homogenates of the visual cortex and S1BF to study the long-term impact of ME on the total 5-HT concentration in these two sensory cortices, known to functionally adapt upon vision loss [15]. Western blotting experiments were conducted to investigate the longitudinal effect of ME on the vesicular monoamine transporter 2 (vMAT2), 5-HTR 1A and 5-HTR 3A protein expression levels. Different time-points post-ME were chosen to enable distinction between the earlier effects occurring in the Bz, during the unimodal open-eye potentiation phase, and those occurring in the Mmz, during the subsequent cross-modal phase. We pharmacologically antagonized 5-HTR 1A , 5-HTR 2A and 5-HTR 3A receptor function, to pinpoint via which of these receptors 5-HT mediates ME-induced cross-modal plasticity in the visual cortex and in S1BF. As before, we relied on in situ hybridization for the neuronal activity reporter gene zif268 as a high-throughput read-out to differentiate the distinct post-ME plasticity phases (Fig.  1c, d). We demonstrate brain region-specific and time-dependent alterations in pre-and postsynaptic aspects of 5-HT neurotransmission in the adult brain upon ME. A role for 5-HTR 1A in unimodal open-eye potentiation was confirmed and we provide evidence for the involvement of 5-HTR 2A and 5-HTR 3A but not 5-HTR 1A in ME-induced cross-modal plasticity. The potential of a defined pharmacological and spatiotemporal control on cross-modal plasticity holds promise towards future refinements of rehabilitation strategies to treat acquired sensory loss.

Animals
In total 54 C57Bl/6 J mice (Janvier Elevage, Le Genest-St-Isle, France) of either sex (32 male/22 female) were used in this study. All mice were housed under standard laboratory conditions with constant room temperature and humidity, an 10/14-h dark/light cycle with food and water available ad libitum. All experiments have been approved by the Ethical Research Committee of KU Leuven and were in strict accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and with the Belgian legislation (KB of 29 May 2013). Every effort was made to minimize animal suffering and to reduce the number of animals. Figure 1 illustrates the experimental manipulations and the number of mice used per condition (Fig. 1b, d). The different phases of cortical plasticity under study have been determined previously based on the impact of either visual stimulation via the spared eye, or somatosensory deprivation/stimulation based on whisker clipping/natural whisker use during the exploration of new toys in complete darkness, on neuronal activity in the visual cortex of adult ME mice [15]. Specifically, 1 week post-ME (1wME) mice are in an ongoing unimodal open-eye potentiation phase. Mice with a 3 week post-ME recovery period (3wME) are at the end of the open-eye potentiation phase, which restores normal visually driven activity levels in an extended binocular zone (Bz). 5 weeks post-ME (5wME) mice are in an ongoing cross-modal phase whereas mice with a 7 week post-ME recovery period (7wME) have undergone maximal cross-modal visual cortex reactivation in which normal activity levels are restored in the monocular zone of the visual cortex, especially medial to the Bz (Mmz), only now relying on whisker inputs. Cortical regions of interest therefore are the visual cortex, Bz and Mmz, and the primary somatosensory barrel field (S1BF).

Monocular enucleation paradigm and tissue preparation
The removal of the right eye, or monocular enucleation (ME), was performed as described previously [14]. Briefly, adult (P120) mice were anaesthetized by intraperitoneal injection of a mixture of ketamine hydrochloride (75 mg/kg, Dechra Veterinary Products, Eurovet) and medetomidine hydrochloride (1 mg/kg Orion Corporation, Janssen Animal Health). Eye ointment (Tobrex, Alcon) was administered to the left eye to prevent dehydration. The right eye was carefully removed and the orbit was filled with hemostatic cotton wool (Qualiphar, Bornem, Belgium) in case of bleeding. Analgesics were injected subcutaneously (Metacam, 2 mg/kg, 0.1 mL) and atipamezol hydrochloride was administered to reverse anaesthesia (1 mg/kg i.p., Orion Corporation, Elanco Animal Health). Following ME, the mice were housed in their home cages under standard laboratory conditions for a 1 to 7 week recovery period. Control mice are age-matched (AMC) to the 7 week ME mice, the time point of maximal recovery of neuronal activity [56]. At the end of the ME period, the mice were sacrificed by cervical dislocation. For HPLC-based determination of the total 5-HT concentration in visual and somatosensory tissue samples, the brains were rapidly extracted and immediately frozen in 2-methylbutane (Merck, Overijse, Belgium) at a temperature of − 40°C. All brains were stored at − 80°C until sectioning. A tissue blotting device (Model PA 002 Mouse Brain Blocker, 1 mm, David KOPF instruments, California) was used to prepare approximately 1 mm-thick coronal slices to subsequently isolate the whole visual cortex and the primary somatosensory cortex (S1BF) with sterile scalpels. Separate analysis of medial monocular and binocular cortex was technically impossible with this method. For Western blotting experiments, 100 μm-thick coronal cryosections were collected on baked glass slides and stored at − 80°C. Specifically for radioactive in situ hybridization (ISH) experiments, the mice were placed overnight in their home cages in a dark room to reduce zif268-expression to basal levels. The following day, the mice were placed in a high-lit environment to upregulate sensory driven zif268-mRNA expression. After 45 min, at peak zif268-mRNA expression levels, the mice were sacrificed by cervical dislocation. The brains were rapidly removed, immersed in 2-methylbutane at a temperature of − 40°C (Merck, Overijse, Belgium) and stored at − 80°C until sectioning. Serial sections with a thickness of 25 μm were prepared on a cryostat (HM 500 OM, Microm, Thermo Scientific, Walldorf, Germany), mounted on 0.1% poly-L-Lysine-coated (Sigma-Aldrich) slides, and stored at − 20°C until further processing.

Immunoblotting
To obtain the optimal primary antibody working concentration, a protein dilution series ranging from 5 to 30 μg was analyzed. A concentration within the linear range of the detection system that resulted in a good signal to noise ratio was chosen for monocular, binocular and somatosensory samples separately. For vMAT2, 5-HTR 1A and 5-HTR 3A analysis, this resulted in 15 μg for samples of all three regions. 5-HTR 2A was excluded from the analyses due to the lack of a specific antibody. Reference sample (pool) consisting of a mixture of equal amounts of each prepared tissue sample was run for monocular, binocular and somatosensory cortex, with the same optimal amount of protein on each gel to gauge blot-to-blot variability. After the addition of 5 μl reducing agent (10×, Invitrogen, Paisley, United Kingdom) and 2 μl LDS sample buffer (4×, Invitrogen), the samples were denatured (10 min, 70°C). The protein samples were separated on 4-12% Bis-Tris Midi-gels in the XCell4 SureLock Midi-Cell (Invitrogen). The Spec-tra™ Multicolor Broad range protein ladder (Thermo-Scientific) was used as molecular weight standard. Subsequently, the samples were transferred to a polyvinylidene fluoride (PVDF) membrane. After 1-2 h incubation in a 5% ECL blocking agent (GE Healthcare, Buckinghamshire, UK) in Tris-saline (0.01 M Tris, 0.9% NaCl, 0.1% TX-100, pH 7.6), the membrane was incubated overnight with a primary antibody rabbit anti-vMAT2 (1:1000, R&D Systems, Novus Biologicals), with rabbit anti-5-HTR 1A (1:200, Alomone Labs), with rabbit anti-5-HTR 3A (1:200, Alomone Labs). The next The peaks were numbered from 1 to 7 and represent noradrenaline; 3,4-dihydroxybenzylamine; 3,4-dihydroxyphenylacetic acid; dopamine; 5-hydroxy-indoleacetic acid; homovanillic acid and serotonin (5-HT). c In the visual cortex (VC) of 7wME mice (n = 6), a significantly decreased total 5-HT concentration was observed compared to AMC mice (n = 9, p = 0.018, Mann-Whitney test). d The primary somatosensory barrel field (S1BF) did not show a significant difference (p = 0.114, Mann-Whitney test). These results indicate an ME-induced effect on 5-HT signaling in sensory deprived cortex. The number of animals (n) is represented in the bars. *P < 0.05 day, the blots were successively washed in Tris-saline (4 × 5 min), 30 min incubated with HRP-conjugated secondary antibody (goat anti-rabbit IgG, 1:50.000, Dako, Glostrup, Denmark), rinsed in Tris-saline (5 × 5 min) and Tris-stock (1 × 5 min) (0.05 M Tris, pH 7.6). The immunoreactive bands were visualized using a chemiluminescent reaction (1 × 5 min, Super Signal West Dura, ThermoScientific, Pierce) combined with the BIO-RAD ChemiDoc™ MP Imaging System. In order to correct for intra-and inter-gel variability and to normalize the concentration of the specific detected proteins to the total amount of protein present, we performed a total protein stain (TPS) with Swift Membrane Stain (G-Biosciences) according to manufacturer's instructions. Immediately after the staining, blots were scanned with the BIO-RAD ChemiDoc™ MP Imaging System.

Semi-quantitative Western analysis
The immunostained protein bands were semi-quantitatively evaluated by densitometry (Image Lab™ software) separately for monocular, binocular and somatosensory cortex samples ( Figs. 3 and 4). First, to account for intra-gel and inter-gel variability including loading differences or incomplete transfer onto the membrane, a TPS was employed rather than the use of a single reference protein [53,66,67]. For each protein of interest, the optical density value per mouse was normalized to its corresponding TPS. Also, to compare samples between different gels, normalized data were expressed relative to the region-specific reference sample (pool).
Pharmacology ME mice (pharmacology total n = 15, saline: n = 6; WAY-100635: n = 3; ketanserin: n = 3; ondansetron: n = 3) received daily injections (i.p.), at 2 pm exactly, of a specific 5-HT receptor antagonist at a dose based on prior literature, and specifically during the 3-week period of cross-modal plasticity from week 5 to 7 post-ME (Fig. 1d). Systemic drug delivery was chosen because a long-term treatment with a locally implanted slow release system (e.g. Alzet minipump) would lead to cortical tissue damage and scar formation, potentially influencing the local neuromodulator levels [68]. Drugs used were the 5-HTR 1A antagonist WAY-100635 maleate (Abcam Ab120550, 1 mg/kg, 0.2 mL i.p.) [37,36,69,70], the 5-HTR 2A antagonist ketanserin tartrate (R&D Systems, Tocris Bioscience, 5 mg/kg, 0.2 mL i.p.) [71][72][73][74] and the 5-HTR 3A antagonist ondansetron hydrochloride (R&D Systems, Tocris Bioscience, 5 mg/kg, 0.2 mL i.p.) [75]. Control animals were housed under the same standard conditions and received saline injections (0.9%, i.p.) following the same injection regimen. All animals were sacrificed by cervical dislocation. Brains were rapidly removed and stored as described above. Time-dependent effect of ME on cortical area-specific vMAT2 expression levels. Western blot OD values are illustrated as the ratio of the expression levels of each ME group relative to the expression of the AMC group to judge the effect of post-ME recovery time. Below each graph, representative Western blot bands are depicted, as well as the expected molecular weight (~75 kDa) and an example total protein stain. The different conditions are indicated with numbers above the respective bands: 1 = 3wME, 2 = 5wME, 3 = AMC, 4 = 7wME and P = region-specific tissue sample pool. a At 1 week post-ME (1wME), the expression of vesicular monoamine transporter 2 (vMAT2) in the medial monocular zone (Mmz) was significantly reduced compared to the AMC mice (dashed line) and the vMAT2 expression levels remain low, even after 7 weeks. b In the binocular zone (Bz), a significant ME-effect was detected in 1wME and 3wME mice, showing increased vMAT2 expression levels immediately after ME. c In the primary somatosensory barrel field (S1BF), vMAT2 expression was increased in 1wME mice compared to AMCs, and was significantly higher in 1wME and 3wME mice compared to 5wME mice. The number of animals (n) is represented in the bars. Significant pair-wise differences between ME mice and the AMC group are indicated with a '*' within the corresponding bar. Differences between ME conditions due to post-ME recovery time are indicated as a '*' above the respective ME conditions. *P < 0.05, **P < 0.01 In situ hybridization for zif268-mRNA High-throughput radioactive ISH experiments were performed on series of coronal brain sections between Bregma levels − 1.5 mm and − 5 mm and changes in the mRNA expression level of the immediate early gene (IEG) zif268, a proven excellent activity reporter gene in the mammalian brain, were quantified (mouse: [15,16,51,53,56,[76][77][78], cat: [79][80][81][82]. As such, the spatial extent and the exact anatomical location of experienceinduced, predominantly excitatory [6,[83][84][85][86][87], neuronal activity changes were analyzed and compared throughout all cortical layers of the visual and somatosensory neocortex. This high-throughput approach allows the molecular visualization of cortical reactivation patterns in response to ME. ISH for zif268-mRNA was performed with a mouse-specific synthetic oligonucleotide probe (Eurogentec, Seraing, Belgium) with sequence 5′-ccgtt gctcagcagcatcatctcctccagtttggggtagttgtcc-3′. As described previously [51,53,77,88], each probe was 3′-end labeled with [ 33 P] dATP using terminal deoxynucleotidyl transferase (Invitrogen, Carlsbad, CA). Unincorporated nucleotides were separated from the labeled probe by means of miniQuick Spin Oligo Columns (Roche Diagnostics, Vilvoorde, Belgium). The cryostat sections were fixed, dehydrated and delipidated. The radioactively labeled probes were added to a hybridization cocktail (50% (v/v) formamide, 4× standard SSC buffer, 1× Denhardt's solution, 10% (w/v) dextran sulfate, 100 μg/ml herring sperm DNA, 250 μg/ml tRNA, 60 mM dithiothreitol, 1% (w/v) N-lauroyl sarcosine, and 20 mM NaHPO 4 , pH 7.4) and applied to the cryostat sections (10 6 cpm/section). After an overnight incubation at 37°C in a humid chamber, the sections were rinsed in 1× standard SSC buffer at 42°C, dehydrated, air-dried and exposed to an autoradiographic film (Biomax MR; Kodak, Rochester, NY). After 7 days, the films were developed in EMS replacement for Kodak developer D-19 (Electron Microscopy Sciences, Hatfield) and fixed in Rapid fixer (Ilford Fig. 4 Time-dependent effect of ME on cortical area-specific 5-HTR 1A and 5-HTR 3A expression levels. a, b Analysis of the Western blot OD values, illustrated as the ratio of the expression level of each ME group relative to the expression of the age-matched control group (AMC, dashed line) to judge the effect of post-lesion recovery time, revealed no significant impact of ME on 5-HTR 1A and 5-HTR 3A receptor expression, and also no time-dependent modulations in the medial monocular zone (Mmz) of the visual cortex of ME mice. c In the binocular zone (Bz), post-ME 5-HTR 1A expression levels were increased in 1wME and 3wME mice and gradually decreased over time, reaching AMC levels in 5wME and 7wME mice. d The 5-HTR 3A receptor levels in the Bz, were significantly higher in 3wME mice compared to AMCs and decreased gradually over time to reach AMC levels at 5 and 7 weeks post-ME. e In the primary somatosensory barrel field (S1BF), the 5-HTR 1A expression was increased in 1wME and 3wME mice, decreased over time to reach its lowest expression levels at 5 weeks post-ME (5wME) and again increased at 7 weeks post-ME. f Although similar ME-induced and time-dependent modulations in 5-HTR 3A expression were observed in S1BF, they did not reach statistical difference, possibly due to larger inter-individual variation. The number of animals (n) is represented in the bars. Significant pair-wise differences between ME mice and the AMC group are indicated with a '*' within the corresponding bar. Differences between ME conditions due to post-ME recovery time are indicated as a '*' above the respective ME conditions. *P < 0.05, **P < 0.01 Hypam; Kodak). Autoradiographic images of adjacent sections per examined cortical area per mouse were scanned at 1200 dpi (CanoScan LIDE 600F; Canon, Tokyo, Japan), and all images were similarly adjusted for brightness and contrast in Adobe Photoshop Elements 2018 (version 16.0, × 64, Adobe Systems Incorporated).

Quantitative analysis of ISH results
To quantify the optical density (OD; mean gray value per pixel) of the ISH autoradiograms, a custom-made Matlab (Matlab R2017a; Mathworks) script was used as described previously [51,53]. We analyzed at least three mice per condition. Per mouse, three ISH sections with an inter-distance of 100 μm were investigated (for visual cortex: − 3.5-(− 3.7); 1-4; 1, relative to Bregma; for somatosensory cortex: − 1.7-(− 1.9); 2.5-4; 1, relative to Bregma). The region of interest in the left hemisphere was demarcated by determining the top edge of the cortex, the boundary between the supra-and granular layers (II-III and IV) and the infragranular layers (V and VI), and the border between the infragranular layers and the corpus callosum. The region of interest was then divided equally into 24 segments from lateral to medial to create two lattices of 24 quadrangles, corresponding to the upper (II-IV) and lower (V-VI) layers. To compensate for possible variation in brain size and morphology, the lattices were translated on each autoradiogram over the cortical curvature, fixing the border of a specific segment to an areal border (border segment 20/21 is the area border V1 m/V2M). For each segment created this way, the relative OD was calculated as the mean gray value of all pixels contained within a particular quadrangle and was normalized to the mean gray value of a square measured in the corpus callosum (a defined region with no zif268-mRNA expression above background) in order to compare autoradiograms across experiments. Relative neuronal activity was expressed in percentages based on the following formula: 1 -(cortical zif268/background) × 100. Results are presented as profiles of neuronal activity per brain area and are illustrated in subregion-specific bar graphs. Pseudo-color maps were generated through a second custom-made Matlab script (Matlab R2017a; Mathworks, Natick, MA) and represent a false color coding of the gray values ranging from black (0) to white (225); high gray values are represented in white/yellow on the false color scale bar (0-50), medium high gray values are represented in red  or blue (100-150) and low gray values are represented in green/black (150-255).

Statistics
All HPLC results, Western blotting data and relative OD-values in ISH-sections were presented as mean ± SEM. Normal distribution and in parallel, equal variance between groups was tested. A non-parametric test (Mann-Whitney) was applied for pairwise comparison. A Kruskal-Wallis test followed by a Dunn's multiple comparisons post-hoc test was used to determine the recovery time-dependent modulations of protein expression levels upon ME. For all tests, a probability level (α level was set to 0.05) of < 0.05 was accepted as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). Statistical analyses were performed using GraphPad Prism 5.01 (GraphPad Software, Inc).

Reduced visual cortex 5-HT concentration in response to monocular enucleation
To investigate if 5-HT is involved in adult ME-induced cross-modal plasticity and to better understand its role therein, we first examined whether the total 5-HT concentration was affected in the two cortices that functionally adapt upon ME. The total 5-HT concentration was analyzed with High-Performance Liquid Chromatography (HPLC) on whole tissue homogenates of the visual cortex and S1BF 7 weeks after performing ME in adult mice (7wME) (Fig. 2a, b). At this 7wME endpoint, when the cross-modal recruitment of the initially deprived visual cortex is completed [15], we observed a decreased 5-HT concentration in the visual cortex (mean ± SEM, AMC: 82.54 ± 4.77; 7wME: 65.50 ± 4.77, Mann-Whitney test, p = 0.018, Fig. 2c). In S1BF, the cortical area in which compensatory plasticity is triggered by vision loss, no significant difference was reached (AMC: 113.40 ± 8.06; 7wME: 86.41 ± 10.49; Mann-Whitney test, p = 0.114, Fig. 2d). These results suggest brain region-specific modulations of total 5-HT content in response to visual deprivation.

ME induces pre-and postsynaptic changes in proteins related to 5-HT signaling
Parallel to the ME-induced decrease in total 5-HT concentration in the visual cortex, the impact of partial vision loss might manifest at the level of 5-HT loading into readily releasable vesicles or at the level of 5-HT receptors. To investigate possible brain region-specific expression changes in these pre-and postsynaptic proteins involved in 5-HT signaling, we performed Western blotting experiments on time-course samples from the visual Mmz and Bz separately and from S1BF. Post-ME recovery periods of respectively 1 week (1wME: ongoing open-eye potentiation phase), 3 weeks (3wME: end of the open-eye potentiation phase) and 5 weeks (5wME: ongoing cross-modal phase) were chosen as intermediate time points towards the time point of maximal visual cortex reactivation (7wME) for the evaluation of possible post-ME recovery time-dependent modulations in protein expression in addition to endpoint evaluation ( Fig. 1) [15]. We chose to analyze vMAT2 protein expression levels since they provide information on the presynaptic loading of vesicles with 5-HT and because vMAT2 expression levels positively correlate with the total 5-HT concentration present in a given brain region of interest [60][61][62]. vMAT2 expression levels were significantly decreased in the Mmz in 1wME, 5wME and 7wME mice (represented as * within the bar, bar graphs in Figs. 3 and 4) compared to AMC (represented as a dashed line in Figs. 3 and 4, mean ± SEM, 1wME: 0.34 ± 0.08, p = 0.0159; 3wME: 0.80 ± 0.56, p = 0.4127; 5wME: 0.45 ± 0.02, p = 0.0159; 7wME: 0.53 ± 0.28, p = 0.0317, Mann-Whitney test). No statistically significant modulations occurred as a function of post-ME recovery time (Kruskal-Wallis test: p = 0.0159, with no statistical difference in the Dunn's post-hoc test for pairwise comparison) (Fig. 3a). Western analysis of the vMAT2 expression in the Bz indicated increased vMAT2 expression in 1wME and 3wME mice compared to AMC mice (mean ± SEM, 1wME: 1.87 ± 0.19, p = 0.0317; 3wME: 1.45 ± 0.06, p = 0.0159; 5wME: 1.07 ± 0.07, p = 0.4127; 7wME: 1.01 ± 0.04, p = 0.7302, Mann-Whitney test). As for the Mmz, also for the Bz no significant recovery time-dependent changes were observed (Kruskal-Wallis test: p = 0.0095, with no statistical differences in the Dunn's post-hoc test for pairwise comparison). In this case, however, a trend of decreasing vMAT2 expression was observed over the 1w to 7w post-ME time course (Fig. 3b). In S1BF, vMAT2 expression was significantly increased in 1wME mice compared to AMC (mean ± SEM, 1wME: 1.58 ± 0.11, p = 0.0317; 3wME: 1.50 ± 0.10, p = 0.0635; 5wME: 0.77 ± 0.09, p = 0.1905; 7wME: 1.29 ± 0.11, p = 0.4127, Mann-Whitney test, Fig. 3c). Furthermore, in 1wME and 3wME mice, the vMAT2 expression levels were higher compared to 5wME mice (Kruskal-Wallis test: p = 0.0081). These results point towards a return to baseline vMAT2 expression levels specifically at the time when the cross-modal whisker take-over of the deprived visual cortex starts to occur [15].

5-HTR 2A and 5-HTR 3A but not 5-HTR 1A antagonists suppress ME-induced cross-modal plasticity
We hypothesized that, in case 5-HT receptor function is involved in the whisker-mediated reactivation of the deprived visual cortex, the ME-induced cross-modal recruitment could be suppressed by systemic administration of the 5-HTR 1A antagonist WAY-100635 maleate, the 5-HTR 2A antagonist ketanserin tartrate, or the 5-HTR 3A antagonist ondansetron hydrochloride during the last 3 weeks of recovery of the Mmz in ME mice ( Fig. 5a-e). As the 5-HT receptors influence the excitability of brain circuits by modulating excitatory and inhibitory neurotransmission and thus by mediating the cortical E/I balance, we expected to observe effects of these drugs on cortical activity, and ultimately on cortical plasticity [36,54,55]. Because it was previously established that the most pronounced impact of whisker inputs on neuronal activity involves the infragranular layers of the visual cortex, as based on the expression of neuronal activity reporter gene zif268, these layers were interrogated separately from the supra-and granular layers. Suppression of the 5-HTR 1A receptor function by long-term administration of WAY-100635 from week 5 to 7 post-ME did not alter the neuronal activity levels reached throughout the Mmz of 7wME mice, compared to saline-injected age-matched control ME mice (Mmz, supra-and granular layers: p = 0.9048, infragranular layers: p = 0.9048, Bz, supra-and granular layers: p = 0.0476, infragranular layers: p = 0.2619, Mann-Whitney test, Fig. 5f ). However, it did decrease the neuronal (See figure on previous page.) Fig. 6 5-HTR antagonist treatment from week 5-7 post-ME affects neuronal activity levels in S1BF. a-d Images of 3 adjacent sections (− 1.7-(− 1.9); 2.5-4; 1, relative to Bregma) from adult 7wME mice, injected during the last 3 weeks of the 7wME recovery period with (a) saline, (b) 5-HTR 1A antagonist WAY-100635, (c) 5-HTR 2A antagonist ketanserin, (d) 5-HTR 3A antagonist ondansetron. Corresponding pseudocolor representations are displayed next to their respective ISH sections. e Line graphs illustrating the relative zif268-mRNA expression level, measured as the average ODvalue/segment, for saline-injected 7wME mice. For supra-and granular layers II/III-IV (e, left panel, dark gray line) and for infragranular layers V-VI (e, right panel, light gray line) the expression levels are displayed along the 3 predefined somatosensory subdivisions (black arrowheads are in accordance with the small arrowheads in a-d). Error bars represent the SEM of the mean OD value in each segment. Relative zif268-mRNA expression levels are shown as OD-values averaged over the primary somatosensory barrel cortex (S1BF) for supra-and granular layers (f-h, left panel) and infragranular layers (f-h, right panel). Comparison of the OD-values of saline-injected (light gray bars) and WAY-100635-injected 7wME mice (orange bars) indicates no post-ME changes in neuronal activity in S1BF due to long-term i.p. injection of 5-HTR 1A antagonist (f). Comparison of the OD-values of saline-injected (light-gray bars) and ketanserin-treated 7wME mice (green bars) indicates increased neuronal activity across all layers of the S1BF (g). Comparison of the OD-values of saline-injected (light-gray bars) and ondansetron-treated 7wME mice (blue bars) indicates increased neuronal activity across all layers of the S1BF (h). The number of animals (n) is represented in the bars. *P < 0.05 activity in the supra-and granular layers of the Bz (Mean ± SEM, supra-and granular layers, saline, Bz: 71. 58 Fig. 5f ), indicative of Bz-specific 5-HTR 1A expression changes during the preceding open-eye potentiation phase.
Blocking 5-HTR 2A receptor function by ketanserin administration from week 5 to 7 post-ME significantly decreased the neuronal activity across all layers of the Mmz (Mmz, supra-and granular layers: p = 0.0476, infragranular layers: p = 0.0238; Bz, supra-and granular layers: p = 0.0952, infragranular layers: p = 0.2619, Mann-Whitney test, Fig. 5g) Fig. 5g). Suppression of the 5-HTR 3A receptor function by administration of ondansetron only reduced the neuronal activity specifically in the supra-and granular layers of the Mmz (Mmz, supra-and granular layers: p = 0.0476, infragranular: p = 0.5476; Bz, supra-and granular layers: p = 0.7143, infragranular layers:  Fig. 5h). Taken together, these results point out that modulation of the 5-HTR 2A and 5-HTR 3A receptor function, influences the late cortical response to ME, suggesting that these receptors play an important role in the cross-modal reactivation phase of the visual cortex following ME.
Previous work has shown that ME induces increased neuronal activity in the spared sensory brain areas adjacent to the deprived visual cortex. For somatosensory cortex, the zif268-mRNA expression levels were found to be significantly higher in adult 7wME mice compared to AMC mice [16,53]. In order to assess the impact of pharmacological modulation of serotonergic neurotransmission on S1BF neuronal activity, next to mapping the extent of visual cortex reactivation, we compared S1BF activity levels between saline-injected 7wME mice and 7wME mice that were treated with the specific 5-HTR 1A , 5-HTR 2A or 5-HTR 3A antagonists.

Discussion
In humans, dysfunction of 5-HT signaling in the brain, is best described in mental illnesses such as depression and anxiety disorders [30,92]. Drugs that target the serotonergic system, for instance the selective serotonin reuptake inhibitors (SSRIs), are amongst the most-often prescribed first-line treatments, predominantly because low 5-HT levels in the brain were considered to be the main cause of such diseases [93]. However, the observed low efficacy and only very late-onset mood and behavioral improvements suggest large scale, functional and structural neuroplastic changes in the brain, rather than immediate, direct effects of those drugs on their targets [94][95][96][97][98]. Neuropsychological illnesses thus seem more complex than first considered and may in fact be neurological disorders with an underlying plasticity deficit. From research using sensory deprivation models, it is known that monoamine system-targeting drugs like SSRIs elicit plastic adaptations in the mammalian cortex [26,99,100]. Evidence for such cortical plasticity brought about by long-term treatment with an SSRI was already provided ten years ago and is based upon the discovery that fluoxetine can reinstate juvenile-like ocular dominance plasticity in the rodent adult visual cortex [26]. Genes involved in synaptic plasticity and chromatin structure remodeling, excitatory/inhibitory neurotransmission, transcription factors and proteolytic enzymes that degrade the extracellular matrix were found to underlie this type of plasticity [101]. It is likely that similar plastic changes occur in human patients in order to be relieved from their neuropsychological illness and to again express a positive mood and behavior.
Late-onset ME modulates 5-HT and vMAT2 levels in adult visual cortex Our HPLC data imply a lowered whole tissue 5-HT concentration specifically in the visual cortex of long-term ME mice. Since 5-HT release is triggered by neuronal activity in the sensory cortex, the observed decrease in 5-HT concentration in the visual cortex of 7wME mice is possibly due to the ME-induced loss of visual input in the Mmz [102]. A similar decrease in total 5-HT levels was also observed in the deprived visual cortex of adult cats in response to retinal lesions, an animal model for age-related macular degeneration [59]. For more in depth interpretation of brain region-specific alterations in 5-HT signaling, we still depend on new methodological developments that would allow reliable longitudinal analysis of the true local 5-HT release from only 1 mm thick cortical tissue, such as the Bz or the Mmz in our mouse model. For now, predictions based on longitudinal vMAT2 expression changes allow to formulate some possibilities. Tong et al., (2011) indeed already showed that vMAT2 levels are positively correlated with the tissue concentrations of total monoamines (5-HT, dopamine and noradrenaline), as measured in human tissue by means of HPLC analysis [62]. By deduction, the significantly decreased vMAT2 protein expression already shortly after the introduction of ME, may indicate decreased 5-HT concentrations in the Mmz immediately upon the loss of all its visual input. Opposite to the Mmz, the Bz displayed only a temporary increase in vMAT2 expression from week 1 to 3 weeks post-ME, at a time when its remaining open-eye inputs become potentiated. Spared cortical territory seems to share such an early 5-HT mediated response, in relation to compensating for the sensory deficit. Indeed, a similar pattern of early increased vMAT2 expression in S1BF, could go hand in hand with a fast, initial increase in 5-HT concentration and might indicate a compensatory, cortical plasticity mechanism similar to the one described by Jitsuki et al., (2011) after only 2 days of visual deprivation in S1BF of young rats [32]. Once a new functional cortical balance is established for the spared sense, it may be able to subsequently recruit nearby sensory deprived cortical territory. In our model of acquired blindness, somatosensation indeed seems to be capable of recruiting the Mmz once fine-tuned whisker-information processing has been established in S1BF.

5-HTR 1A mediates unimodal open-eye potentiation in the Bz of the visual cortex
Cortical plasticity depends on both excitation and inhibition levels, which is determined by the distribution of excitatory and inhibitory receptors. An established E/I balance defines the stability and efficacy of neuronal circuits and is therefore required for proper processing of sensory information [36,43,103,104]. A study, investigating the serotonergic regulation of the two predominant types of inhibition, respectively the short intermittent bursts of phasic inhibition and the constant long-lasting tonic inhibition, aside from its effect on the pyramidal neurons in the rat visual cortex, showed that 5-HT suppressed tonic inhibition through the 5-HTR 1A receptor and Protein Kinase A signaling while phasic inhibition was enhanced through 5-HTR 2A receptor function and CaM Kinase II [105,106]. Clearly, alterations in 5-HT receptor expression and function can exert a major impact on the excitability of brain circuits involved in brain plasticity, as this would influence the inhibitory neurotransmission and the regulation of the cortical E/I balance [36,54,55].
Our observations indicate ME-induced increases in 5-HTR 1A receptor expression in the spared cortices, the Bz and S1BF, confirming a regionally restricted and modulatory role in maintaining cortical excitability immediately upon insult. In the Bz, the increase in 5-HTR 1A expression occurred at the start of the open-eye potentiation phase, pointing towards an early involvement of 5-HTR 1A -mediated tonic inhibition, which implies the consistent activation of extra-and perisynaptic GABA A R δ1 receptors on pyramidal and inhibitory neurons by ambient GABA, to regulate the overall cortical excitability during this thalamocortical plasticity process [107]. Of note, such enhanced tonic inhibition also triggers ocular dominance plasticity in the mouse visual cortex during the critical period early in life [108].
Many studies previously reported on 5-HT-mediated induction of unimodal brain plasticity in adulthood. For example, adult rats were again susceptible for monocular deprivation-induced ocular dominance plasticity after cortical infusion of 5-HT directly into the visual cortex [37]. Also in rodents, treatment with the SSRI fluoxetine, was found to induce both hippocampal synaptic plasticity and unimodal ocular dominance plasticity in the visual cortex [26,37,109,110]. In line with this, in the human visual system, long-term systemic treatment with the SSRI sertraline was shown to induce synaptic plasticity by increasing the amplitude of stimulus-induced visually evoked potentials [111]. Fluoxetine even emerged as a possible treatment for persistent amblyopia, or lazy eye, in adult humans [112,113]. Maya-Vetencourt et al., (2011) could previously also show that the ocular dominance shift, normally occurring in the Bz shortly after monocular deprivation, can be prevented by blocking this receptor with WAY-100635 [37]. Furthermore, local application of this same selective 5-HTR 1A antagonist in V1 by means of reverse-phase microdialysis was found to facilitate long-term potentiation (LTP) after theta-burst stimulation (TBS) in adult rats, while LTP was inhibited in juvenile rats, confirming an age-dependent role for 5-HTR 1A in gating V1 plasticity [27].
We were not surprised by the observation that 5-HTR 1A expression levels remained close to the AMC level in the Mmz, as our lab previously described that well-defined levels of GABA A R α1 -mediated phasic inhibition, but not GABA A R δ1 -mediated tonic inhibition, are crucial for the establishment of the cross-modal reactivation of the Mmz in adult ME mice [53]. Our interpretation, that 5-HTR 1A is of central importance to ME-induced open-eye potentiation in the Bz but not to the late cross-modal plasticity phase in the Mmz, is further supported by the observed downregulation of 5-HTR 1A receptors in the Bz from the moment that open-eye potentiation is completed, and by the absence of suppressive drug-effects in the Mmz itself during the cross-modal phase. Indeed, the normal zif268-mRNA expression levels observed across the upper and lower cortical layers of the Mmz argue against a substantial contribution of this receptor subtype to the cross-modal take-over of the deprived cortical territory.
Antagonism of 5-HTR 2A and 5-HTR 3A to spatiotemporally control cross-modal brain plasticity In this study, next to WAY-100635, we also chronically injected ketanserin or ondansetron to respectively block 5-HTR 2A and 5-HTR 3A receptor function during the entire time window of cross-modal plasticity, and following a normal period of open-eye potentiation. Although applying a local slow drug-release approach might have allowed a better discrimination of true cortical events from for example subcortical contributions to the observed plasticity phenomena, we still chose the i.p. injection approach because this systemic approach holds a greater prediction value with regards to human clinical trials than an invasive cortical infusion system. Also, long-term cannula implantation may have led to cortical tissue damage and glial scar formation. Such side-effects would have influenced the local neuromodulator levels [63] hampering the read-out as well as the interpretation of the results.
By blocking the 5-HTR 2A receptor with ketanserin, we successfully suppressed adult cross-modal visual cortex reactivation across all layers of the Mmz. This observation accords with literature describing a role for 5-HTR 2A in compensatory cross-modal plasticity [32] and with our previous observations, that post-ME cross-modal reactivation of the Mmz only occurs under well-defined levels of GABA A R α1 -mediated phasic inhibition [53], which is considered to be strictly regulated by upstream 5-HTR 2A function [105].
5-HTR 3A interneurons constitute a heterogenous population. One type of interneurons dependent on 5-HT signaling are the Vasoactive Intestinal Peptide (VIP)expressing 5-HTR 3A interneurons. VIP-mediated disinhibition is a well-described mechanism in which VIP interneurons inhibit somatostatin and parvalbumin expressing interneurons and may underlie ME-induced cortical plasticity [44, 46-48, 114, 115]. VIP interneurons exert an important function in controlling sensory processing and are target cells of long-range projections that integrate sensory information originating from different brain regions [116,117]. This interneuron type thus constitutes an ideal candidate for 5-HT to shape cross-modal brain circuits and plasticity patterns via the 5-HTR 3A receptor. In a recent study [4], convergence of sensory and neuromodulatory information onto 5-HTR 3A cells was shown to vastly contribute to the shaping of critical period plasticity in the primary auditory cortex of the mouse. This research group discovered that a topographic map is formed already early in life, regulated by the non-VIP expressing 5-HTR 3A cell population in cortical layer I. As predicted based on the 5-HTR 3A cortical distribution pattern, which is predominantly in L1 of the cortex [4], our results indeed indicate that 5-HTR 3A receptor antagonism with ondansetron during the cross-modal reactivation phase, specifically suppressed the neuronal activity in the upper layers of the Mmz. It will be interesting to further investigate how and to what extent both the VIP and non-VIP 5-HTR 3A cells can spatiotemporally regulate adult cross-modal plasticity as this may enable steering brain plasticity towards the desired outcome as a future therapy, being complete functional recovery of the lost primary sense. Ex-vivo and in vivo electrophysiology and fast-scan voltammetry, should allow dissecting the local molecular cascade and cellular circuit involved in the different cortical plasticity phenomena.
A possible strategy to improve the success rate of bionic implants The success of bionic implants in restoring sensory function depends on our understanding of how the brain responds to sensory loss. Often, by the time the neuro-electric device is implanted, the brain has already compensated for the loss of sensory input through mechanisms of cross-modal plasticity, thereby possibly impairing the cortical translation of inputs, transmitted by the device, into meaningful sensory information [118]. During the last decades, the attempt to fully restore primary sensory function received much attention, giving rise to very promising neurobionics such as the cochlear implants to restore auditory function [119][120][121], visual prostheses [122][123][124][125][126] and brain-computer interfaces [127][128][129]. In addition, patients suffering from progressive vision loss, as caused by the neurodegenerative diseases Retinitis pigmentosa or age-related macular degeneration, can nowadays benefit from the recently developed, fully organic, subretinal prosthesis [130], which tackles some of the common mechanical, manufacturing and technical difficulties generally occurring in epiretinal [131], subretinal [132] and suprachoroidal [133] prosthetics.
Given the fact that 5-HT modulates sensory information processing and integration [134] and based on our findings that chronic, systemic administration of the 5-HTR 2A antagonist ketanserin and 5-HTR 3A antagonist ondansetron can suppress the cross-modal reorganization after partial vision loss in a cortical brain region-and layer-specific manner, we predict that a pharmacological monoamine-oriented strategy in combination with a bionic implant may significantly increase their success rate, as this approach would better allow to spatiotemporally control different forms of cortical plasticity that precede and co-occur with its functional integration.

Conclusions
We report ME-induced molecular and functional adaptations in the visual and somatosensory cortex of adult mice. We observed brain region-specific changes in 5-HT levels, presynaptic vMAT2 expression and postsynaptic 5-HT receptor levels in function of post-ME recovery time. We showed that 5-HTR 2A and 5-HTR 3A are involved in the cross-modal reactivation of the deprived visual cortex, as pharmacological antagonism of the 5-HTR 2A and 5-HTR 3A but not 5-HTR 1A receptor function hampered this plasticity process, specifically at the time when whiskers normally recruit the Mmz in adult ME mice. Our findings significantly add to the current understanding of the brain plasticity phenomena, occurring in response to chronic treatment with 5-HT-system targeting drugs. In future, promising strategies to restore primary sensory functions, might become spatiotemporally controllable through 5-HT-assisted neurobionics.