All chemical reagents were purchased from Sigma (St Louis, MO), unless otherwise stated.

Male Sprague–Dawley rats were housed in groups of three to four animals in their home cages and kept under a 12-h light/dark cycle (light on at 7 am) cycle at 22 ± 1°C. Food and water were available ad libitum. At the beginning of the experiment, rats were 78 days old, and their average weight was 266.5 ± 42 g (control group) and 269 ± 40 g (fluoxetine group). At the end of the treatment period, rats were 106 days old and weighted 392 ± 51 and 372 ± 53 g on average, respectively.

As in our previous studies, fluoxetine at a dose of 0.7 mg/kg (Ely-Lilly Co., Indianapolis, USA) or saline solution (control) was administered by intraperitoneal (i.p.) injection once daily between 9 and 10 am for 28 days. This dose induces increased BDNF expression and TrkB-dependent signaling at synapses, as well as remodeling glutamate neurotransmission and GluN2A over GluN2B switch of NMDA-R subunits (Wyneken et al., 2006; Ampuero et al., 2010; Rubio et al., 2013). This study was carried out in accordance with the Guide for the Care and the Use of Laboratory Animals from the National Institutes of Health (Ed 8; http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf) and the Bioethical Guidelines for the Use of Laboratory Animals from Universidad de los Andes Bioethical Committee. The protocol was supervised by the Universidad de los Andes Bioethical Committee.

To perform Golgi staining, the adult rats were sacrificed under ketamine (50 mg/kg) and xylazine (5 mg/kg) anesthesia, 24 h following the last fluoxetine or saline dose (n = 10–11 per group). The rats were perfused intracardially with saline solution followed by 300 ml of 4% buffered paraformaldehyde solution. Immediately after perfusion, the brains were removed and processed for Golgi staining using GolgiStainTM kit as previously described (Ampuero et al., 2007).

Coronal sectioning at 150 µm was performed to analyze dendritic morphology. The morphometric analysis of pyramidal neurons was restricted to stereotaxic coordinates (in mm): for the prelimbic (PrL) cortex to: interaural 13.20 and Bregma −4.20, for the secondary motor cortex (M2) to: interaural 5.86 and Bregma −3.14, for retrosplenial granular b cortex (RSGb) to: interaural 5.20 and Bregma −3.8 mm and for primary auditory cortex (AUD1) to: interaural 4.70 and Bregma −4.30 (Paxinos and Watson, 1998).

Golgi-stained images were obtained with a Nikon Eclipse TE2000-U inverted epifluorescence microscope with a Plan Fluor 20x/1.25 numeric aperture attached to a cooled monochrome camera DS-2MBWc.

Pyramidal neurons were defined by the presence of basilar dendrites, a distinctive, single apical dendrite, and dendritic spines (Spruston, 2008). The observer, blind to experimental conditions, selected randomly at least 10 neurons from each animal that fulfilled the following selection criteria: 1) absence of truncated dendrites, 2) consistent and dark impregnation along the entire dendritic field, and 3) spatial separation from neighboring impregnated neurons to avoid overlap. Camera lucida tracings from selected neurons (500×, BH-2, Olympus Co., Tokyo, Japan) were performed and then scanned (eight-bit grayscale TIFF images with 1,200 dpi resolution; EPSON ES-1000C) along with a calibrated scale for subsequent computerized image analysis. For morphometric analysis of digitized images, we used Image J software (National Institutes of Health). In each selected neuron, the total, basal, and apical dendritic lengths were determined.

For in utero electroporation, five pregnant young female rats at embryonic day 16.5 were anesthetized with xylazine (5 mg/kg) and ketamine (50 mg/kg). After shaving the rat’s belly at the location of the desired incision and disinfecting the abdominal skin with povidone solution, an approximately 2-cm midline skin incision was made followed by another incision along the linea alba. Then, both uterine horns were drawn out through the incision. To inject the plasmid, glass capillaries (pulled with the P97 Pipet Puller, Sutter Instruments, USA) were filled with the plasmid solution (1 μg/μl), mixed with Fast Green (<0.75 mg/ml, Sigma Aldrich, USA), and delivered into the left lateral ventricle with a pressure Pico pump (PV830, World Precision Instruments, USA). The plasmid containing the CAG promoter (i.e., driven by the strong ubiquitous promoter cytomegalovirus early enhancer and chicken beta actin) and the monomeric red fluorescent protein (mRFP) sequence, kindly shared by Joseph LoTurco, was used (LoTurco et al., 2009) ( Supplementary Figure 1 ).

Electric pulses were applied with an electroporation system built with a power supply, a resistor, pulse/charge power switch, capacitor, and a foot pedal for providing the pulse. To introduce the plasmid into a larger region of the cortex, the cathode was placed on the location corresponding to the left temporal lobe, and the anode was placed on the location corresponding to the right temporal lobe. Voltage pulses were adjusted to the developing stage of the fetuses (65 mV for E16). Only one pulse lasting 3 s was applied. The embryos closest to the cervix were not injected nor electroporated because the possible death of these animals could block the delivery. After the procedure was completed, the two horns were reinserted into the abdominal cavity with an additional 5 ml of isotonic saline solution, and then, the abdominal wall and skin were closed with nylon and silk sutures, respectively. After surgery, the rats gave birth normally. Only male pups (n = 18) underwent further induction and treatment to circumvent hormonal differences influencing the results. At 78 days of age rats began fluoxetine treatment. Overall, the effectiveness of the electroporation procedure was 41%, and from these, 30% expressed mRFP in the primary auditory cortex (AUD1) resulting in six rats for 3-dimensional analysis.

To prepare brain slices, animals were quickly decapitated and brains removed, immersed in ice-cold dissection buffer (in mM; 212.7 sucrose; 5 KCl; 1.25 NaH₂PO₄; 3 MgSO₄; 1 CaCl₂; 26 NaHCO₃; 10 glucose; pH 7.4), and coronal slices (400 µm thickness) were obtained using a vibratome (Ted Pella Inc., USA). Slices were transferred to an interface chamber containing artificial cerebrospinal fluid (aCSF) saturated with 95% O₂–5% CO₂ at 36°C, left at these conditions for 45 min, and then maintained at 24°C for 1 h.

Field excitatory postsynaptic potentials (fEPSP) recorded from auditory cortex were evoked as described in Hefti and Smith (Hefti and Smith, 2000). Briefly, electrical stimulation (biphasic, constant current, 200 ms stimuli) was delivered every 15 s to the layer I in the primary auditory cortex of control and fluoxetine-injected rat brain slices, using a concentric bipolar electrode (FHC Corporate and Manufacturing, Bowdoin, ME, USA) and recorded in layers II–III. Recording electrodes comprised glass micropipettes (1–3 MΩ) filled with aCSF. Baseline recordings were performed using oxygenated (5% CO₂–95% O₂) Mg²⁺-free aCSF at 32°C on the recording chamber continually perfused at a flow of 2 ml/min. After 10 min, the recording solution was replaced by Mg²⁺-free aCSF containing 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 10 µM Picrotoxin, to block α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and gamma-aminobutyric acid receptors (GABA_A) receptors. After incubation of 20 min, the NMDA-R-mediated component of the fEPSP was recorded for 10 additional minutes. Finally, the recording chamber solution was replaced a second time by an aCSF containing 20 µM CNQX, 10 µM Picrotoxin, and 150 nM ifenprodil, to block GluN2B-containing NMDA receptors, and incubated again for 20 min to record the remaining GluN2A-mediated NMDA-R component for 10 additional minutes. Data recorded from pharmacological experiments were acquired using an extracellular amplifier (Model EX 4-400 Differential Extracellular Amplifier, Dagan Corporation, USA) and a data acquisition board (National Instruments, USA) controlled through Igor Pro software (WaveMetrics Inc, Lake Oswego, Oregon, USA). All the electrophysiological experiments were performed using tissues from at least eight different animals for each condition.

To determine differences between fEPSP obtained from control and fluoxetine-treated slices, acquired data were transferred to the scientific graphing and data analysis software Origin 9.0. Thereafter, the rising and decay portions of each curve were independently adjusted to a Boltzmann curve, obtaining from the fitted rising part the peak and rise time parameters, whereas decay time was obtained from the latter. The area under curve was calculated using the first 30 ms of the curve, taking as time 0 the beginning of the fEPSP curve.

The values are shown as the means ± SEM. Statistical significance was evaluated by Mann–Whitney U-test for morphological data (Golgi staining and 3D reconstruction) and two way-ANOVA followed by post hoc Bonferroni correction for electrophysiological data (GraphPad Prism software, San Diego, USA), as indicated in the figure legends. A probability level of 0.05 or less was considered significant.

We had previously shown that repetitive fluoxetine treatment augmented the spine density and the number of mature, mushroom-type spines and favored GluN2A-containing NMDARs and GluA2-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPA-Rs) (Ampuero et al., 2010; Ampuero et al., 2013; Rubio et al., 2013). Consistently, this switch in glutamate receptor subunits was associated with decreased calcium influx, synaptic plasticity, and hippocampus-dependent learning (Ampuero et al., 2013; Rubio et al., 2013; Beshara et al., 2016). Taking into consideration that glutamate receptor subunits determine dendritic morphology in neurons, we then examined morphological changes in dendritic length and complexity (Bustos et al., 2017; Keith et al., 2019). Using the Golgi staining method, we measured the dendritic length of apical and basal branches of pyramidal neurons in several cortical areas. In two limbic cortices, e.g., the prelimbic cortex (PrL) and retrosplenial granular b cortex (RSGb), a decrease in the total dendritic length after fluoxetine treatment was observed, and this resulted from a reduction in the basal dendritic lengths in layer II–III neurons of the PrL cortex (in µm, control = 998 ± 53; fluoxetine = 802 ± 108, p = 0.045) and in layer V neurons (in µm, control = 986 ± 204; fluoxetine = 624 ± 72, p = 0.038). Similarly, the basal dendritic length decreased in layer V neurons of the RSGb cortex (in µm, control = 821 ± 134; fluoxetine = 499 ± 54, p = 0.020) ( Figure 1 and Supplementary Table 1 ). In the RSGb, we did not obtain reliable staining of layer II–III neurons. Similarly, in cortical areas not directly involved in antidepressant action, e.g., pyramidal neurons of the II–III layer of the secondary motor cortex (M2) and primary auditory cortex (AUD1), a reduction in the dendritic length was shown: in M2, apical branches were reduced (in µm, control = 1,043 ± 129; fluoxetine = 598 ± 60, p = 0.014), while in AUD1, the basal dendritic length was affected (in µm, control = 1,661 ± 138; fluoxetine = 1,086 ± 99, p = 0.0025) ( Figure 2 and Supplementary Table 1 ).

Although the Golgi method has been used for decades to assess dendritic arbor morphology, this technique does not allow a 3-dimensional reconstruction. We thus used mRFP expression in cortical neurons to analyze the dendritic arbor of pyramidal neurons from layer V of AUD1 ( Figure 3 and Supplementary Table 2 ). In Figure 3A , an overview of the electroporation procedure, including expression of the fluorescent protein in isolated neurons, is shown. In Figure 3B , a representative image of the 3-dimensional reconstructed branches of neurons obtained from control or repetitive fluoxetine-treated animals is shown. Representative images at different levels of analysis are indicated, showing the raw data in the upper panel, the skeletonized data in the middle panel, and the reduced skeleton in the lower panel. The analysis of the reduced skeleton revealed a significant reduction in both the apical (in µm, control = 2,739 ± 272; fluoxetine = 1,460 ± 315, p = 0.008) and the basal (in µm, control = 3,178 ± 302; fluoxetine = 1,786 ± 242, p = 0.008) dendritic length after fluoxetine ( Figure 3C ). When comparing both experimental approaches to assess the dendritic length, we found (as expected) greater dendritic length when the 3-dimensional reconstruction was performed, as well as a significant reduction of basal dendrites lengths after fluoxetine, in contrast to the Golgi method with which this difference was not significant and thus was not detectable with the 2-dimensional analysis. Similarly, decreased complexity of the dendritic tree was detected after fluoxetine when using the 3-dimensional method but not the Golgi method ( Figure 3D ). This resulted from a decrease in the number of secondary and tertiary processes, while primary processes were not affected. This result is consistent with the morphological consequences observed in neuronal cultures in which GluN2A/GluN2B ratios are manipulated (Bustos et al., 2014; Bustos et al., 2017). Now, we also show that repetitive fluoxetine treatment induces a general reduction in the dendritic arbor in limbic and nonlimbic cortical areas, which is indicative of increased maturation. These results are also consistent with previous observations of increased dendritic spine density and of mushroom-type synapses after fluoxetine (Ampuero et al., 2010; Rubio et al., 2013).

To assess whether the evoked field potentials in the auditory cortex are consistent with increased synaptic GluN2A levels (Ampuero et al., 2010), we evaluated the NMDA-mediated responses by blocking AMPA receptors with CNQX. Additionally, we blocked GluN2B-containing NMDA-Rs with ifenprodil in a sequential manner, using slices obtained from control and fluoxetine-treated animals ( Supplementary Table 3 ). Figure 4A shows representative traces of these experimental conditions, while in Figure 4B–E , different parameters of the fEPSP are evaluated. The peak amplitude of the total or NMDA-mediated response did not change ( Figure 4B ). However, ifenprodil was ineffective in reducing the peak amplitude of the field excitatory postsynaptic potential (fEPSP) after fluoxetine treatment (in mV, control = −0.247 ± 0.066; fluoxetine = −0.499 ± 0.087, p = 0.038 in the condition CNQX plus ifenprodil), consistent with a GluN2B-to-GluN2A switch. In the same line, control responses were blocked by CNQX plus ifenprodil (in mV, aCSF = −0.758 ± 0.019; CNQX + ifen = −0.247 ± 0.066; p = 0.017), while in slices of fluoxetine-treated rats, CNQX plus ifenprodil did not block the response with respect to aCSF. Moreover, Figure 4C shows a significant decrease in the rise time when the AMPA and GluN2B components of the fEPSP were removed (i.e., in the presence of CNQX plus ifenprodil) (in ms, control = 0.264 ± 0.02; fluoxetine = 0.17 ± 0.02, p = 0.008), consistent with an increase in GluN2A receptor subtypes, which are known to present faster rise time kinetics than other GluN2 subunits (Hansen et al., 2018). Interestingly, the integrated response over 30 ms (area under the curve, mV*ms) increased in all conditions after fluoxetine compared to control animals ( Figure 4D ). In this case, the total aCSF response was control = 3.4 ± 0.3; fluoxetine = 5.0 ± 0.6 (p = 0.029); the NMDA-mediated response after AMPA-R blockade was of control = 1.2 ± 0.2; fluoxetine = 3.0 ± 0.5 (p = 0.029); and the GluN2A-mediated response was of control = 0.80 ± 0.1; fluoxetine = 1.5 ± 0.3 (p = 0.031). These results are consistent with previous data showing a larger proportion of mushroom-like spines and increased spine density after fluoxetine administration (Ampuero et al., 2010). In addition, the percentage of blockade of the integrated NMDA-mediated response (i.e., in the presence of CNQX) after ifenprodil decreased in treated animals, revealing a relative reduction in the GluN2B-mediated response ( Figure 4E ). The following percentages of blockade were observed: in control slices, the blockade by CNQX was of 37.93 ± 4.9% and increased to 72.86 ± 14% when ifenprodil was added (p = 0.035). In contrast, in slices from fluoxetine-treated rats, blockade by CNQX was 57.68 ± 3.8%, and after ifenprodil, it was only 50.79 ± 4.5%, revealing (as in Figure 4B ) a failure of ifenprodil to block the synaptic response; this is consistent with a reduction in the GluN2B-mediated response after fluoxetine treatment.

We and several other groups had previously reported that fluoxetine increased spine density as well as the proportion of mushroom-type (i.e., mature) spines in several forebrain regions (Hajszan et al., 2005; Guirado et al., 2009; Ampuero et al., 2010; Kitahara et al., 2016). A spine increment in the somatosensory cortex with the use of the rapid acting antidepressant ketamine has been proposed as a common and necessary mechanism involved in effective antidepressant treatment (Pryazhnikov et al., 2018). However, the functional consequences of such changes for neuronal network activity remain uncertain. In our experiments, we also observed increased spine density but decreased dendritic length. To estimate whether fluoxetine changes the total number of synapses, we calculated whether a mean increase of 21.4 ± 3.4% in spine density (Ampuero et al., 2010; Rubio et al., 2013) and a mean decrease in the dendritic length of 25.7 or 41.3% (Golgi method or 3-dimensional reconstruction, respectively) affects the total synapse number. With both estimations, it could be concluded that the number of total synapses does not change (Golgi method or 3-dimensional reconstruction, respectively). Note that as spine density varies along primary and higher order dendrites, these estimations are simple approximations based on the mean percentage of spine increase. However, as reported by us and by others, the synaptic contacts shift to a more stable type (i.e., mushroom type), putatively affecting in a positive manner the strength of certain synaptic connections but causing the pruning of others.

In patients, it has been shown that auditory cortex activation and auditory perception and processing are impaired in depression (Zwanzger et al., 2012; Bonetti et al., 2017; Zweerings et al., 2019). Moreover, age-related hearing loss has been associated with late life depression (Husain et al., 2014; Rutherford et al., 2018; Scinicariello et al., 2019), and in animals, chronic restraint stress decreased glucose metabolism in the auditory cortex (Wei et al., 2018). It would be relevant to know whether antidepressant medication reverts auditory dysfunction. In our study, we addressed the effects of fluoxetine in naïve (nondepressed) rats.

The electrophysiological recordings of fEPSPs in the auditory cortex were compatible with increased glutamatergic synaptic response and increased GluN2A levels in synapses (Ampuero et al., 2010; Rubio et al., 2013). In fact, the amplitude of the evoked and integrated response increased in all the examined conditions (total, NMDA-mediated and GluN2A-mediated), revealing that, independent of the postsynaptic glutamate receptor composition, the glutamatergic response increased. In addition, the ineffectiveness of ifenprodil in reducing the NMDA-mediated response after fluoxetine administration indicated a loss in the contribution of GluN2B receptors to synaptic fEPSPs, and the fEPSP rise time decreased when the GluN2B component was removed with ifenprodil, unmasking in such a way the characteristic GluN2A fast rise time kinetics, as reported previously (Amico-Ruvio et al., 2012).

Consistent with the idea of more mature and less plastic synapses at AUD1 intracortical synapses, the field postsynaptic potential augmented after fluoxetine while long-term potentiation (LTP) was impaired (Dringenberg et al., 2014). Similar effects of fluoxetine were observed by us and others in the hippocampal CA1 region (Rubio et al., 2013; Van Dyke et al., 2019). These changes, indicating more mature but less plastic synapses, were corroborated using glutamatergic markers which also extended to GABAergic markers of synaptic maturation in the visual cortex (Beshara et al., 2016). We postulate that the plasticity-inducing effect of fluoxetine described by others (Maya Vetencourt et al., 2008; Kobayashi et al., 2010; Guirado et al., 2014; Ruiz-Perera et al., 2015) might be compatible, at different time points, with the synaptic stabilization and dendritic pruning observed in our rat model. However, it is not yet known which temporal changes during the treatment period may sequentially induce a plasticity period, followed by a stabilization period characterized by a “mature” network. The time course of these changes might be related to region-specific differences in serotonin receptor expression types and/or levels (Charnay and Leger, 2010). Auditory processing is impaired in major depression, and this is reverted by SSRIs (Frodl, 2017). We have focused here on the auditory cortex because the participation of serotonin in the auditory processing is well documented, and it can be modulated after repetitive administration of the SSRI fluoxetine (Ji and Suga, 2007; Hurley and Hall, 2011).

Our results indicate that the SSRI fluoxetine induced dendritic and synaptic pruning in areas of the cerebral cortex not directly implicated in the main symptoms of depression. In principle, these pro-maturational effects could be useful for restoring stable synaptic networks in pathologies where a shift towards immature circuits and/or to GluN2B-dependent neurotoxicity occurs. Thus, in animal models of temporal lobe epilepsy, the GluN2B/GluN2A ratio increased in several forebrain regions (Amakhin et al., 2017; Zubareva et al., 2018), and synapses enriched in GluN2B subunits favored seizure susceptibility (Okuda et al., 2017), whereas blockade of GluN2B receptors during experimental status epilepticus with compounds that included the rapid acting antidepressant ketamine (a non-selective NMDA-R antagonist) reduced neuronal damage (Loss et al., 2019). Moreover, fluoxetine and other SSRIs are considered to be safe in the treatment of depression in epileptic patients, although exceptions (e.g., in some epilepsy subtypes) to this general rule might exist (Kanner, 2016a; Kanner, 2016b). However, the use of fluoxetine to favor GluN2A-associated structural and functional synaptic changes in nondepressed epileptic patients or even in preventing oxidative stress associated to neurological disorders is open to discussion and additional experimental work (Zhu et al., 2018).

Interestingly, the neuroprotective potential of direct and selective GluN2B blockade has extensively been addressed in several brain disorders (Gielen et al., 2009; Mony et al., 2009). Moreover, uncoupling of GluN2B C-terminal-dependent signaling or selective GluN2B blockade has antidepressant effects (Li et al., 2018) and might exert neuroprotective properties in Alzheimer’s disease, Huntington’s disease, and after stroke (Dau et al., 2014; Wu and Tymianski, 2018; Xu et al., 2019). In all these cases, fluoxetine could complement therapeutic strategies aimed at preventing or reducing neurotoxicity. In additional observations, fluoxetine reduces β-amyloid levels and toxicity, an effect that may depend on subunit-specific NMDA-R blockade and increased GluN2A-dependent brain-derived neurotrophic factor (BDNF) and transforming-growth-factor-β1 (TGF-β1) signaling (Tucci et al., 2014; Schiavone et al., 2017; Caraci et al., 2018). Moreover, fluoxetine displays direct GluN2B antagonism, an effect that results convergent with the GluN2B to GluN2A subunit switch and the morphological consequences described by us (Kiss et al., 2012). This selectivity would be advantageous over the nonselective NMDA-R antagonist ketamine, a promising novel antidepressant drug with unwanted side effects that have yet to be overcome (Duman, 2018; Orhurhu et al., 2019). Interestingly, several mechanisms underlying the therapeutic action of ketamine are shared with fluoxetine, but with a different time scales. In such a way, GluN2B subunits are essential for the antidepressant effect of ketamine and the downstream activation of mTOR signaling (Wang et al., 2011; Miller et al., 2014) and ketamine regulates positively spinogenesis (Moda-Sava et al., 2019).

Taken together, the morphological changes observed by us after repetitive fluoxetine administration, consistent with a GluN2B subunit reduction in several forebrain areas, may have promising applications in brain disorders beyond depression.