Increasing Adiponectin Signaling by Sub-Chronic AdipoRon Treatment Elicits Antidepressant- and Anxiolytic-Like Effects Independent of Changes in Hippocampal Plasticity

(1) Background: Adiponectin is an adipocyte-secreted hormone that has antidepressant- and anxiolytic-like effects in preclinical studies. Here, we investigated the antidepressant- and anxiolytic-like effects of sub-chronic treatment with AdipoRon, an adiponectin receptor agonist, and its potential linkage to changes in hippocampal adult neurogenesis and synaptic plasticity. (2) Methods: Different cohorts of wild-type C57BL/6J and CamKIIα-Cre male mice were treated with sub-chronic (7 days) AdipoRon, followed by behavioral, molecular, and electrophysiological experiments. (3) Results: 7-day AdipoRon treatment elicited antidepressant- and anxiolytic-like effects but did not affect hippocampal neurogenesis. AdipoRon treatment reduced hippocampal brain-derived neurotrophic factor (BDNF) levels, neuronal activation in the ventral dentate gyrus, and long-term potentiation of the perforant path. The knockdown of N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B in the ventral hippocampus did not affect the antidepressant- and anxiolytic-like effects of AdipoRon. (4) Conclusions: Increasing adiponectin signaling through sub-chronic AdipoRon treatment results in antidepressant- and anxiolytic-like effects independent of changes in hippocampal structural and synaptic function.


Introduction
Depression has a lifetime prevalence of nearly 15% [1] and is the leading cause of disability in the world [2]. Even though antidepressants have been developed over the past 60 years [3,4], advancements were made in terms of increased tolerability rather than efficacy [5,6]. Noteworthy, monoaminergic antidepressants' efficacy hardly goes beyond 50% [5], with a delayed therapeutic onset ranging from 4 to 8 weeks [5]. Adiponectin is the most abundant plasma protein [7,8] that, when downregulated, has been associated with depression and anxiety-related disorders [9][10][11]. On the other hand, increasing adiponectin signaling in preclinical studies leads to rapid antidepressant-and anxiolyticlike effects [12][13][14][15][16]. Despite that, the potential antidepressant and anxiolytic properties of AdipoRon, a potent adiponectin receptor agonist [17], have been scarcely explored. Many adipokines can cross the blood-brain barrier (BBB) and regulate mood and cognition [18]. The adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) are widely expressed in brain regions associated with emotional regulation, including the hippocampus, amygdala, and medial prefrontal cortex (mPFC) [16,19,20]. When peripheral adiponectin levels rise in response to energetic challenges, such as physical exercise [21,22], the lowmolecular-weight isoforms of adiponectin cross the BBB [23] and modulate the activity of such brain regions [12,14]. We have previously shown that adiponectin signaling is a key mediator of the neurogenic and antidepressant properties of voluntary wheel running [12,24]. Moreover, our group has also shown that chronic treatment with AdipoRon, a synthetic and orally available adiponectin receptor agonist [17], increases hippocampal neurogenesis in a dose-dependent manner [25] and mimics the effects of physical exercise on restoring hippocampal neuroplasticity and cognitive deficits in diabetic mice [26].
AdipoRon treatment for three weeks prevents the depressive phenotype in association with increased hippocampal neurogenesis and reduced neuroinflammation in corticosteronetreated mice [13]. Additionally, increased adiponectin signaling has been associated with antidepressant-and anxiolytic-like effects in shorter time windows [16,27,28]. Increasing activity of the peroxisome proliferator-activated receptor-γ (PPARγ), an upstream positive regulator of adiponectin synthesis, through systemic administration of rosiglitazone (a PPARγ agonist) increases peripheral adiponectin levels and reduces antidepressant-and anxietylike behavior in 24 h, which is not observed in adiponectin knockout mice [27]. Moreover, infusion of adiponectin in the ventricles [16] or ventral tegmental area [28] results in rapid antidepressant and anxiolytic responses. Such pre-clinical evidence holds the promise for exogenously increasing adiponectin signaling through its receptor agonist (i.e., AdipoRon) as a more fast-acting and efficient antidepressant and anxiolytic intervention compared to classical serotonergic drugs [9].
The hippocampus has been long implicated in depression pathogenesis [29][30][31]. The N-methyl-D-aspartate (NMDA) receptors play a significant role in hippocampal information processing [32][33][34], and NMDA receptor-associated structural and functional changes in the ventral hippocampal plasticity have been associated with mood impairments [35,36]. Although previous work has linked adiponectin's therapeutic effects with increased hippocampal neuroplasticity [12,13,[24][25][26], new evidence suggests that short-term antidepressant response might not rely on the hippocampus [37][38][39]. Inactivating the ventral hippocampus does not affect the ketamine's rapid antidepressant properties [37]. Moreover, acute activation of the ventral hippocampus does not elicit antidepressant or anxiolytic responses [37,39]. Common serotonergic antidepressants normally require chronic treatment (14 days) to elicit a therapeutic response in stressed mice [40]. Since increasing adiponectin signaling has been previously shown to elicit a rapid antidepressant response [12][13][14][15][16], we aimed at investigating whether short-term (7-day) AdipoRon treatment would elicit antidepressant-and anxiolytic-like effects in a shorter treatment period than the conventional antidepressants and to test whether these effects would be linked to changes in hippocampal neuroplasticity.

Animals and Experimental Design
All experimental procedures were approved and followed the Animal Subjects Ethics Sub-Committee's guidelines at The Hong Kong Polytechnic University. Eight-week-old C57BL/6J and CamKIIα-Cre male mice with the same genetic background were grouphoused (3-5 per cage) in a holding room with controlled temperature (22 ± 2 • C) and kept under a 12 h light-dark cycle. Mice were fed with standard chow and water ad libitum and could habituate to the housing conditions for 7 days before experiments started. Wild-type animals were randomly assigned to a 7-day treatment protocol with AdipoRon (20 mg/kg, i.p.) or vehicle. The day after the last injection, different cohorts of animals were submitted to behavioral (n = 11-12/group), molecular (n = 5-10/group), or electrophysiological (n = 5/group) experiments (Figure 1a). For knockdown experiments, CamKIIα-Cre mice were randomly assigned to intrahippocampal injection of vehicle (PBS, Control) or adeno-associated viruses (AAV) driving the expression of shRNA targeting the N-methyl-D-aspartate (NMDA) subunits GluN2A or GluN2B (n = 7-9/group). After fourteen days of recovery from the surgery, animals were submitted to AdipoRon treatment followed by a battery of behavioral tests (Figure 1b).

Animals and Experimental Design
All experimental procedures were approved and followed the Animal Subjects Et Sub-Committee's guidelines at The Hong Kong Polytechnic University. Eight-week-C57BL/6J and CamKIIα-Cre male mice with the same genetic background were gro housed (3-5 per cage) in a holding room with controlled temperature (22 ± 2 °C) and k under a 12 h light-dark cycle. Mice were fed with standard chow and water ad libit and could habituate to the housing conditions for 7 days before experiments started. W type animals were randomly assigned to a 7-day treatment protocol with AdipoRon mg/kg, i.p.) or vehicle. The day after the last injection, different cohorts of animals w submitted to behavioral (n = 11-12/group), molecular (n = 5-10/group), electrophysiological (n = 5/group) experiments (Figure 1a). For knockdown experime CamKIIα-Cre mice were randomly assigned to intrahippocampal injection of veh (PBS, Control) or adeno-associated viruses (AAV) driving the expression of shR targeting the N-methyl-D-aspartate (NMDA) subunits GluN2A or GluN2B (n = 9/group). After fourteen days of recovery from the surgery, animals were submitted AdipoRon treatment followed by a battery of behavioral tests (Figure 1b). (a) Wild-type animals were randomly assigned to a 7-day treatm protocol with AdipoRon (20 mg/kg, i.p.) or vehicle. The day after the last injection, different coh of animals were subjected to behavioral, molecular, or neurophysiological experiments. The co subjected to behavioral analysis received an AdipoRon injection after the last behavioral test before euthanasia. (b) CamKIIα-Cre mice were randomly assigned to microsurgery intrahippocampal injection of PBS (control) or adeno-associated viruses (AAV) driving expression of shRNA targeting the N-methyl-D-aspartate (NMDA) subunits NR2A or NR Fourteen days after surgery, animals were submitted to AdipoRon treatment followed by a bat of behavioral tests.

Drugs
AdipoRon (MedChemExpress, Princeton, NJ, USA) was dissolved in 0 carboxymethylcellulose sodium salt (CMC) (Sigma-Aldrich, St. Loius, MO, USA) at 70 in 5% DMSO, as previously performed (26). Animals were treated with AdipoRon mg/kg, i.p.) or vehicle (CMC in 5% DMSO). For adult neurogenesis study, 5-bromo deoxyuridine (BrdU, Abcam, Cambridge, UK) was dissolved in 0.9% saline solution administered for three consecutive days (100 mg/kg) before AdipoRon treatment. (a) Wild-type animals were randomly assigned to a 7-day treatment protocol with AdipoRon (20 mg/kg, i.p.) or vehicle. The day after the last injection, different cohorts of animals were subjected to behavioral, molecular, or neurophysiological experiments. The cohort subjected to behavioral analysis received an AdipoRon injection after the last behavioral test, 2 h before euthanasia. (b) CamKIIα-Cre mice were randomly assigned to microsurgery for intrahippocampal injection of PBS (control) or adeno-associated viruses (AAV) driving the expression of shRNA targeting the N-methyl-D-aspartate (NMDA) subunits NR2A or NR2B. Fourteen days after surgery, animals were submitted to AdipoRon treatment followed by a battery of behavioral tests.

Behavioral Tests
Behavioral tests were performed during the afternoon period (1-5 PM) in a procedure room with controlled temperature (22 ± 2 • C) and bright light by an experienced researcher blinded to the treatment conditions. The behavioral analysis was equally performed by a blinded researcher. Animals could habituate to the room conditions for at least 2 h before experiments started.

Sucrose Preference Test
Sucrose preference, a behavior that relies on the rodent's natural preference for sweetened solutions and of relevance for the screening of antidepressant-like properties [41], was evaluated by the sucrose preference test (SPT) as previously performed [12]. Briefly, mice were habituated to having access to two water bottles for two consecutive days before the test. On the testing day, mice were single housed for 24 h and allowed access to two standard bottles of water, one containing a 2% sucrose solution (w/v) and another filled with filtered tap water. The positions of the two bottles were swapped after 12 h to avoid any selection bias effect. Liquid consumption was measured by weighing the bottles before and after the test. Sucrose preference was determined by the ratio of sucrose solution intake in comparison to total liquid consumption over the 24 h period.

Novelty-Suppressed Feeding Test
The novelty-suppressed feeding test (NSFT) was used to evaluate the expression of approach-avoidance behaviors that are of relevance for the screening of anxiety-like properties, as previously described [42]. Mice were deprived of food for 16 h, including the overnight period, before the test. A squared arena (L × W × H: 40 × 40 × 30 cm) was used as a novel environment, with the floor covered by 2 cm of wooden bedding and a single food pellet placed on a filter paper at its center. On the testing day, one mouse at a time was placed in the arena randomly facing one of its corners and let explore it freely for up to 10 min, while the latency to feed was recorded by an experienced researcher in real-time. Once the animal bit the food pellet, or after 10 min had passed, the test ended. Wooden bedding was changed, and the arena was cleaned with 70% ethanol solution in between animals.

Light-Dark Box Test
The light-dark box (LDB) test was used as an additional measure of approachavoidance behavior [43]. The LDB consisted of an apparatus with two chambers of equal dimensions (L × W × H: 20 × 40 × 30 cm) but enclosed either with transparent (light compartment) or black Plexiglas (dark compartment). Both compartments were interconnected by a sliding door. The light compartment was brightly illuminated with a table lamp, while the dark compartment was covered by a removable ceiling. During the test, a mouse was introduced into the black compartment and let freely explore both compartments for 5 min once the interconnecting door was opened [28]. The entire session was video recorded, and the number of visits and the time spent in the light compartment were manually recorded. Entering and exiting compartments were considered when the mouse crossed the door into the opposite compartment with the four paws. The apparatus was thoroughly cleaned with 70% ethanol solution in between animals.

Forced Swim Test
To evaluate behavioral despair, a behavior that has been commonly associated with antidepressant-like properties [44], mice were subjected to the forced swim test (FST) [45]. Mice were individually placed in a transparent cylinder (height: 30 cm; diameter: 15 cm) filled with two-thirds of water (24 ± 2 • C) for 6 min, as previously performed [12]. The session was video recorded and the duration the animal spent immobile in the last 4 min of the test was manually scored by an experienced researcher. Immobility was defined as no limb or body movements except those necessary for keeping the head above water.

Immunofluorescence Staining
Immunofluorescence double staining of c-Fos, an immediate early gene marker induced by neural activation [46,47], and NeuN, a neuronal marker [48], was performed in free-floating brain sections. Sections were rinsed in 0.01 M PBS, incubated with 10 mM citric acid buffer for 10 min at 95 • C, and rinsed with 2N HCl for 10 min at 37 • C to unmask antigen. After PBS wash, sections were incubated overnight with mouse anti-c-Fos (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-NeuN antibodies (1:1000, Millipore Sigma, Burlington, MA, USA) at room temperature, followed by a 2 h incubation with goat anti-mouse IgG AlexaFluor-568 and goat anti-rabbit IgG AlexaFluor-488 (1:200, Invitrogen, Waltham, MA, USA). After PBS washes, the sections were coverslipped with fluorescent mounting medium (Dako, Santa Clara, CA, USA).
Immunofluorescence double staining of BrdU as a marker of surviving newborn cells [49] and doublecortin (DCX), a marker of immature neurons [50], was performed following the same steps. After antigen retrieval, sections were incubated overnight at room temperature with rat anti-BrdU

Immunoperoxidase Staining
Immunoperoxidase staining of Ki-67, a proliferative cellular marker [51], and DCX was performed in free-floating brain sections as previously performed [25]. Briefly, sections were rinsed in 0.01 M PBS and incubated with 10 mM citric acid buffer for 10 min at 95 • C for antigen unmasking. After PBS wash, sections were incubated overnight at room temperature with rabbit anti-Ki-67 (1:1000, Abcam, Cambridge, UK) or mouse anti-DCX (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). After PBS wash, sections were then incubated for 2 h with goat anti-rabbit or goat anti-mouse (1:200; Vector, Newark, CA, USA), followed by a 2 h incubation with Vectastain Elite ABC solution (Vector, Newark, CA, USA) and 5-10 min in 3,3 -diaminobenzidine (DAB) peroxidase kit (Vector, Newark, CA, USA) for visualization. Sections were then mounted onto coated slides, dehydrated with ethanol and xylene baths, and coverslipped with DPX Mountant.

Cell Quantification
Immunofluorescence images were obtained using a confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) at 200× magnification in intervals of 4 µm in the z-focal plane with 256 × 256 pixels resolution. Images were z-stacked with maximum intensity projection using the ZEN (blue edition) software (Carl Zeiss Microscopy, Jena, Germany). Immunoperoxidase images were visualized at 400× using a microscope (Nikon series Eclipse H600L, Tokyo, Japan).
c-Fos+ cells were counted automatically in ImageJ (National Institutes of Health, Bethesda, MD, USA) using the "Analyze particles" function after background subtraction, as previously described [42]. After confirming co-labeling with NeuN, c-Fos+ cell count per unit area (mm2) in the DG was obtained through the average of 3 to 4 sections in the dorsal (−1.34 to −2.54 mm from Bregma) and the ventral hippocampus (−2.54 to −3.80 mm from Bregma) per animal.
BrdU-, Ki-67-, and DCX-positive cells located in the dentate subgranular zone and granular cell layer were manually counted in 5 to 6 coronal sections per animal along the anteroposterior hippocampal plane (−1.34 to −3.80 mm from Bregma). The total number of positive cells was estimated by multiplying the average by the expected number of 30 µ coronal sections obtained along the referred plane, as adapted from previous works [52,53].
Fifty BrdU+ cells were randomly selected per animal from 5 to 6 coronal sections along the anteroposterior hippocampal plane (−1.34 to −3.80 mm from Bregma), and the ratio of BrdU-labeled cells that co-labeled with DCX was quantified as a marker of neuronal differentiation, as previously performed [12,25].

Hippocampal Synaptoneurosome and Homogenate Preparations
To purify a crude synaptoneurosome, animals were sacrificed approximately 2 h after the last AdipoRon injection and hippocampi were isolated and homogenized in ice-cold Syn-PER synaptic protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's manual. After sonication, hippocampal homogenates were centrifuged at 1200× g at 4 • C for 10 min, and cytosolic fraction was collected and centrifuged at 15,000× g, 4 • C, for 30 min. Crude synaptosomal fraction was resuspended in Syn-PER reagent. To extract total proteins from hippocampal homogenate, the hippocampi were homogenized in ice-cold radioimmunoprecipitation assay buffer (Abcam, Cambridge, UK) containing Halt© phosphatase/protease cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and phenylmethanesulfonyl fluoride (Thermo Fisher Scientific, Waltham, MA, USA). Samples were then sonicated for 20 s with a 50% pulse, followed by centrifugation at 14,000× g at 4 • C for 30 min. The protein concentrations of hippocampal synaptoneurosome and hippocampal homogenates were quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were stored at −80 • C until use.

Immunoassays for Adiponectin and BDNF Levels
Levels of adiponectin and BDNF in the total hippocampal lysates were determined 22 to 24 h after the last AdipoRon injection by using the commercial ELISA kits, including the mouse adiponectin ELISA kit (AdipoGen Life Sciences, San Diego, CA, USA) for measuring adiponectin levels in the hippocampus and the total BDNF Quantikine ELISA kit (R&D system, Minneapolis, MN, USA) for measuring BDNF levels in the hippocampus according to the manufacturer's instructions.

Field Recording
Recordings of field excitatory postsynaptic potentials (fEPSPs) were performed in a multi-electrode array system (Alpha MED Scientific Inc., Japan), as previously described [26]. A recording probe with extracellular electrodes (P515A, Alpha MED Scientific Inc., Osaka, Japan) was used to stimulate granule neurons in the middle molecular layer of the suprapyramidal blade of the hippocampal dentate gyrus. Slices were perfused at a rate of approximately 2 mL/min. fEPSPs were acquired using the MED-A64MD1 and MED-A64HE1S recording amplifiers (Alpha MED Scientific Inc., Osaka, Japan) and Mobius software. For each slice, stimulus intensity (20-30 µA) was adjusted to yield 40-50% of the maximal response slope without population spikes. Baseline fEPSP measurements were obtained by delivering single-pulse stimulation at 15 s interstimulus intervals. After obtaining a steady baseline of at least 20 min, a highfrequency tetanic stimulation (HFS) was used to induce long-term potentiation (LTP) in the presence of 5 µM bicuculline methiodide. The HFS protocol consists of 4 trains of 50 pulses delivered at 100 Hz with a 30 s intertrain interval. After field recording has finished, input/output curves were recorded with increasing stimulation intensity (10 µA steps).

Surgical Procedure for Adeno-Associated Virus Injection
Adeno-associated viruses (AAV2-psico-CMV-mCherry-NR2A-shRNA and AAV2psico-CMV-mCherry-NR2B-shRNA) were purchased from Virovek (2.1 × 1013 vg/mL, USA). CamKIIα-Cre male mice were anesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) and positioned in a stereotaxic apparatus (RWD, Shenzhen, China). Bilateral craniotomies were performed over the hippocampus and a Hamilton syringe (33-gauge needle, Reno, Hamilton, USA) mounted in an automated pump (11 Elite Nanomite, Harvard Apparatus, Holliston, MA, USA) was used to inject a viral bolus or vehicle (PBS, control) of 0.5 µL/site at a rate of 0.8 µL/min in the ventral hippocampus (3 mm posterior, 3.3 mm lateral, and 3.6 mm ventral to Bregma). After injection, the needle was left in place for 5 min before retraction. Animals were treated with Flunixin (2.5 mg/kg, s.c.) for three consecutive days and allowed to recover for 14 days before experimental procedures.

Statistical Analyses
Data were shown as mean ± SEM. The unpaired t-test was employed to compare the effects of AdipoRon versus vehicle treatments in behavioral assessments, histological analyses, fEPSP analyses, western blotting analyses, and ELISA. The Mantel-Cox (log-rank) overall test was employed to examine the survival percentage in the novelty-suppressed feeding test. Two-way ANOVA with Sidak's post-hoc test was used to evaluate the effects of AdipoRon and stimulus intensity in the input-output response in field electrophysiological recordings. One-way ANOVA with Tukey's post-hoc test was used to evaluate the effects of AdipoRon on behavioral changes in CamKIIα-specific NR2A and NR2B knockdowns. Statistical analyses were performed in Prism 9.0 software (GraphPad Software, Boston, CA, USA). p < 0.05 was considered statistically significant.

Sub-Chronic AdipoRon Treatment Induced Antidepressant-and Anxiolytic-Like Effects
We first determined whether sub-chronic (7 days

Sub-Chronic AdipoRon Treatment Did Not Change Adult Hippocampal Neurogenesis
Chronic (14 days) AdipoRon treatment has been associated with increased cell proliferation in the hippocampal DG [25,26]. We investigated whether sub-chronic AdipoRon treatment would also change adult hippocampal neurogenesis. Seven-day AdipoRon treatment did not change the number of hippocampal proliferative cells (Ki67+

Sub-Chronic AdipoRon Treatment Did Not Change Adult Hippocampal Neurogenesis
Chronic (14 days) AdipoRon treatment has been associated with increased cell proliferation in the hippocampal DG [25,26]. We investigated whether sub-chronic AdipoRon treatment would also change adult hippocampal neurogenesis. Seven-day AdipoRon treatment did not change the number of hippocampal proliferative cells (Ki67+ cells: Figure 3a Overall, these data suggest that sub-chronic AdipoRon treatment does not affect hippocampal neurogenesis, suggesting its antidepressant and anxiolytic effects are not linked to adult neurogenesis.

Acute AdipoRon Treatment Suppressed Neuronal Activation in the Ventral Hippocampus
Hippocampal DG neuronal activation has been associated with antidepressant effects [54]. To further investigate whether the AdipoRon antidepressant-and anxiolyticlike effects could be due to increased neuronal activation in the hippocampal DG, we quantified the c-Fos-immunopositive cells (Figure 4a) 2 h after delivering a single dose of AdipoRon. We used a single dose for this experiment because c-Fos is an acute marker of neuronal activation, which peaks around 2 h after stimulation and does not accumulate upon consecutive treatment [47]. Our results showed that acute AdipoRon administration significantly reduced neuronal activation in the ventral DG (Figure 4c; t10 = 3.974, p = 0.0026), although there was no significant change in the number of c-Fos-positive cells in

Acute AdipoRon Treatment Suppressed Neuronal Activation in the Ventral Hippocampus
Hippocampal DG neuronal activation has been associated with antidepressant effects [54]. To further investigate whether the AdipoRon antidepressant-and anxiolytic-like effects could be due to increased neuronal activation in the hippocampal DG, we quantified the c-Fos-immunopositive cells (Figure 4a) 2 h after delivering a single dose of AdipoRon. We used a single dose for this experiment because c-Fos is an acute marker of neuronal activation, which peaks around 2 h after stimulation and does not accumulate upon consecutive treatment [47]. Our results showed that acute AdipoRon administration significantly reduced neuronal activation in the ventral DG (Figure 4c; t

Sub-Chronic AdipoRon Treatment Reduced Protein Levels of Brain-Derived Neurotrophic Factor but Not Synaptic NMDA Receptor Subunits in the Hippocampus
We further investigated whether the level of neurotrophic support in the hippocampus was altered after subchronic AdipoRon treatment. Results showed that AdipoRon treatment reduced the hippocampal BDNF levels (Figure 5a

Sub-Chronic AdipoRon Treatment Reduced Protein Levels of Brain-Derived Neurotrophic Factor but Not Synaptic NMDA Receptor Subunits in the Hippocampus
We further investigated whether the level of neurotrophic support in the hippocampus was altered after subchronic AdipoRon treatment. Results showed that AdipoRon treatment reduced the hippocampal BDNF levels (Figure 5a Overall, these results suggest that sub-chronic AdipoRon treatment reduces neurotrophic support without significantly changing NMDA receptor subunits and adiponectin protein expression in the hippocampus. We further investigated whether the level of neurotrophic support in the hippocampus was altered after subchronic AdipoRon treatment. Results showed that AdipoRon treatment reduced the hippocampal BDNF levels (Figure 5a; t18 = 2.227, p = 0.039). Of note, AdipoRon treatment did not alter the level of adiponectin (Figure 5b; t18 = 0.6939; p = 0.4966) as well as the expressions of AdipoR1 (Figure 5c,d; t18 = 0.6372, p = 0.5320) or AdipoR2 (Figure 5c,e; t18 = 0.241; p = 0.9026) in the hippocampus.

Sub-Chronic AdipoRon Treatment Reduced Synaptic Plasticity in the Hippocampal DG
To further confirm whether changes in neurotrophic support would affect hippocampal synaptic plasticity, we examined LTP formation at the perforant path of the hippocampal DG region after sub-chronic AdipoRon treatment. Treatment reduced the HFS-induced potentiation of synaptic responses in the perforant path (Figure 7a), as

Sub-Chronic AdipoRon Treatment Reduced Synaptic Plasticity in the Hippocampal DG
To further confirm whether changes in neurotrophic support would affect hippocampal synaptic plasticity, we examined LTP formation at the perforant path of the hippocampal DG region after sub-chronic AdipoRon treatment. Treatment reduced the HFS-induced potentiation of synaptic responses in the perforant path (Figure 7a), as indicated by a significant reduction in the LTP of AdipoRon-treated mice when compared to vehicletreated mice (Figure 7b; t 18 = 2.607, p = 0.0173). In agreement with that, basal synaptic transmission efficiency after LTP was different between AdipoRon-and vehicle-treated animals, as depicted by a significant reduction in the input-output response in AdipoRontreated mice (Figure 7c; effect of AdipoRon: F 1,19 = 11.97; p = 0.0026; effect of stimulus intensity: F 1.403,26.66 = 162.9, p < 0.0001; effect of interaction: F 8,152 = 12.64, p < 0.0001). Post-hoc analysis revealed that such reduction in synaptic transmission efficiency was significant for stimulation intensities above 50 µA (p < 0.05). Collectively, these results indicate that sub-chronic AdipoRon treatment inhibits LTP formation, suggesting its action in suppressing hippocampal DG synaptic plasticity in healthy wild-type mice. Post-hoc analysis revealed that such reduction in synaptic transmission efficiency was significant for stimulation intensities above 50 μA (p < 0.05). Collectively, these results indicate that sub-chronic AdipoRon treatment inhibits LTP formation, suggesting its action in suppressing hippocampal DG synaptic plasticity in healthy wild-type mice.

Antidepressant-and Anxiolytic-Like Effects of AdipoRon Were Independent of Hippocampal NMDA Receptors
Finally, to confirm whether the antidepressant-and anxiolytic-like effects of subchronic AdipoRon treatment were independent of the hippocampal NMDA receptors, we used AAV-shRNA in CamKIIα-Cre mice to specifically knockdown the expression levels of hippocampal GluN2A and GluN2B subunits. Results showed that neither GluN2A nor GluN2B subunit knockdown affected the behavioral profile indicative of antidepressantlike properties of AdipoRon on sucrose preference in the SPT (Figure 8a

Discussion
Antidepressants have a delayed therapeutic onset [55], which contributes to the disease burden and vulnerability to suicidal behavior [56]. Here, we demonstrated that 7 days of AdipoRon treatment at a dosage of 20 mg/kg effectively modulate behaviors indicative of antidepressant-and anxiolytic-like properties in naïve mice, which is a shorter onset of antidepressant response compared to conventional antidepressants [40], whereas our molecular and electrophysiological results suggest that the hippocampus is not a key structure mediating such responses.
We observed a significant reduction in behaviors associated with depression-and anxiety-like phenotypes after 7 days of AdipoRon administration. Previously, Nicolas and colleagues [13] showed that chronic AdipoRon treatment (21 days) at a dosage of 1 mg/kg prevented corticosterone-induced depression-and anxiety-like behaviors. However, it did not elicit antidepressant-or anxiolytic-like effects in naïve control animals (not exposed to corticosterone). Moreover, we have previously demonstrated that AdipoRon treatment for 14 days at a dosage of 20 mg/kg restored the cognitive deficits and anxiogenic phenotype in an animal model of diabetes. Nonetheless, AdipoRon's potential therapeutic properties in terms of depression and anxiety remained unclear. Our current data shows that behavioral changes associated with such effects are observable after 7 days of treatment. Noteworthy, this is about half of the time required for classical antidepressants to elicit a therapeutic response [40], suggesting activating adiponergic signaling can elicit a faster antidepressant-like response.
We also observed that the antidepressant-and anxiolytic-like effects of AdipoRon were not associated with increased hippocampal neurogenesis. Specifically, sub-chronic AdipoRon administration did not change the levels of adult hippocampal cell proliferation, survival, or neuronal differentiation, nor did it affect the amount of hippocampal immature neurons. Even though our previous investigations demonstrated

Discussion
Antidepressants have a delayed therapeutic onset [55], which contributes to the disease burden and vulnerability to suicidal behavior [56]. Here, we demonstrated that 7 days of AdipoRon treatment at a dosage of 20 mg/kg effectively modulate behaviors indicative of antidepressant-and anxiolytic-like properties in naïve mice, which is a shorter onset of antidepressant response compared to conventional antidepressants [40], whereas our molecular and electrophysiological results suggest that the hippocampus is not a key structure mediating such responses.
We observed a significant reduction in behaviors associated with depression-and anxiety-like phenotypes after 7 days of AdipoRon administration. Previously, Nicolas and colleagues [13] showed that chronic AdipoRon treatment (21 days) at a dosage of 1 mg/kg prevented corticosterone-induced depression-and anxiety-like behaviors. However, it did not elicit antidepressant-or anxiolytic-like effects in naïve control animals (not exposed to corticosterone). Moreover, we have previously demonstrated that AdipoRon treatment for 14 days at a dosage of 20 mg/kg restored the cognitive deficits and anxiogenic phenotype in an animal model of diabetes. Nonetheless, AdipoRon's potential therapeutic properties in terms of depression and anxiety remained unclear. Our current data shows that behavioral changes associated with such effects are observable after 7 days of treatment. Noteworthy, this is about half of the time required for classical antidepressants to elicit a therapeutic response [40], suggesting activating adiponergic signaling can elicit a faster antidepressantlike response.
We also observed that the antidepressant-and anxiolytic-like effects of AdipoRon were not associated with increased hippocampal neurogenesis. Specifically, sub-chronic AdipoRon administration did not change the levels of adult hippocampal cell proliferation, survival, or neuronal differentiation, nor did it affect the amount of hippocampal immature neurons. Even though our previous investigations demonstrated that 14 days AdipoRon treatment (20 mg/kg) increased adult hippocampal cell proliferation [25,26], short-term antidepressant responses are unlikely to rely on neurogenic mechanisms due to the time course of the neurogenic cycle [57][58][59]. As demonstrated in vitro, ketamine has no significant effects on gene expression of neurogenesis and proliferative markers [60]. Moreover, it has been shown that the adiponectin-dependent antidepressant and anxiolytic effects of an enriched environment also do not rely on increased neurogenesis [14]. Therefore, our results support the view that compounds with faster antidepressant-like responses can be neurogenesis-independent.
AdipoRon also acutely reduced neuronal activation in the ventral dentate gyrus and sub-chronically reduced hippocampal neurotrophic support (BDNF). Such neurochemical changes were associated with a downregulation of functional plasticity in the perforant path, as observed by reduced LTP in AdipoRon-treated animals. These results agree with the adiponectin's neurophysiological properties previously reported in the literature [28,61,62]. Of note, adiponectin infusion reduced the intrinsic excitability of dopaminergic neurons in the ventral tegmental area [28], whereas bath-perfusing acute hippocampal slices with AdipoRon reduces dentate granule neurons' intrinsic excitability [62] and impairs CA1 LTP formation [60].
Our investigation also suggests that the hippocampus may not be a key structure in the antidepressant-and anxiolytic-like effects of sub-chronic AdipoRon treatment. The relevance of the hippocampus in antidepressant effects has also been questioned by others, where it was demonstrated that chemical inactivation of the ventral hippocampus did not interfere with the rapid antidepressant effects of ketamine [37], and acute stimulation of the ventral hippocampus projecting fibers failed to induce antidepressant effects [37,39]. Indeed, activation of the hippocampus (either optogenetically or chemogenetically) is rather associated with increased stress resilience than with antidepressant response [63]. Therefore, our findings support recent evidence that the hippocampus may have a more significant role in depression pathogenesis [29,31] than in antidepressant response [37][38][39].
Hippocampal functional plasticity is substantially dependent on NMDA-associated glutamatergic transmission [32][33][34]. Although we did not observe a significant reduction in the activity of the NMDA receptor subunits GluN2A and GluN2B after subchronic AdipoRon treatment, we conducted knockdown experiments of such sub-units to confirm whether the ventral hippocampal glutamatergic transmission was necessary for the AdipoRon therapeutic effects. Remarkably, AdipoRon antidepressant-and anxiolytic-like effects were independent of ventral hippocampal NMDA receptor-dependent synaptic function, as knockdown of the NMDA receptor subunits did not interfere with the effects of AdipoRon.
Overall, our investigation indicates that increasing adiponectin signaling through its receptor agonist, AdipoRon, changes the behavioral profile indicative of antidepressantand anxiolytic-like effects, which is already evident after 7 days of treatment. Moreover, we observed that such properties are likely independent of increased hippocampal activity, although the investigation of synaptogenic markers in the hippocampus has not been excluded and warrants further exploration. The involvement of other brain regions requires further investigation. The mPFC expresses high levels of both adiponectin receptors [19,20] and is one of the key brain regions implicated in rapid antidepressant response [42,64]. Moreover, other brain regions that have been associated with rapid antidepressant response, including the amygdala [42], dorsal raphe nucleus [65], and ventral tegmental area [66], express high levels of adiponectin receptors [16,28,67].