https://orcid.org/0000-0003-0922-4900
Figures & data
Figure 1. Antidepressant-like actions of NPY1R agonist and ketamine co-administration in the forced swimming test (FST).
Behavioral outcomes observed following the intracerebroventricular (icv) delivery of a neuropeptide Y1 receptor (NPY1R) agonist and Ketamine, administered both individually and in combination, in the Forced Swimming Test (FST). The graph quantitatively depicts behavioral measures including total immobility time (a) and swimming duration (b) across different experimental conditions. Data points represent mean ± SEM for groups of six animals each. Statistical annotations are as follows: *p < 0.05 denotes significant differences from the aCSF and NPY1R agonist alone groups; ★P < 0.05 indicates significant differences from both the Ketamine alone and NPY1R+Ketamine+BIBP3226 groups; ***p < 0.001 significantly differs from the aCSF and NPY1R agonist alone groups. Statistical significance was evaluated using one-way ANOVA and the Newman-Keuls post-hoc test, with lines above bars indicating comparisons between groups (n = 6). Abbreviations: aCSF = artificial cerebrospinal fluid (control group), NPY1R agonist = Neuropeptide Y1 receptor agonist [Leu31, Pro34]NPY (0.1 nmol); Ketamine = Ketamine administration (10 mg/kg); NPY1R+Ketamine = combined administration of NPY1R agonist and Ketamine; NPY1R+Ketamine+BIBP3226 = combined administration of NPY1R agonist, Ketamine, and NPY1R antagonist BIBP3226 (0.1 nmol).
Figure 2. Impact of intranasal NPY1R agonist and ketamine Co-administration on FST behavioral outcomes.
Effects of intranasal (i.n.) delivery of a neuropeptide Y1 receptor (NPY1R) agonist and Ketamine, individually and in combination, on depressive-like behaviors in the Forced Swimming Test (FST). The graph quantitatively displays the behavioral measures assessed, specifically the total immobility duration (a) and the active swimming duration (b), for different treatment groups. Each point denotes the mean ± SEM for groups comprising six animals each. Statistical annotations include: *p < 0.05, indicating significant differences from the control (distilled water) and NPY1R agonist groups; ★P < 0.05, marking significant differences from the Ketamine alone and NPY1R+Ketamine+BIBP3226 groups; ⌘ p < 0.05, signifying a significant difference from the Ketamine alone and NPY1R+Ketamine+ANA-12 groups; ***p < 0.001, demonstrating a significant difference from the control and NPY1R agonist groups (n = 6). Statistical analysis was conducted using one-way ANOVA and Newman-Keuls post-hoc test, with lines above bars indicating inter-group comparisons. Abbreviations include: Control = distilled water; NPY1R agonist = Neuropeptide Y1 receptor agonist [Leu31, Pro34]NPY (68 mcg); Ketamine = administration of Ketamine (10 mg/kg); NPY1R+Ketamine = co-administration of NPY1R agonist and Ketamine; NPY1R+Ketamine+BIBP3226 = co-administration of NPY1R agonist, Ketamine, and NPY1R antagonist BIBP3226 (68 mcg); NPY1R+Ketamine+ANA-12 = co-administration of NPY1R agonist, Ketamine, and TrkB antagonist ANA-12 (0.5 mg/kg).
Figure 3. Visualization of NPY1R/TrkB heteroreceptor complex formation in the ventral hippocampal DG via proximity ligation assay.
Identification of NPY1R/TrkB heteroreceptor complexes within the ventral hippocampal dentate gyrus (DG) through the application of the in situ Proximity Ligation Assay (PLA). This innovative technique allows for the direct observation of protein-protein interactions in close proximity, utilizing highly specific primary antibodies targeted at each protein. The assay highlights: (a) The presence of PLA-positive signals (shown as red dots) primarily within the subgranular zone (SGZ) of the DG, delineating the interface between the granule cell layer (GCL) and the polymorphic layer (PL) in the ventral hippocampus. These signals extend into the PL, with the molecular layer (ML) characterized by the absence of PLA signals (indicated by blue dots). The anatomical landmarks correspond to the Bregma -5.6 mm coordinate according to the Paxinos and Watson [0.59] stereotaxic atlas. (b) A quantitative analysis of PLA signals in the SGZ involved counting the red PLA-positive spots per nucleus in each field, carried out by an analyst blind to the experimental conditions. (c,d) Representative images showcase a marked elevation in the density of red PLA blobs indicative of NPY1R/TrkB heteroreceptor complexes in the SGZ following co-administration of NPY1R agonist and Ketamine, relative to the control group. These complexes are depicted as red PLA blobs (clusters) with high densities per cell across numerous neurons, visualized using confocal laser microscopy. Statistical annotations include: **p < 0.01, indicating significant differences from the Control and NPY1R agonist alone; ★★P < 0.01, denoting significant differences from Ketamine alone; ***p < 0.001, highlighting significant differences from the Control and NPY1R agonist; ★★★P < 0.001, signifying significant differences from the NPY1R+Ketamine+ANA-12 group, as determined by one-way ANOVA and Newman-Keuls post-hoc test (n = 4). Vertical lines above bars facilitate inter-group comparisons. Data are presented as mean ± SEM for four rats per group, in duplicates. White arrows point to PLA-positive clusters. Dashed lines outline the GCL of the DG. Nuclei are counterstained in blue with DAPI. Abbreviations: Control = distilled water; NPY1R agonist = Neuropeptide Y1 receptor agonist [Leu31, Pro34]NPY (68 mcg); Ketamine = administration of Ketamine (10 mg/kg); NPY1R+Ketamine = combined administration of NPY1R agonist and Ketamine; NPY1R+Ketamine+ANA-12 = co-administration of NPY1R agonist, Ketamine, and TrkB antagonist ANA-12 (0.5 mg/kg).
Figure 4. Enhancing neurogenesis in the ventral dentate gyrus through intranasal Co-administration of NPY1R agonist and ketamine.
Impact of co-administering an NPY1R agonist and Ketamine on neurogenesis within the ventral dentate gyrus in adult rats, as evidenced by BrdU immunostaining. (a,d) Predominantly, BrdU-positive cells localize to the subgranular zone (SGZ) at the boundary of the granule cell layer (GCL) and the polymorphic layer (P) within the ventral hippocampus, often forming clusters. This area corresponds to the Bregma -5.6 mm location as per the Paxinos and Watson stereotaxic guide [0.59]. (b) Presents a quantitative analysis of BrdU-immunoreactive (IR) cells within the SGZ, showing differences across groups treated with control conditions, NPY1R agonist [Leu31-Pro34]NPY, Ketamine, or their co-administration with or without ANA-12. (d) Highlights the increased BrdU labeling in the SGZ following the combined treatment compared to control conditions (c), with arrows pointing to clusters of BrdU-positive neurons and dashed lines outlining the GCL. (e) Quantifies BrdU-IR cells that are also double-labeled with DCX, indicating that the co-administration specifically targets neuroblast proliferation. **p < 0.01 indicates significant enhancement versus control, analyzed using Student’s t-test. (f) A representative image showcases DCX+/BrdU+ cells (white arrows), DCX-/BrdU+ cells (white arrowheads), and DCX+/PCNA- cells (white asterisks) in the group receiving both NPY1R agonist and Ketamine. Statistical significance is marked as: *p < 0.05 versus control and NPY1R agonist alone; ★P < 0.05 versus Ketamine alone and combined treatment with ANA-12; ***p < 0.001 versus control and NPY1R agonist, determined through one-way ANOVA and Newman-Keuls post-hoc analysis. Vertical and horizontal lines above the bars enable comparison between groups (n = 4). Each experimental group consisted of four animals. Abbreviations: Control = distilled water; NPY1R agonist = Neuropeptide Y1 receptor agonist [Leu31, Pro34]NPY (68 mcg); Ketamine = administration of Ketamine (10 mg/kg); NPY1R+Ketamine = co-administration of NPY1R agonist and Ketamine; NPY1R+Ketamine+ANA-12 = co-administration of NPY1R agonist, Ketamine, and TrkB antagonist ANA-12 (0.5 mg/kg).
Figure 5. Enhancing BDNF expression in the ventral dentate gyrus through Co-administration of NPY1R agonist and ketamine.
Effects of co-administering an NPY1R agonist and Ketamine on the distribution of brain-derived neurotrophic factor immunoreactive (BDNF-IR) cells within the ventral dentate gyrus (DG) of the hippocampal region. The focus here is on the synergistic impact of NPY1R agonist and Ketamine. Panel (a) highlights that BDNF-IR cells are predominantly found in the granule cell layer (GCL) of the DG, with sparse presence in the polymorphic layer (P), corresponding to the Bregma -5.6 mm location as per the Paxinos and Watson stereotaxic atlas [0.59]. Panel (b) quantitatively demonstrates a significant upsurge in BDNF-IR cells in the ventral DG following icv co-administration of NPY1R agonist and Ketamine. The application of the TrkB antagonist ANA-12 is shown to counter this effect. Statistical significance is indicated by *p < 0.05 compared to control, NPY1R alone, and NPY1R+Ketamine+ANA-12; **p < 0.01 versus NPY1R+Ketamine, as established by one-way ANOVA and subsequent Newman – Keuls post-hoc testing (n = 4 per group). The graph’s vertical lines above bars facilitate inter-group comparison. Data are depicted as mean ± SEM. Panels (c) and (d) provide representative images showcasing the notable enhancement in BDNF-positive cells in the DG after co-administration of NPY1R agonist and Ketamine (d), relative to the control group treated with distilled water (c). Dashed lines mark the GCL boundaries within the DG. Abbreviations include: Control = distilled water; NPY1R agonist = Neuropeptide Y1 receptor agonist [Leu31, Pro34]NPY (68 mcg); Ketamine = administration of Ketamine (10 mg/kg); NPY1R+Ketamine = combined treatment with NPY1R agonist and Ketamine; NPY1R+Ketamine+ANA-12 = combined treatment with NPY1R agonist, Ketamine, and TrkB antagonist ANA-12 (0.5 mg/kg).
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