Role of neuropeptide Y Y₁ and Y₂ receptors on behavioral despair in a rat model of depression with co-morbid anxiety
Abstract
Background
A substantial and growing body of evidence, primarily derived from animal studies conducted in otherwise healthy and unmanipulated subjects, strongly suggests that neuropeptide Y (NPY) plays a significant and intricate role in the pathophysiology of various emotional disorders. This hypothesized involvement is believed to be mediated through NPY’s actions on its two primary receptor subtypes, Y(1) and Y(2) receptors. However, it is crucial to note that much of this foundational evidence has not been systematically investigated within established animal models of depression, which are specifically designed to mimic the complex symptomatology of human affective disorders. One such highly relevant and widely accepted animal model is the olfactory bulbectomized (OBX) rat. The OBX rat model faithfully reproduces a wide array of behavioral and neurochemical alterations that closely mimic several key aspects of both human depression and anxiety disorders, making it an invaluable tool for studying potential therapeutic interventions. Given this gap in the literature, our study aimed to directly address whether the previously observed effects of NPY in normal animals translate to a compromised emotional state, using the OBX rat model as our primary experimental platform.
Methodology/Principal Findings
In the present comprehensive study, our primary objective was to thoroughly investigate the effects of sustained intracerebroventricular (ICV) administration of NPY Y(1) and Y(2) receptor agonists and antagonists. The sustained administration, lasting for a period of two weeks, was carefully chosen to reflect potential chronic therapeutic interventions rather than acute modulations. This targeted drug delivery into the cerebral ventricles allowed for direct pharmacological manipulation of NPY receptor activity within the central nervous system, bypassing peripheral effects. The behavioral outcomes were meticulously assessed using a battery of well-validated behavioral tests designed to evaluate both depressive- and anxiogenic-like behaviors in OBX rats. These tests included the open field test, which assesses locomotor activity and exploratory behavior, the forced swim test (FST), a widely used paradigm for measuring behavioral despair, and the social interaction (SI) test, which probes social exploratory drive and anxiolytic-like effects. Additionally, to gain insights into the neurobiological underpinnings of any observed behavioral changes, the expression levels of Y(1) and Y(2) receptors were quantitatively evaluated in two key brain regions critically involved in emotional processing: the hippocampus and the basolateral amygdala (BLA).
The results of our extensive behavioral and neurobiological assessments yielded several key findings. Treatment with [Leu(31)Pro(34)]PYY, a selective Y(1)-like receptor agonist, consistently led to a significant decrease in both depressive-like and anxiogenic-like behaviors in the OBX rats. This suggests that activation of Y(1) receptors can exert a beneficial, mood-stabilizing effect under conditions of emotional challenge. Conversely, the administration of BIIE0246, a selective Y(2) receptor antagonist, produced a reduction in the immobility time in the forced swim test specifically in OBX animals, indicating an antidepressant-like effect. Furthermore, in sham-operated (non-depressed) rats, this Y(2) receptor antagonist increased active social contacts in the social interaction test, pointing to anxiolytic-like properties under control conditions. In contrast to the beneficial effects of Y(2) antagonism, treatment with PYY3-36, a selective Y(2) receptor agonist, led to an increase in the immobility time in the forced swim test in OBX rats, further supporting the notion that Y(2) receptor activation may exacerbate depressive-like states in this model.
Beyond behavioral observations, our neurobiological evaluations provided crucial correlative evidence. We quantified significantly increased levels of Y(2) receptor binding in both the dorsal hippocampus and the basolateral amygdala in OBX rats compared to sham controls. These brain regions are known to play pivotal roles in the regulation of mood and anxiety. Taken together, these autoradiographic results provide compelling additional evidence that the NPYergic system, particularly the Y(2) receptor subtype, undergoes significant alterations in disturbed emotional states, such as those mimicked by the OBX model. Moreover, our comprehensive study distinctly demonstrates a differential and critical role for NPY Y(1) and Y(2) receptors in modulating emotional processes, with their precise contributions varying significantly between control (sham) and challenged (OBX) conditions. These findings advance our understanding of the NPY system’s involvement in emotional regulation and highlight its potential as a target for novel therapeutic interventions. This article is published as part of a Special Issue dedicated to advancing research in the complex fields of ‘Anxiety and Depression’.
Introduction
Neuropeptides, a diverse class of signaling molecules in the brain, have progressively emerged as critical neuromodulators of both physiological functions and the pathological dysfunctions intimately associated with various emotional disorders. Prominent among these are corticotrophin releasing factor (CRF), VGF (a non-acronymic neuropeptide), and neuropeptide Y (NPY). Of particular interest, NPY, a 36 amino acid peptide first identified by Tatemoto and colleagues in 1982, stands out for its remarkable evolutionary conservation and its widespread distribution throughout the central nervous system (CNS). This abundant neuropeptide orchestrates its diverse biological effects through the activation of at least four distinct G protein-coupled receptors, which are conventionally known as Y1, Y2, Y4, and Y5 receptors. Intriguingly, the Y1 and Y2 receptor subtypes are highly expressed within the CNS, with particularly high concentrations found in key brain regions such as the cortex, hippocampus, and amygdala. These specific brain areas are well-established to play pivotal roles in the intricate processing and regulation of emotional states, suggesting a direct involvement of NPY and its receptors in mood and anxiety.
Clinical investigations have provided compelling evidence that NPY levels may be significantly decreased in individuals suffering from depression. This observation, coupled with a growing body of cumulative data, strongly suggests a direct role for NPY and NPY-related molecules in modulating emotional processes in rodent models. For example, groundbreaking work by Heilig and colleagues in 1989 reported that NPY exhibits distinct anxiolytic properties in the elevated plus maze test, a widely used behavioral paradigm for assessing anxiety in otherwise healthy, or “naïve,” normal rats. Concurrently, our research group and others have demonstrated that the acute, exogenous administration of NPY itself, as well as the selective Y1-like receptor agonist [Leu31Pro34]PYY, can induce significant antidepressant-like effects in these naïve normal animals. Furthermore, pharmacological antagonism of the Y2 receptor subtype in naïve normal rats, or its genetic ablation in mice, has been shown to induce both antidepressant- and anxiolytic-like behaviors, further solidifying the inverse relationship between Y2 receptor activity and emotional well-being. However, a critical limitation of these earlier studies is that they were predominantly conducted in animals without any induced emotional disturbance. A more clinically relevant and robust strategy, therefore, necessitates the utilization of well-established animal models of emotional disorders to more thoroughly characterize the potential anxiolytic- and antidepressant-like effects of NPY and its related molecules in a compromised emotional state.
Animal models serve as powerful and indispensable tools for exploring the complex pathology of depression and, crucially, for the effective screening of novel therapeutic agents. Among these, the olfactory bulbectomized (OBX) rat model is particularly valuable. OBX rats consistently display a wide array of behavioral, neurochemical, endocrine, and immunological alterations that remarkably mimic those observed in human mood disorders. Interestingly, NPY levels themselves have also been demonstrated to be altered within the OBX rat model, indicating a direct perturbation of the NPYergic system in this context. Additionally, research by Wang and colleagues in 2007 suggested that OBX animals exhibit both depressive- and anxiogenic-like behaviors. More recently, our group proposed the strategic use of a comprehensive battery of behavioral tests in OBX animals as a refined approach to more robustly establish the antidepressant- and anxiolytic-like properties of candidate therapeutic molecules. Given that this animal model presents unique disturbances in anxiety- and depression-related behaviors, coupled with known alterations in the NPY system, it represents an exceptionally suitable platform to comprehensively evaluate the precise role of this neuropeptide and its receptor subtypes in the intricate processes of emotional regulation.
Accordingly, the present study was meticulously designed to achieve two primary objectives. Firstly, we aimed to definitively demonstrate the respective roles of the NPY Y1 and Y2 receptor subtypes in modulating depression- and anxiety-related behaviors specifically within the OBX rat model. Secondly, we sought to determine if the apparent levels of Y1 and Y2 receptor binding are indeed altered in this animal model, thereby providing crucial additional neurobiological support for the fundamental role of NPY in emotional behaviors under conditions of pathology.
Methods
A comprehensive series of meticulously planned behavioral and autoradiographic studies were systematically executed to thoroughly evaluate the specific role of NPY Y1 and Y2 receptors in the olfactory bulbectomized (OBX) rat model. Generally, all experimental animals were afforded a critical two-week recovery period following either OBX surgery or sham surgical procedures, a timeframe established to allow for post-operative recuperation and the development of OBX-induced behavioral changes. Since the therapeutic administration of antidepressant drugs in clinical settings typically spans approximately two weeks, our behavioral experiments and subsequent receptor autoradiography were deliberately performed four weeks after the surgical removal of the olfactory bulbs, providing a physiologically relevant timeline for assessing sustained drug effects.
Animals
Male Sprague Dawley rats, obtained from Charles River Canada, Montréal, QC, Canada, with an initial body weight ranging from 150 to 170 g at the commencement of treatment, were utilized for this study. Animals were housed two per cage under meticulously controlled standard laboratory conditions, including a 12-hour light/dark cycle (with lights on at 8:00 AM) and continuous *ad libitum* access to standard rodent chow (Purina Lab Chow) and water. These animals were then assigned to either behavioral studies or quantitative receptor autoradiographic studies. For behavioral investigations, all animals underwent two distinct surgical procedures: (1) OBX or sham lesions, performed on day 1, and (2) osmotic minipump implantation, carried out on day 14. Animal body weights were meticulously recorded once a week until the behavioral tests commenced on days 28-30, and their overall health status was constantly monitored. For autoradiographic studies, animals underwent either sham or OBX surgery and were subsequently sacrificed four weeks post-lesion, with their brains collected at this same four-week time point. A detailed flow chart illustrating the precise procedural order of these experiments is provided in an accompanying figure. All experimental procedures received explicit approval from the McGill Animal Care Committee and were conducted in strict accordance with the guidelines set forth by the Canadian Council on Animal Care and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023). Every effort was consistently made to minimize animal suffering and to reduce the total number of animals utilized, adhering to the highest ethical standards.
OBX Surgery
Bilateral olfactory ablation was precisely performed following established protocols from previous reports. Briefly, anesthesia was initially induced using 5% isoflurane (Baxter, Mississauga, ON) and then consistently maintained at 2.5% throughout the entirety of the surgical procedures. A cranial window, accurately positioned 5.2 mm anterior to the bregma, was meticulously created in the frontal bone. Through this opening, the olfactory bulbs were carefully transected and aspirated out. Sham operations were conducted in an identical manner, with the crucial distinction that the olfactory bulbs were left intact, serving as a surgical control. To prevent blood loss from the cranial window, the open space was effectively filled with a haemostatic sponge (Gelfoam, Pfizer Canada Inc., Montréal, QC). Post-surgery, all rats received a subcutaneous administration of carprofen (0.1 mL/100 g animal) (Pfizer Animal Health, Montréal, QC) for pain management, along with a saline solution (0.9% NaCl) (Hospira Healthcare Corporation, Montréal, QC) for rehydration. Animals were then returned to their respective cages, housed in pairs, to recover for a period of two weeks. Only data obtained from animals confirmed to have undergone complete removal of both olfactory bulbs and showing no discernible damage to the frontal cortex (verified by macroscopic examination following brain removal) were included in the final analysis, ensuring the integrity and relevance of the experimental model.
Osmotic Minipump Implantation
For the behavioral test phase, an osmotic minipump (Alzet, model 2002, Durect Cupertino, CA, USA) was surgically implanted. This minipump was meticulously connected to an indwelling intracerebroventricular (ICV) cannula, designed to deliver NPY analogs at a constant flow rate of 0.5 μL/hour for a duration of two weeks in both OBX and sham-operated rats. Previous studies have consistently demonstrated the stability of NPY-related molecules within Alzet pumps for periods extending up to 28 days, ensuring the integrity of the infused compounds. The surgical procedure for osmotic pump implantation commenced with the induction of anesthesia using 5% isoflurane, subsequently maintained at 2.5% throughout. Animals were then secured in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA), and a cannula was precisely implanted into the left lateral ventricle, guided by stereotaxic coordinates (anterior-posterior: 0.8 mm from bregma, lateral: 1.3 mm from bregma, and vertical: 4.5 mm from dura), as referenced in the Paxinos and Watson atlas (1986). The cannula was firmly sealed with dental cement and connected to the osmotic pump via medical-grade vinyl tubing. The pump itself was then subcutaneously positioned within a pocket created in the dorsal region of the animal. Rats from the same treatment and condition group were housed in pairs until the commencement of behavioral testing. To rule out any localized effects of the long-term infusion, we additionally examined the lateral ventricles and the dentate gyrus. No discernible differences were observed between the ventricle that received the cannula and the contralateral ventricle, nor between the dentate gyri, ensuring that any behavioral effects were due to the infused compounds rather than cannula placement.
Typically, NPY or NPY Y1 agonists and Y2 antagonists are administered in the picomolar range for targeted microinjections into specific brain nuclei, and in the nanomolar (nmol) range for ICV infusions, as previously reported. In this study, the vehicle solution comprised 0.9% NaCl for agonists, or a 1% DMSO/0.9% NaCl mixture for antagonists. For OBX animals, the osmotic pumps were filled with either vehicle or specific NPY analogs: BIBO3304 (3 nmol/day), [Leu31Pro34]PYY (at concentrations of 0.1, 0.3, and 1 nmol/day), BIIE0246 (at 1 and 10 nmol/day), and PYY3-36 (1 nmol/day), all administered continuously for 14 days. For sham-operated animals, the pumps contained either vehicle or BIBO3304 (3 nmol/day), [Leu31Pro34]PYY (1.0 nmol/day), BIIE0246 (10 nmol/day), or PYY3-36 (1 nmol/day), also for 14 days. To ensure optimal drug delivery from the onset of implantation, all pumps were filled one day prior to surgery and subsequently incubated overnight at 37°C in a sterile saline solution for priming. Human (h) [Leu31Pro34]PYY and hPYY3-36 were custom-synthesized at the Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montréal, Canada, following previously established methods. BIBO3304 and BIIE0246 were generously provided by Boehringer Ingelheim, Ingelheim, Germany.
Behavioral Tests
A comprehensive series of three distinct behavioral tests was systematically performed on three consecutive days, a protocol previously established and validated in another model of depression-related behaviors. Similar protocols have also been successfully applied within the OBX model. Each test was conducted separately to avoid confounding influences. The open field (OF) test was carried out on day 27 post-surgery, the forced swim test (FST) was performed on day 28, and the social interaction (SI) test was conducted on day 29, following either OBX or sham surgery. All three behavioral tests were consistently performed during the light phase of the light-dark cycle, specifically between 9:00 AM and 1:00 PM. Throughout the entire testing period, rats were maintained in their consistent housing conditions. Each experimental group comprised between eight and twenty animals, ensuring robust statistical power.
Open Field Test
The open field test, a classical paradigm originally introduced for assessing emotionality in rats, was conducted in a spacious arena measuring 100 × 100 × 45 cm. The apparatus comprised a black wooden box with a grey floor, open at the top, and the floor was divided into 64 equally sized squares. The testing environment was maintained under a consistent illumination of 300 LUX. The behavior of the animals was meticulously recorded for a period of 10 minutes, with an observer blind to the treatment conditions. Parameters measured included locomotion during the initial exposure (termed horizontal behavior) and the frequency of rearing and grooming activities (termed vertical behaviors). The testing apparatus was thoroughly cleaned with peroxigard solution (Bayer Healthcare, Toronto, ON) after each individual trial to eliminate olfactory cues. In OBX animals, hyperlocomotion, along with enhanced rearing and grooming, are commonly observed and are associated with depression-related behavior and stress-coping mechanisms, respectively, and are typically reversed by repeated antidepressant treatment.
Forced Swim Test (FST)
The forced swim test is a widely recognized and well-characterized tool for screening potential antidepressant compounds. In the current study, we employed a modified version of the test where animals perform this task only once, a protocol that minimizes habituation effects. This behavioral test was conducted in a white cylindrical tank, measuring 29 cm in diameter and 43 cm in height, without a top lid. The tank was filled with water maintained at a temperature of 25°C ± 2°C. Rats were individually placed into the swimming tank for a 10-minute period. A camera, mounted 1 meter above the tank, recorded their behavior, and immobility time was subsequently evaluated by an observer blind to the experimental conditions. Immobility time was defined as the duration when the animal made only the minimum movements necessary to keep its body afloat. Increased time spent immobile, reflecting a lack of motivated escape behavior from the water, was interpreted as a depression-related behavior. Following the test, rats were promptly removed from the cylinder, thoroughly dried with a towel, and placed under a red lamp until their fur was completely dry.
Social Interaction (SI) Test
The social interaction test has been rigorously pharmacologically validated as an experimental paradigm for quantitatively measuring anxiety-like behaviors. This test was conducted in a familiar environment, utilizing the same apparatus as the open field test, but under brightly lit conditions. A pair of rats, comprising animals that had previously undergone the same treatments but were housed in different cages, were simultaneously placed in opposing corners of the open field arena and allowed to freely explore for 10 minutes. Active social behavior was meticulously recorded when animals engaged in direct physical contact such as running towards, grooming, mounting, or crawling under the other rat. The open field apparatus was thoroughly cleaned with peroxigard after each trial to eliminate any residual scent cues. The total duration of active social contacts and the total number of contacts were precisely measured for each pair of animals by a person blind to the treatment conditions. A reduction in social contacts in OBX animals was interpreted as an indicator of an anxiogenic-like behavior.
Quantitative Receptor Autoradiography
To ascertain changes in NPY Y1 and Y2 receptor levels in OBX rats relative to sham-operated controls, quantitative receptor autoradiography was performed following previously established methodologies. Radiolabeling of both [125I][Leu31Pro34]PYY (for Y1 receptor assessment) and [125I]PYY3-36 (for Y2 receptor assessment) was carried out using the chloramine T method, as described in prior reports. The specific activity of these radioligands was assumed to be their theoretical value (2000 Ci/mmol).
Rats designated for autoradiographic studies were humanely sacrificed by decapitation on day 28 after either olfactory bulb ablation or sham surgery. Their brains were rapidly removed from the skull, immediately frozen in 2-methylbutane at -40°C for 15 seconds, and then stored at -80°C until further processing. Coronal sections, 16 micrometers thick, were obtained using a cryomicrotome maintained at -17°C. These sections were mounted onto super frost plus slides, dried overnight in a desiccator at 4°C, and then stored at -80°C until use. On the day of the experiment, adjacent coronal sections were pre-incubated for 60 minutes at room temperature in Krebs Ringer Phosphate (KRP) buffer at pH 7.4. This was followed by incubation in a freshly prepared KRP buffer containing 0.1% bovine serum albumin (BSA) and 0.05% bacitracin, along with the respective radioligands, [125I]PYY3-36 and [125I][Leu31Pro34]PYY. To define specific binding, parallel sections were incubated in the presence or absence of 100 nM BIBO3304 (to block Y1 receptors) or 1000 nM BIIE0246 (to block Y2 receptors). After a 2-hour incubation period, sections were subjected to four washes, each lasting 1 minute, in ice-cold KRP buffer. Subsequently, they were briefly dipped in deionized water to remove residual salts, followed by rapid drying. Nonspecific binding was determined using 1 µM [Leu31Pro34]PYY for Y1 receptor assays, or 1 µM PYY3-36 for Y2 receptor assays. Finally, the processed sections were placed alongside radioactive standards on Kodak Biomax MR films (Eastman Kodak, Rochester, NY) for an exposure period ranging from 5 to 14 days, and the resulting autoradiographs were then quantitatively analyzed.
Statistical Analysis
Data acquired from the behavioral tests were systematically analyzed using a one-way ANOVA, which was subsequently followed by Dunnett’s test for post hoc comparisons, allowing for detailed examination of specific group differences. Results obtained from the autoradiographic experiments were analyzed through a two-way ANOVA, followed by Bonferroni post-test for multiple comparisons. In all statistical analyses performed throughout the study, a p-value of less than 0.05 was consistently considered to indicate statistical significance, thereby ensuring the reliability of the observed findings.
Results
The rigorous initial assessments confirmed that neither the olfactory bulbectomy (OBX) lesion itself nor the subsequent treatment with NPY-related compounds exerted any significant effect on the body weight of the animals throughout the entire four-week observation period, ensuring that observed behavioral or neurobiological changes were not confounded by alterations in general health or feeding. Across the experimental groups, horizontal and vertical activities in the open field (OF) apparatus, immobility time in the forced swim test (FST), and active social contacts in the social interaction (SI) paradigm were meticulously measured following sustained exposure to agonists and antagonists of NPY Y1 and Y2 receptors, delivered via osmotic minipumps in both OBX and sham-operated rats. Furthermore, as a crucial neurobiological component of the study, the expression levels of Y1 and Y2 receptor binding were also thoroughly investigated in both OBX and sham animals.
Effect of Sustained Administration of Agonists and Antagonists of NPY Y1 and Y2 Receptors in Hyperlocomotion in Open Field in OBX Rats
Chronic intracerebroventricular (ICV) administration of [Leu31Pro34]PYY, a selective Y1-like receptor agonist, resulted in a statistically significant decrease in hyperlocomotion in OBX animals, as evidenced by F(5, 63) = 9.957, p < 0.0001. Specifically, concentrations of 0.3 nmol (p < 0.001) and 1.0 nmol (p < 0.05) of [Leu31Pro34]PYY produced notable reductions in locomotor activity. Conversely, sustained infusion of BIBO3304 (a Y1 antagonist), PYY3-36 (a Y2 agonist), or BIIE0246 (a Y2 antagonist) did not modulate locomotion in OBX rats in the open field test. Furthermore, chronic ICV infusion of any of the NPY Y1 and Y2 agonists and antagonists tested had no significant effect on locomotion in sham-operated animals, indicating that these compounds primarily exert their modulatory effects under challenged conditions, as represented by the OBX model. Effect of Sustained Infusion of Agonists and Antagonists of NPY Y1 and Y2 Receptors on Rearing and Grooming in Open Field in OBX Rats The surgical removal of the olfactory bulbs consistently led to a notable increase in both grooming and rearing behaviors when compared to sham-operated control animals. Our observations revealed that the Y1-like receptor agonist [Leu31Pro34]PYY (F(5, 66) = 31, p < 0.0001, with 1.0 nmol resulting in p < 0.01), the Y1 receptor antagonist BIBO3304 (F(3, 27) = 62, p < 0.0001, with 3.0 nmol resulting in p < 0.05), and the Y2 receptor antagonist BIIE0246 (F(4, 59) = 53, p < 0.0001, with 10 nmol resulting in p < 0.001) all led to a decrease in grooming activity in sham animals within the open field test. In contrast, the Y2 receptor agonist PYY3-36 (F(3, 34) = 58, p < 0.01, with 1 nmol resulting in p < 0.05) increased this activity in sham animals. Notably, none of the NPY-related molecules had a significant effect on grooming in OBX rats, suggesting a complex and context-dependent modulation of this specific behavior. On the other hand, BIIE0246 (F(4, 59) = 60.57, p < 0.0001, with 10.0 nmol resulting in p < 0.05) effectively attenuated the enhanced rearing behavior that was typically produced by the OBX lesions. Conversely, [Leu31Pro34]PYY, BIBO3304, and PYY3-36 had no discernible effect on rearing behavior in either sham or OBX rats. These findings highlight a differential modulation of stress-related vertical behaviors by NPY receptor subtypes. Effect of Sustained Administration of NPY Y1 Agonist and Y2 Antagonist on Immobility in the Forced Swim Test in OBX Rats As clearly illustrated, intracerebroventricular (ICV) administration of [Leu31Pro34]PYY, a Y1-like receptor agonist, significantly decreased immobility time in the forced swim test (FST) in OBX rats (F(5, 55) = 78, p < 0.0001, with 0.3-1.0 nmol yielding p < 0.05; and 3.0 nmol yielding p < 0.01). Similarly, BIIE0246, a Y2 receptor antagonist, also produced a significant reduction in immobility time in the FST in OBX rats (F(4, 51) = 61, p < 0.0001, with 1.0 nmol yielding p < 0.05; and 10.0 nmol yielding p < 0.001). This indicates that both Y1 receptor activation and Y2 receptor antagonism can exert antidepressant-like effects in this model. In stark contrast, treatment with PYY3-36, a Y2 receptor agonist, led to a significant increase in immobility time in this paradigm in OBX rats (F(3, 35) = 65, p < 0.0001, with 1.0 nmol yielding p < 0.001), suggesting a pro-depressive effect of Y2 receptor activation. Conversely, BIBO3304, a Y1 receptor antagonist, had no discernible effect on immobility in the FST in OBX rats. Furthermore, it is important to note that none of the NPY-related molecules tested here exerted any significant effect in the FST in sham-operated (control) animals, reinforcing the notion that their modulatory effects are primarily evident under conditions of emotional challenge. Role of Sustained Administration of NPY Y1 Agonist and Y2 Antagonist in Active Contacts in the SI Test in OBX Rats In the social interaction (SI) test, a key indicator of anxiety-like behavior, OBX animals consistently exhibited a notable deficit in active social contacts. This deficit, characteristic of anxiogenic-like states, was effectively reversed by a two-week ICV infusion of [Leu31Pro34]PYY, a Y1-like receptor agonist (F(5, 31) = 6.057, p = 0.0008, with concentrations of 0.3-1.0 nmol yielding p < 0.05; and 3.0 nmol yielding p < 0.01). This demonstrates an anxiolytic-like effect of Y1 receptor activation in the OBX model. In contrast, treatment with BIBO3304 (a Y1 antagonist), PYY3-36 (a Y2 agonist), or BIIE0246 (a Y2 antagonist) had no significant effect on active social contacts in OBX rats, suggesting that their primary influence in this paradigm is not on social exploratory behavior in the lesioned animals. However, in sham-operated (control) animals, BIIE0246, the Y2 receptor antagonist, significantly increased social contacts (F(4, 33) = 15.79, p < 0.0001, with 10 nmol yielding p < 0.05), indicating an anxiolytic-like effect under normal conditions. Conversely, [Leu31Pro34]PYY, BIBO3304, and PYY3-36 had no significant effect in the social interaction test in sham animals, further highlighting the context-dependent roles of NPY receptor subtypes in modulating emotional behaviors. OBX Lesion Alters the Level of NPY Y2 Receptor Binding in Hippocampus and Amygdala Beyond the behavioral changes observed, our investigation delved into the neurobiological underpinnings of the olfactory bulbectomized (OBX) rat model, specifically assessing alterations in NPY receptor expression. We found that the level of specific [125I][Leu31Pro34]PYY binding, which serves as a marker for Y1 receptors, remained unaltered in OBX rats when compared to sham-operated animals, both in the presence or absence of the Y1 antagonist BIBO3304. This suggests that OBX lesion does not significantly impact the overall expression or availability of Y1 receptors. In stark contrast, after meticulously subtracting the nonspecific binding using BIIE0246, we observed a statistically significant increase in specific [125I]PYY3-36 binding, indicative of Y2 receptor levels, in two crucial brain regions: the CA1/CA2 subfield of the dorsal hippocampus and the basolateral amygdala (BLA) of OBX rats. This finding highlights a selective upregulation of Y2 receptors in brain areas highly relevant to emotional processing in the OBX model. A rigorous two-way ANOVA, analyzing the effects of OBX lesion and brain region, provided further statistical validation for these observations. This analysis revealed that the levels of Y2 receptor sites were significantly affected by the specific brain region (F7,40 = 119, p < 0.0001), by the OBX lesion itself (F1, 40 = 6.17, p < 0.0172), and critically, by a significant interaction between the OBX lesion and the brain region (F7, 40 = 6.13, p < 0.0001). These statistical results unequivocally confirm that the OBX lesion induces a specific and region-dependent increase in Y2 receptor binding. Accompanying figures illustrate the autoradiographic sections of the dorsal and ventral hippocampus treated with [125I]PYY3-36, providing visual evidence of the altered receptor distribution. Discussion The accumulated evidence, further substantiated by the findings of the present study, strongly supports the notion that the neuropeptide Y (NPY) system is intricately involved in emotional processes. Here, we embarked on a more detailed characterization of the precise roles played by NPY Y1 and Y2 receptors within the context of a well-established animal model of depression, which additionally exhibits comorbid anxiety-like behaviors. Our results provide compelling evidence that treatment with [Leu31Pro34]PYY, a selective Y1-like agonist, effectively reversed several behavioral deficits observed in OBX rats. Specifically, it ameliorated hyperlocomotion in the open field test, decreased immobility time in the forced swim test, and significantly increased active social interactions in the social interaction test. Conversely, treatment with BIIE0246, a selective Y2 antagonist, led to a reduction in the immobility time observed in the forced swim test in OBX animals and, interestingly, increased active social contacts in the social interaction test in sham-operated animals. These diverse effects are summarized in an accompanying table. Furthermore, our autoradiographic receptor studies provided crucial neurobiological corroboration, revealing a significant increase in Y2 receptor binding levels in both the dorsal hippocampus and the basolateral amygdala of OBX rats. Collectively, these data suggest that in the OBX rat model, the exogenous administration of a Y1 agonist effectively reverses symptoms associated with both depression- and anxiety-like behaviors. In parallel, a Y2 antagonist appears to improve depression-like behaviors in OBX animals while simultaneously inducing an anxiolytic effect under normal, unchallenged conditions. Relevance of a Battery of Behavioral Tests in the OBX Rat We have previously advocated for the strategic employment of a comprehensive battery of behavioral tests within animal models of depression and anxiety. This multi-test approach aims to provide a more nuanced and thorough characterization of novel candidate therapeutic molecules, moving beyond the limitations of single-test evaluations. Various research groups have adopted similar methodological approaches in diverse animal models of depression-related behaviors, including the OBX animal model, highlighting the growing recognition of this comprehensive strategy. Moreover, compelling evidence from Mitra and Sapolsky in 2008 demonstrated that acute stressors, when applied for only a single day, generally do not induce long-lasting behavioral changes. Therefore, the sequential performance of a second behavioral test one or two days after the first is expected to exert only limited, if any, anxiogenic effects, a conclusion further supported by previous work from Wang and colleagues. This methodological rigor ensures that the observed effects are genuinely attributable to the experimental manipulations rather than confounding factors from serial testing. The Y1 Receptor Subtype in Depression and Anxiety-Related Behaviors Our study presents a novel finding, being the first to demonstrate that sustained administration of [Leu31Pro34]PYY, a Y1-like receptor agonist, at a dose of 1 nmol/day for 14 days, effectively reverses the characteristic hyperlocomotion observed in OBX rats. This is particularly significant given that hyperlocomotion in the open field test is a well-recognized behavioral trait of the OBX animal model. In agreement with these results, previous research has shown that repeated exogenous intracerebroventricular (ICV) administration of [Leu31Pro34]NPY, another Y1-like agonist, also decreased hyperlocomotion in OBX rats. Interestingly, our current study also observed that [Leu31Pro34]PYY (1 nmol/day) decreased grooming behaviors in the open field in sham animals. Abnormal rearing and grooming in the open field test can be influenced by various factors, including aversive stimuli, strain differences, and the OBX lesions themselves. We also noted a decrease in both rearing and grooming at a higher dose of [Leu31Pro34]PYY (3 nmol), which also resulted in decreased locomotion in sham animals, potentially suggesting a sedative-like effect at this higher concentration. Prior studies have also reported that ICV administration of NPY and [Leu31Pro34]NPY decreased grooming in OBX rats. Beyond its effects on locomotion and grooming, [Leu31Pro34]PYY significantly and dose-dependently reversed the deficits in the social interaction test observed in OBX rats. This test is a widely employed paradigm for screening both anxiolytic and anxiogenic effects of various environmental and physiological factors. In accordance with these findings, previous research has shown that the administration of NPY into the basolateral amygdala and the dorso-caudal lateral septum produced anxiolytic-like effects in the social interaction test in naïve, normal animals. Taken together, these results indicate that chronic infusion of [Leu31Pro34]PYY exerts both anxiolytic and antidepressant-like effects specifically in the OBX rat model, underscoring its therapeutic potential in conditions of emotional dysregulation. In contrast to the pronounced effects of [Leu31Pro34]PYY, treatment with BIBO3304, a Y1 antagonist, at a dose of 3 nmol, exhibited only limited effects in the battery of behavioral tests employed in the present study. These results are consistent with earlier reports where neither BIBO3304 nor BIBP3226 (a first-generation Y1 antagonist) produced significant behavioral effects in the open field or forced swim test in control mice. However, a study by Goyal and colleagues in 2009 reported that BIBP3226 (0.1 nmol) actually enhanced hyperlocomotion in the open field in OBX rats. This finding is somewhat surprising, considering that BIBO3304 generally possesses a tenfold higher affinity for the Y1 receptor compared to BIBP3226, suggesting that this particular effect might be linked to the well-documented neurotoxic properties of BIBP3226 rather than a pure Y1 receptor antagonism. Interestingly, earlier studies have shown that the behavioral effects of NPY were abolished by pre-treatment with BIBO3304 in the forced swim test and social interaction test in control animals, further suggesting a direct role for this receptor subtype in emotional processes. In the current study, due to the inherent technical complexities and limitations of currently available tools, we were unable to investigate the specific effect of co-treatment with both an agonist and an antagonist of the Y1 receptor subtype. Specifically, both peptide agonists and antagonists of NPY Y1 and Y2 receptor subtypes typically exhibit poor penetration into the brain following peripheral injection. Furthermore, the distinct solvent vehicles required for peptide agonists are often incompatible with those needed for antagonists, making their simultaneous co-administration in a single infusion pump logistically challenging at present. It is also noteworthy that very scarce literature exists on the co-administration of an agonist and an antagonist for a given receptor within the same minipump, highlighting the technical novelty and challenges of such an approach. Future studies will necessitate the development of novel molecules that possess the ability to effectively cross the blood-brain barrier to facilitate such comprehensive co-administration studies.
Y2 Receptor Subtype in Depression and Anxiety-Related Behaviors
In the present study, treatment with BIIE0246, a Y2 receptor antagonist, significantly reversed the immobility time in the forced swim test in OBX rats, indicating an antidepressant-like effect. Additionally, this antagonist was observed to decrease grooming behavior in the open field and increase social interaction contacts in sham-operated animals, suggesting anxiolytic properties under normal conditions. The Y2 receptor subtype is known to play a crucial modulatory role in regulating the release of NPY itself, as well as other important neurotransmitters. Given that antidepressant activity has been consistently associated with elevated levels of NPY, while a depressive phenotype is often linked to low NPY levels, the pharmacological blockade of Y2 receptors has been hypothesized as an indirect yet effective strategy to increase endogenous NPY levels, thereby potentially improving emotional processes. In strong accordance with these preclinical data, germinal Y2 knockout mice exhibit a robust antidepressant- and anxiolytic-related phenotype, a phenotype that is independent of sex, age, or the specific behavioral paradigm employed. Furthermore, acute BIIE0246 treatment has also been shown to increase swimming time in the forced swim test in naïve control rats, further supporting its antidepressant-like potential.
In contrast to the beneficial effects of Y2 antagonism, treatment with PYY3-36, a Y2 receptor agonist, led to a notable increase in the immobility time in the forced swim test in OBX rats, suggesting a pro-depressive effect of Y2 receptor activation in this model. PYY3-36 also increased grooming behavior in the open field in sham animals. While we have previously demonstrated that the acute administration of another Y2 agonist, NPY13-36, had no effect in the open field or forced swim test in naïve animals, another Y2 agonist, C2-NPY, was shown to elicit an anxiogenic response in the basolateral amygdala in the social interaction test in control animals. While additional research is undoubtedly warranted, these nuanced findings collectively suggest that the Y2 receptor subtype may exert differential roles, with distinct outcomes, under normal physiological conditions compared to challenged, pathological emotional states. Recent data also suggest a potential role for Y4 and Y5 receptor subtypes in emotional processes, investigated in both naïve and knockout animal models. Therefore, future research should endeavor to thoroughly evaluate their possible contributions in various established animal models of depression-related behaviors to gain a more complete understanding of the NPY receptor family’s involvement.
Effect of OBX Lesion on the Level of NPY Y1 and Y2 Receptors
Given our clear observations of significant changes in behavioral responses following treatments with [Leu31Pro34]PYY and BIIE0246 in OBX animals, we formulated the hypothesis that these behavioral differences could be intrinsically linked to corresponding alterations in the expression levels of NPY receptors. Indeed, our rigorous quantitative autoradiographic studies revealed that specific Y2 receptor binding was significantly increased in both the hippocampus and the basolateral amygdala (BLA) of OBX rats. These particular brain regions were strategically chosen for investigation as preliminary data from our research group had previously suggested that these two areas exhibit disrupted neuronal dendritic arborization in the OBX rat model, indicating structural plasticity associated with the emotional disturbance. In apparent agreement with our current data, Caberlotto and Hurd in 2001 previously observed increased Y2 mRNA levels in post-mortem brain samples from depressive suicide completers, suggesting a similar upregulation in human pathology. Additionally, earlier studies investigating NPY expression in the OBX rat noted increased levels of preproNPY mRNA in the piriform cortex, hippocampus, and amygdala after the surgical removal of the olfactory bulbs. These findings collectively suggest that the decreased levels of NPY often observed in the OBX rat might be induced by the activation of negative presynaptic Y2 autoreceptors, which serve to regulate the subsequent release of this neuropeptide.
Therefore, the increased levels of Y2 receptor binding observed in the hippocampus and amygdala in our study may, at least partially, account for the detrimental effect of the Y2 agonist in the forced swim test in OBX rats, as well as the beneficial anxiolytic action of the Y2 antagonist in the social interaction test in sham animals. Conversely, the observed beneficial effects of the Y1 agonist may be directly related to a postsynaptic compensatory mechanism of NPY signaling, potentially arising from the hyperactivation of these Y2 negative autoreceptors. Our results collectively suggest that the detectable changes in the level of expression of the Y2 receptor in both the hippocampus and the amygdala are likely intrinsically linked with the known alterations in NPY levels within the OBX rat model, providing a more comprehensive understanding of the NPYergic system’s dysregulation in this model of emotional disorders.
Conclusion
In summary, the present study comprehensively explored the possible contribution of the NPY Y1 and Y2 receptor subtypes to the intricate landscape of depression- and anxiety-related behaviors in the olfactory bulbectomized rat model. Our robust data significantly extend earlier findings by strongly suggesting that the NPYergic system is indeed disrupted in processes associated with depression and anxiety. Crucially, our findings highlight a likely differential and distinct role for the Y1 and Y2 receptor subtypes in mediating these complex emotional states, indicating their potential as targets for novel, precise therapeutic interventions.
Acknowledgments
This comprehensive study was made possible through the generous financial support provided by grants from the Canadian Institutes of Health Research (supporting R.Q.). J.C.M.M. is a PhD student who received fellowship support from CONACyT-Mexico. We extend our sincere gratitude to Mira Thakur and Mark Farso for their diligent editing and proofreading of the text, and to Drs. Diksic and Skelin for their invaluable technical assistance. Boehringer Ingelheim (Ingelheim, Germany) is also gratefully acknowledged for generously providing the antagonists utilized in this research.