Neurotensin receptor-1 agonist PD 149163 modulates the lipopolysaccharide-induced behavioral disturbances in mice

Background Neuroendocrine-immune homeostasis is a prerequisite for neurobehavioral performances. Dysregulation of this homeostasis manifested in behavioral dysfunctions and neurodegenerative diseases, including schizophrenia and Parkinson’s disease. The present study aimed to investigate the role of PD 149163 (PD), a neurotensin agonist, in the modulation of behavioral disturbances induced by lipopolysaccharide (LPS) in mice. Thirty-six female mice, 12 weeks old, were divided into 6 groups ( n = 6/group). Group I (control) mice were given intraperitoneal (i.p.) injection of saline. Group II (LPS) received LPS (1 mg/kg, i.p.) for 5 days. Group III (LPS + PD Low) and IV (LPS + PD High) have received an injection of LPS (1 mg/kg, for 5 days) and after that treated with PD 100 µg/kg and 300 µg/kg, i.p., respectively, for 21 days. Group V (PD Low) and VI (PD High) were exposed to PD 100 µg/kg and 300 µg/kg, respectively, for 21 days. Results In the open-field test, the PD attenuated the behavior of LPS-exposed mice by increasing the number of squares crossed, time spent in the central square, rearing and grooming, and decreasing immobility, latency and defecation. Likewise, in the elevated plus-maze test, PD increased the number of entries on open and enclosed arms, time spent on open and enclosed arms, grooming and rearing, and reduced the head dipping and immobility in LPS-challenged mice. The PD enhanced the immobility time in the forced swimming test, and sucrose consumption in the sucrose preference test decreased after LPS exposure. Conclusion This study suggests that PD modulates the LPS-induced anxiety and depression-like behavioral impairments and could be an alternate choice of the atypical antipsychotic drugs (AAPDs) in the future.

the immune and neuroendocrine systems to maintain the homeostasis of animals.The research investigation revealed that immune activation could disrupt the function of neuroendocrine agents, including hormones, cytokines, etc., leading to neuroinflammation, HPA axis activation, neuropeptides imbalance (Ahmad et al., 2021;Holsboer & Ising, 2010).
The lipopolysaccharide (LPS), an endotoxin of the cell wall of gram-negative bacteria such as Escherichia coli, is a well-known disruptor of neuroendocrine-immune homeostasis (Ahmad et al., 2021).The systematic (peripheral) administration of LPS may induce psychopathological conditions such as anxiety, depression and cognitive-like behavioral impairments (Guzman- Martinez et al., 2019;Lee et al., 2008;Moraeset al., 2017).In addition, LPS also causes physiological changes (anorexia and weight loss) and psychiatric arrays (anhedonia, sexual and social).Hence, the LPS is used frequently to study the mechanism of anxiety or depression-like behaviors and develop targeted therapies to treat neurological disorders in the animal model (Banks et al., 2015).As a well-established immune stressor, the LPS induced the peripheral innate immune system leading to proinflammatory cytokines cascade, microglial activation, neuroinflammation and HPA axis activation (Beishuizen & Thijs, 2003;Orihuelaet al., 2016;Pepeet al., 2017).The LPS exposure enhances the secretion of various proinflammatory cytokines such as TNF-α, IFN-γ and IL-1β and nuclear factor-kappaB (NF-κB) system via Toll-like receptor 4 (TLR4) and the stimulation of reactive oxygen species (ROS) response and neuroendocrine components, particularly neurotransmitter and neurohormones (Batista et al., 2019;Bennett & Molofsky, 2019).
The hippocampus, a key area of the brain, is pivotal for learning and cognitive function that is vulnerable to stress (Rubin et al., 2014;Singh & Singh, 2017).The LPS via inflammatory and oxidative stress response impaired the cytoarchitectural design of pyramidal neurons in the hippocampus, resulting in hippocampal atrophy (Mustapha et al., 2019;Valero et al., 2017).The disruption of hippocampus function is exhibited in behavioral deficits like learning and memory impairment (Owen et al., 2019).Thus, the LPS-induced peripheral inflammation, HPA axis activation, hippocampus atrophy and neurotransmitters imbalance collectively lead to behavioral disturbances (Biesmans et al., 2013;Cordeiro et al., 2019).
For therapeutic management of psychiatric disorders, first (typical)-and second-generation (atypical) antipsychotic drugs (APDs) are available in the market.Since classical or typical APDs are associated with extrapyramidal (EPS) side effects, healthcare providers heavily rely on various atypical antipsychotic drugs (AAPDs) considering their efficacy and mechanism of action with the least side effects (Farah, 2015).Despite the availability of several AAPDs, anti-neuroinflammation modulation of psychosis is an emerging approach to treat psychiatric illness with a unique mechanism of action.Earlier reports suggested that antipsychotic drugs modulate the dysregulation of the neuroendocrine-immune axis under stress conditions (Meyer et al., 2011;Pandurangi & Buckley, 2019).AAPDs such as clozapine and risperidone (RIS) paliperidone (PAL) suppressed the pro-inflammatory cytokines induced by LPS exposure (MacDowell et al., 2013(MacDowell et al., , 2015;;Suginoet al., 2009).The PD 149163 (PD) is a modified 8-13 amino acid brain penetrating peptide selected analog of neurotensin receptor-1 (NTR-1), exhibited an antipsychotic drug-like property in the treatment of neuropsychiatric disorders (Petrie et al., 2004;Vadnieet al., 2016).The antipsychotic-like property of PD has been shown to suppress psychomotor agitation and disruptions in prepulse inhibition (PPI) without causing EPS as side effects (Fiefelet al., 2016), indicating the impact of neurotensin (NT) analogs more closely resembles that of AAPDs (Boules et al., 2013).Besides the well-known antipsychotic-like property, the PD's role in preventing behavioral deficits caused by stress or neuroinflammation has not been well studied and documented so far.However, this laboratory has elucidated the anti-inflammatory and anti-oxidative properties of PD (Mishra & Singh, 2021a) and its role in HPA axis regulation (Mishra & Singh, 2021b) against the LPS-challenged regimes in the mice model.Hence, the present study hypothesized that PD's antipsychotic and anti-inflammation activity might ameliorate the LPSinduced behavioral impairment in mice.Therefore, the present study aimed to evaluate the PD's role in modulating behavioral deficits induced by LPS exposure.

Animal and experimental design
Twelve-week-old Swiss albino female mice procured from the Indian Institute of Toxicology Research (IITR), Lucknow, INDIA, were kept in polyvinyl chloride cages (42 × 20 × 20 cm) in standard laboratory conditions, temperature 21 ± 1 °C, humidity 55 ± 5%, 12 h light-dark cycle and free to access food and water.After acclimatization for 2 weeks, mice were randomly divided into six groups (n = 6 per group, total 36 mice were used in this study).Group I (Control) were given intraperitoneal (i.p.) injection of physiological saline (0.9% NaCl).Group II (LPS) mice received LPS (i.p., 1 mg/kg, prepared in water); Escherichia coli serotype 026:B6, L-2654, Sigma-Aldrich, USA) for 5 days.Group III (LPS + PD Low) and IV (LPS + PD High) were received an injection of LPS (1 mg/kg, for 5 days) and after that treated i.p. with PD 149163 (Sigma-Aldrich, USA) 100 µg/kg and 300 µg/kg, respectively, for 21 days.Group V (PD Low) and VI (PD High) were exposed to PD 100 µg/kg BW and 300 µg/kg BW for 21 days.
The stock solution of PD was prepared by first dissolving the amount of peptide needed for the study in 1 mL of sterile water containing 0.1% (weight/volume) bovine serum albumin (BSA) diluted with normal saline containing 1% BSA.The PD solution was divided into equal aliquots of 0.1 ml stored at − 20 °C.During the administration period, each aliquot was further diluted in sterile saline to the required concentration and given, i.p., as a bolus injection of 0.5 ml between 9 and 10 AM.According to our laboratory protocol, after the completion of experiment, the animals were euthanized by phenobarbital anesthesia (Singh et al., 2016).The maintenance and handling of the animals were done according to the Guideline of the Care and Use of Laboratory Animals (National Institutes of Health, 2011).The experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of the University of Allahabad (approval number IAEC/ AU/2017(1)/019).

Behavioral studies
The behavioral test was performed 24 h after the LPS and after that weekly in the light phase (10.00 am-04.00pm) and recorded by a digital camera without the investigator.The entire experimental design and behavioral examination are shown in Fig. 1.

Open-field test (OFT)
The OFT was performed to measure mice's locomotor activity and anxiety-like behavior (Bassiet al., 2012).An open black wooden box (81 cm × 81 cm floor and 28 cm wall height) was used; the box floor was painted white lines (3 mm wide) to form 16 equal squares (20 cm × 20 cm each) (Fig. 2).Illumination was provided by using a LED white light (12 W) fixed at the height of 3 m from the center of the field.Each mouse was placed in the corner of the box and allowed to explore the openfield environment for 5 min.The number of squares crossed (line crossings with both hind paws), time spent in the central square, rearing (vertical activity), grooming (licking, scratching and nibbling), immobility, latency (time spent in the first square) and fecal boli (defecation) The open-field area was cleaned with 90% alcohol to eliminate odors before testing the next mouse.

Elevated plus maze (EPM)
The anxiety-like behavior was tested using a black elevated plus maze made up of Plexiglas.The plus ('+') shape maze has four equal-sized arms (30 × 5 cm), including two enclosed arms (15 cm in height) and two open arms, elevated 90 m above the floor (Fig. 2).The mouse was placed on a central arena (5 × 5 cm) facing an open arm and observed for 5 min.The number of entries into the open and enclosed arms, the time spent in open and enclosed arms, grooming, rearing and immobility were recorded and calculated during a single trial.An arm entry was recorded when four paws of the rat entered into any arm.The maze was cleaned with 90% alcohol to remove odors after each trial (Sulakhiya et al., 2016).

Forced swimming test (FST)
The FST was used to test the depression-like behavioral responses (Cryan et al., 2002).Briefly, each mouse was placed in a cylinder (diameter: 22 cm; height: 30 cm) containing water maintained at 24 ± 1 °C for 5 min (Fig. 2).The water was changed between testing sessions.During the test, the mice were video-recorded, and the duration of immobility was determined over the last 5 min of the test.The period of immobility (remain floating in the water without struggling) during the 5-min test was noted and evaluated.Increased immobility time depicts an increased depressive state.

Sucrose preference test (SPT)
This test was performed to evaluate anhedonia (Cordeiro et al., 2019).The procedure consisted of an acclimatization period 48 h before the test.The mice were trained to adapt with two bottles of 1% (w/v) sucrose solution placed in each cage.After twenty-four hours, sucrose solution in one bottle was replaced with tap water for 24 h.For the sucrose preference test, mice were housed in individual cages with free access to two bottles containing 30 ml of sucrose solution (1% w/v) and 30 ml of water.After 24 h of LPS injection, the volumes of consumed sucrose solution and water were recorded daily, and the sucrose preference was calculated as follows (Cordeiro et al., 2019): × 100.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 statistical software (GraphPad Software Inc., San Diego, CA, USA).All values are presented as mean ± SEM (standard errors of the means) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.The significant level was determined at *P < 0.05, **P < 0.01 and ***P < 0.001.

Behavioral analysis in open-field test (OFT)
The results of OFT are shown in Fig. 3 Further, the number of squares crossed by the animal was significantly increased in LPS + PD High and PD Low (P < 0.01 for both) group compared to LPS-and LPS + PD Low-treated mice (Fig. 3A).The time spent by mice in the central square was substantially decreased in LPS (P < 0.001)-, LPS + PD Low (P < 0.05)-and PD High (P < 0.05)-exposed mice compared to the vehicle group.Compared to LPS-challenged mice, the time spent in the central square was increased in all treatment groups, i.e., LPS + PD Low (P < 0.05), LPS + PD High (P < 0.001), PD Low (P < 0.001) and PD High (P < 0.05).Similarly, the mice's time spent in the central square was also increased in the LPS + PD High and PD Low-exposed group (P < 0.001 for both) compared with the LPS + PD Low group (Fig. 3B).The post hoc analysis showed the decreased and increased rearing frequency in LPS (P < 0.05)-and PD High (P < 0.01)-treated mice, respectively, as compared to control.Further, compared to LPS exposure, the LPS + PD High, PD Low and PD High-exposed mice showed increased rearing frequency (P < 0.001 for all).Likewise, the rearing frequency was also increased in LPS + PD High (P < 0.05), PD Low (P < 0.05) and PD High (P < 0.001) treatment group compared to LPS + PD Low (Fig. 3C).
There was no significant effect of LPS on grooming frequency compared to control, but it increased only in PD High-exposed mice (P < 0.05).After PD treatment, the grooming frequency was substantially increased in LPS + PD High (P < 0.01), PD Low (P < 0.001) and PD High group (P < 0.001) in comparison with LPS exposure.The PD High group also showed increased grooming (P < 0.05) compared with the LPS + PD Low group (Fig. 3D).The immobility in LPS-exposed mice was significantly decreased (P < 0.05) than the control, whereas increased in PD Low (P < 0.01)-and PD High (P < 0.01)-exposed group compared to LPS (Fig. 3E).
The latency time was significantly increased in LPS (P < 0.001)-and PD High (P < 0.01)-treated mice compared to control.Further, the latency was decreased in LPS + PD Low, LPS + PD High and PD Low treatment group (P < 0.001 for all) compared to LPS exposure (Fig. 3F).The frequency of fecal boli was substantially increased in only LPS-exposed mice (P < 0.01) in comparison with control, whereas deceased in LPS + PD Low (P < 0.05), LPS + PD High (P < 0.01), PD Low (P < 0.01) and PD High (P < 0.01) group compared to LPS-exposed mice (Fig. 3G).

Behavioral analysis in elevated plus maze (EPM) test
The  At the same time, the time spent on open arms was increased in LPS + PD High (P < 0.01)-exposed mice than in the LPS (Fig. 4B).Likewise, the number of entries in enclosed arms was increased in the LPS (P < 0.01) and LPS + PD Low (P < 0.05) group, in comparison with the control; other groups showed similar results as in control.Further, compared to the LPS-and LPS + PD Lowexposed group, none of the treatment groups showed any significant changes regarding the entry to open arms (Fig. 4C).The time spent in enclosed arms was significantly increased in the LPS (P < 0.001)-, LPS + PD Low (P < 0.05)-and PD High (P < 0.05)-treated group compared to the control.Compared to LPS-and LPS + PD Low-exposed mice, other groups showed no significant effect in time spent in closed arms (Fig. 4D).The grooming frequency of mice was significantly increased in LPS (P < 0.05)-and PD High (P < 0.001)-treated mice compared to control.Compared to LPS-challenged mice, the LPS + PD High-, PD Low-and PD High-exposed mice revealed the increased (P < 0.001 for all) grooming frequency, whereas the PD High group (P < 0.001) also showed increased grooming compared to LPS + PD Low treatment (Fig. 4E).The rearing frequency was significantly decreased in the LPS (P < 0.01)-and LPS + PD Low (P < 0.05)-exposed mice compared to control, while it was increased in LPS + PD High-treated mice compared to LPS-and LPS + PD Low-challenged mice (Fig. 4F).
The number of head dipping was increased only in LPS (P < 0.01) compared to the control group, whereas compared to LPS, it was decreased in all treatment groups, i.e., LPS + PD Low (P < 0.001), LPS + PD High (P < 0.001), PD Low (P < 0.001) and PD High (P < 0.01) (Fig. 4G).The immobility in LPS-induced mice was increased (P < 0.01) compared to control.After PD treatment, the LPS + PD High-and PD Low-exposed group showed increased immobility (P < 0.001 and P < 0.01, respectively) compared to LPS (Fig. 4H).

Measurement of immobility time in forced swimming test (FST)
The one-way ANOVA showed that the immobility was significantly changed [F (5, 60) = 6.62,P < 0.001] in the treatment group compared to the control.In Tukey's multiple comparison tests, immobility was significantly increased in the LPS (P < 0.001)-, LPS + PD Low (P < 0.05)-and PD High (P < 0.05)-treated group compared with control (Fig. 5A).After PD treatment, the LPS + PD Low, LPS + PD High and PD Low group revealed decreased immobility (P < 0.001 for all) relative to LPS-challenged mice.At the same time, compared to LPS + PD Low-exposed group exhibited reduced immobility in the LPS + PD High (P < 0.001)-and PD Low (P < 0.01)-treated group (Fig. 5A).

Analysis of sucrose consumption in sucrose preference test (SPT)
The sucrose preference test studied the anhedonic behavior of mice.The result of one-way ANOVA showed that the treatment group exhibited significant changes in sucrose consumption [F (5, 68) = 5.42, P < 0.001], in comparison with the control.The Tukey post hoc test showed that the sucrose intake was significantly reduced only in LPS-exposed mice (P < 0.01) when compared to control (Fig. 5B).After PD treatment, sucrose preference in the LPS + PD High-and PD Low-exposed group was substantially increased (P < 0.001 and P < 0.05, respectively) than the LPS-challenged mice (Fig. 5B).

Discussion
The reciprocal bidirectional interaction between the neuroendocrine and immune systems provides a finely tuned regulatory system required to maintain mental health (Bennett & Molofsky, 2019).Psychologically, peripheral inflammation can aggravate ongoing neurological damage and exaggerate motor and cognitive impairments in patients with neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD).The peripherally generated inflammatory mediators induced by systemic challenge with LPS have increased brain inflammatory cytokines and oxidative stress response, HPA axis activation and hippocampus atrophy resulting in progressive neurotoxicity and behavioral deficit (Banks et al., 2015;Jamali-Raeufy et al., 2019;Zhao et al., 2019).The present study has been investigated the protective role of NT agonist PD in the LPS-induced behavioral deficit in mice.
In the present study, the OFT revealed a significantly decreased number of square crossed and time spent in the central square in LPS-challenged mice, as reported by others (Bassi et al., 2012;Lee et al., 2018).The rearing and grooming frequency were decreased, whereas immobility, latency and defecation were increased after LPS exposure which confirms the anxiety-like behavior in animals (Bassi et al., 2012).On the other hand, the PD exposure enhanced the number of squares crossed, time spent in the central square, rearing and grooming frequency, whereas it decreased the immobility, latency time and defecation might be corroborated with the antipsychotic property of PD against the psychotic illness.The previous research reports have been suggested that antipsychotic drugs such as aripiprazole and paliperidone modulate the LPS-induced impairment like the motor deficit, rearing, grooming, immobility and defecation in OFT (Kumar & Mohanty, 2015;Viana et al., 2013).Thus, the antipsychotic-like activity of PD could be linked with the attenuation of LPS-induced anxiety-like behavior in mice.In the EPM test, the decreased entries of mice in open and enclosed arms and time spent on open arms in the LPS-exposed group showed the anxiety-like behavior as suggested by others (Kumar et al., 2020;Sulakhiya et al., 2016).As reported earlier, the grooming and rearing frequency decreased after LPS exposure (Sulakhiya et al., 2016).In addition, the increased immobility of LPSinduced mice in EPM is also a manifestation of anxietylike behavior (Bassi et al., 2012).After the PD exposure on LPS-challenged mice, the number of entries in open and enclosed arms and time spent on open arms along with grooming and rearing frequency was increased.In contrast, the immobility time and the number of head dipping were decreased.Thus, these results showed the LPS-induced anxiety-like behavior was prevented and reversed to control by NT agonist PD.
In FST, the immobility time was assessed to evaluate the LPS-induced depressive-like behavior of mice.In the present study, the LPS-enhanced immobility time of mice was reversed by PD.The previous study showed that the increased immobility time after LPS injection is a result of depression (Zhang et al., 2019), which is further ameliorated by antipsychotic drugs like clozapine and risperidone (MacDowell et al., 2013;Todorović & Filipović, 2017).Likewise, a recent study also suggested that the PD reduced immobility time in the rodent model (Carey et al., 2017).The anhedonia represents the decreased ability to experience pleasure is a sign of depression (Scheggi et al., 2018).The anhedonia was measured by the SPT, which is a reward-based test.The result showed that the LPS-exposed mice reduced the preference for sucrose consumption, whereas LPS-induced sucrose intake was reversed and normalized to control by PD.Additionally, the previous reports have also been revealed that LPSinduced systemic inflammation is one of the potent contributors to anhedonia, a depressive-like behavior (Yeh et al., 2018).Some antipsychotic and anti-depression drugs, such as leptin, modulated the LPS-induced immobility (in FST) and anhedonia (in SPT) (Cordeiro et al., 2019).
The neuroendocrine and immune systems intimately link to each other; any disturbance to neuroendocrineimmune communication leads to animals' behavioral deficits (Krishnan & Nestler, 2011;Meyer et al., 2011).The endotoxin LPS can induce behavioral alterations through diverse effects on the neuroendocrine-immune axis (Sugino et al., 2009).The peripheral LPS administration induced inflammation, oxidative stress and hyperactivation of the HPA axis (Turnbull & Rivier, 1999;Utsuyama & Hirokawa, 2001).The LPS injection accelerates the release of pro-inflammatory cytokines such as IL-6 and TNF-α and oxidative stress response, resulting in neuroinflammation characterized by the HPA axis activation, neurotransmitters and neuropeptides, particularly dopamine and NT, imbalances and microglial activation (Lee et al., 2018;Mac-Dowell et al., 2013).The previous study evaluated the LPSinduced inflammation, oxidative stress, NT and NTR-1 intensity (Mishra & Singh, 2021a), and neuroinflammation, including HPA axis activation and hippocampus atrophy (Mishra & Singh, 2022).We found that the LPS exposure evoked an increase in the basal ACTH and corticosterone level, indicating a stimulated HPA axis response (Mishra & Singh, 2022).The level of anxiety-related behavior might be corroborated with the significantly increased plasma ACTH and corticosterone level as shown in the LPS group, but not in the PD co-treated group.However, the PD co-treatment maintained the HPA axis homeostasis and counteracted the behavioral disturbances accompanied by LPS.Additionally, in our previous study, the PD attenuated the LPS-induced systemic inflammation and oxidative stress, which could be linked to reducing anxiety and depression.The PD modulates the neuroinflammation and cytoarchitectural disruption of the hippocampus region, particularly the cornu Ammonis (CA) 1 and CA3 area of hippocampus (Mishra & Singh, 2022).Moreover, PD inhibited amphetamine-induced hyperactivity in mice, indicating that NTR-1 activation inhibits psychomotor agitation (Boules et al., 2013;Fiefel et al., 2016).PD also increased prepulse inhibition, suggesting that the NTR-1 activation reduces prepulse inhibition deficits which often co-occur with psychosis in humans.In addition, the PD, like NT, acts upon the NTR-1 in the neuronal cells and regulates dopamine signaling.The PD inhibits dopamine activity, which contributes to neuropsychiatric disorders such as bipolar disorder and schizophrenia (Vadnie et al., 2016).In addition, PD can also regulate other neurotransmitter functions, especially GABA and serotonin, to maintain mental health.Therefore, it may be concluded that PD acts as a neuroimmunomodulator and prevent LPSinduced behavioral dysfunctions, including anxiety and depression.
Furthermore, in the present study, the PD exposure was given to the mice in two doses: 100 µg/kg BW (low) and 300 µg/kg BW (high), to evaluate which dose is more appropriate to prevent behavioral deficits against LPS exposure.The results showed that the LPS effect on psychopathology is also extended to some degree in LPS + PD Low-treated mice (although much less than LPS-challenged), whereas a high dose of PD reversed the effect of LPS to the control level.Thus, these results suggested that the PD modulated the LPS-induced behavioral impairment in mice in a dose-dependent Mishra and Singh The Journal of Basic and Applied Zoology (2024) 85:27 manner.Moreover, the only PD High-treated mice also revealed behavioral abnormalities/psychopathology to some quantity might be due to enhanced inflammation and oxidative stress by PD exposure as reported in our previous study.

Conclusions
This study elucidated the protection/reversal of LPSchallenged behavioral deficits by the NTR-1 agonist PD 149163 in a dose-dependent manner.Hence, the antiinflammatory, anti-oxidative, HPA axis modulating and antipsychotic-like properties of PD might protect the behavioral deficits in LPS-exposed mice.The PD high dose is also exhibited some behavioral dysfunctions, which reflects the drug-induced side effect.Thus, the present study suggests that PD may play a significant role in ameliorating neuropsychiatric illness, including behavioral impairments.