The antipsychotic agent sulpiride microinjected into the ventral pallidum restores positive symptom-like habituation disturbance in MAM-E17 schizophrenia model rats.

Dysfunction of subcortical D2-like dopamine receptors (D2Rs) can lead to positive symptoms of schizophrenia, and their analog, the increased locomotor activity in schizophrenia model MAM-E17 rats. The ventral pallidum (VP) is a limbic structure containing D2Rs. The D2R antagonist sulpiride is a widespread antipsychotic drug, which can alleviate positive symptoms in human patients. However, it is still not known how sulpiride can influence positive symptoms via VP D2Rs. We hypothesize that the microinjection of sulpiride into the VP can normalize hyperactivity in MAM-E17 rats. In addition, recently, we showed that the microinjection of sulpirid into the VP induces place preference in neurotypical rats. Thus, we aimed to test whether intra-VP sulpiride can also have a rewarding effect in MAM-E17 rats. Therefore, open field-based conditioned place preference (CPP) test was applied in neurotypical (SAL-E17) and MAM-E17 schizophrenia model rats to test locomotor activity and the potential locomotor-reducing and rewarding effects of sulpiride. Sulpiride was microinjected bilaterally in three different doses into the VP, and the controls received only vehicle. The results of the present study demonstrated that the increased locomotor activity of the MAM-E17 rats was caused by habituation disturbance. Accordingly, larger doses of sulpiride in the VP reduce the positive symptom-analog habituation disturbance of the MAM-E17 animals. Furthermore, we showed that the largest dose of sulpiride administered into the VP induced CPP in the SAL-E17 animals but not in the MAM-E17 animals. These findings revealed that VP D2Rs play an important role in the formation of positive symptom-like habituation disturbances in MAM-E17 rats.

The role of intraamygdaloid oxytocin in spatial learning and avoidance learning.

The goal of the present study is to investigate the role of intraamygdaloid oxytocin in learning-related mechanisms. Oxytocin is a neuropeptide which is involved in social bonding, trust, emotional responses and various social behaviors. By conducting passive avoidance and Morris water maze tests on male Wistar rats, the role of intraamygdaloid oxytocin in memory performance and learning was investigated. Oxytocin doses of 10 ng and 100 ng were injected into the central nucleus of the amygdala. Our results showed that 10 ng oxytocin significantly reduced the time required to locate the platform during the Morris water maze test while significantly increasing the latency time in the passive avoidance test. However, the 100 ng oxytocin experiment failed to produce a significant effect in either of the tests. Wistar rats pretreated with 20 ng oxytocin receptor antagonist (L-2540) were administered 10 ng of oxytocin into the central nucleus of the amygdala and were also subjected to the aforementioned tests to highlight the role of oxytocin receptors in spatial- and avoidance learning. Results suggest that oxytocin supports memory processing during both the passive avoidance and the Morris water maze tests. Oxytocin antagonists can however block the effects of oxytocin in both tests. The results substantiate that oxytocin uses oxytocin receptors to enhance memory and learning performance.

Intraamygdaloid Oxytocin Increases Time Spent on Social Interaction in Valproate-Induced Autism Animal Model.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder that affects about 1.5% of children worldwide. One of the core symptoms is impaired social interaction. Since proper treatment has not been found yet, an investigation of the exact pathophysiology of autism is essential. The valproate (VPA)-induced rat model can be an appropriate way to study autism. Oxytocin (OT) may amend some symptoms of ASD since it plays a key role in developing social relationships. In the present study, we investigated the effect of the intraamygdaloid OT on sham and intrauterine VPA-treated rats' social interaction using Crawley's social interaction test. Bilateral guide cannulae were implanted above the central nucleus of the amygdala (CeA), and intraamygdaloid microinjections were carried out before the test. Our results show that male Wistar rats prenatally exposed to VPA spent significantly less time on social interaction. Bilateral OT microinjection increased the time spent in the social zone; it also reached the level of sham-control animals. OT receptor antagonist blocked this effect of the OT but in itself did not significantly influence the behavior of the rats. Based on our results, we can establish that intraamygdaloid OT has significantly increased time spent on social interaction in the VPA-induced autism model, and its effect is receptor-specific.

The antipsychotic drug sulpiride in the ventral pallidum paradoxically impairs learning and induces place preference.

Sulpiride, as a D2-like dopamine (DA) receptor (D2R) antagonist, is an important antipsychotic drug in the treatment of schizophrenia. Recently, we have shown that the activation of D2Rs in the ventral pallidum (VP) modulates the activity of the ventral tegmental area (VTA) DAergic neurons. According to our hypothesis, intra-VP sulpiride can influence the motivational and learning processes, pervasively modifying the behavior of examined animals. In the present study, sulpiride was microinjected into the VP of male Wistar rats in three different doses. Morris water maze (MWM) test was applied to investigate the effects of sulpiride on spatial learning, while conditioned place preference (CPP) test was used to examine the potential rewarding effect of the drug. In order to show, whether the animals can associate the rewarding effect with an area which can be recognized only on its spatial location, we introduced a modified version of the CPP paradigm, the spatial CPP test. Our results show that the intra-VP sulpiride dose-dependently impairs learning processes. However, the largest dose of sulpiride induces place preference. Results of the spatial CPP paradigm demonstrate that the animals cannot associate the rewarding effect of the drug with the conditioning area based on its spatial location. In the CPP paradigm, locomotor activity decrease could be observed in the sulpiride-treated rats, likely because of a faster habituation with the conditioning environment. In summary, we can conclude that intra-VP sulpiride has a dual effect: it diminishes the hippocampus-dependent spatial learning processes, in addition, it has a dose-dependent rewarding effect.

Effect of D1- and D2-like Dopamine Receptor Antagonists on the Rewarding and Anxiolytic Effects of Neurotensin in the Ventral Pallidum.

BACKGROUND: Neurotensin (NT) acts as a neurotransmitter and neuromodulator in the central nervous system. It was shown previously that NT in the ventral pallidum (VP) has rewarding and anxiolytic effects. NT exerts its effect in interaction with dopamine (DA) receptors in numerous brain areas; however, this has not yet been investigated in the VP. The aim of this study was to examine whether the inhibition of D1-like and D2-like DA receptors of the VP can modify the above mentioned effects of NT. METHODS: Microinjection cannulas were implanted by means of stereotaxic operations into the VP of male Wistar rats. The rewarding effect of NT was examined by means of a conditioned place preference test. Anxiety was investigated with an elevated plus maze test. To investigate the possible interaction, D1-like DA receptor antagonist SCH23390 or D2-like DA receptor antagonist sulpiride were microinjected prior to NT. All of the drugs were also injected independently to analyze their effects alone. RESULTS: In the present experiments, both the rewarding and anxiolytic effects of NT in the VP were prevented by both D1-like and D2-like DA receptor antagonists. Administered on their own, the antagonists did not influence reward and anxiety. CONCLUSION: Our present results show that the activity of the D1-like and D2-like DA receptors of the VP is a necessary requirement for both the rewarding and anxiolytic effects of NT.

The Role of Intraamygdaloid Oxytocin and D2 Dopamine Receptors in Reinforcement in the Valproate-Induced Autism Rat Model.

BACKGROUND: autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting around 1 out of 68 children and its incidence shows an increasing tendency. There is currently no effective treatment for ASD. In autism research, the valproate (VPA)-induced autism rodent model is widely accepted. Our previous results showed that intraamygdaloid oxytocin (OT) has anxiolytic effects on rats showing autistic signs under the VPA-induced autism model. METHODS: rats were stereotaxically implanted with guide cannulae bilaterally and received intraamygdaloid microinjections. In the present study, we investigated the possible role of intraamygdaloid OT and D2 dopamine (DA) receptors on reinforcement using VPA-treated rats in a conditioned place preference test. OT and/or an OT receptor antagonist or a D2 DA antagonist were microinjected into the central nucleus of the amygdala (CeA). RESULTS: valproate-treated rats receiving 10 ng OT spent significantly longer time in the treatment quadrant during the test session of the conditioned place preference test. Prior treatment with an OT receptor antagonist or with a D2 DA receptor antagonist blocked the positive reinforcing effects of OT. The OT receptor antagonist or D2 DA antagonist in themselves did not influence the time rats spent in the treatment quadrant. CONCLUSIONS: Our results show that OT has positive reinforcing effects under the VPA-induced autism rodent model and these effects are OT receptor-specific. Our data also suggest that the DAergic system plays a role in the positive reinforcing effects of OT because the D2 DA receptor antagonist can block these actions.

Intraamygdaloid Oxytocin Reduces Anxiety in the Valproate-Induced Autism Rat Model.

BACKGROUND: Autism spectrum disorder (ASD) is a lifelong neurodevelopmental disorder affecting about 1.5% of children, and its prevalence is increasing. Anxiety is one of the most common comorbid signs of ASD. Despite the increasing prevalence, the pathophysiology of ASD is still poorly understood, and its proper treatment has not been defined yet. In order to develop new therapeutic approaches, the valproate- (VPA) induced rodent model of autism can be an appropriate tool. Oxytocin (OT), as a prosocial hormone, may ameliorate some symptoms of ASD. METHODS: In the present study, we investigated the possible anxiolytic effect of intraamygdaloid OT on VPA-treated rats using the elevated plus maze test. RESULTS: Our results show that male Wistar rats prenatally exposed to VPA spent significantly less time in the open arms of the elevated plus maze apparatus and performed significantly less head dips from the open arms. Bilateral OT microinjection into the central nucleus of the amygdala increased the time spent in the open arms and the number of head dips and reduced the anxiety to the healthy control level. An OT receptor antagonist blocked the anxiolytic effects of OT. The antagonist by itself did not influence the time rats spent in the open arms. CONCLUSIONS: Our results show that intraamygdaloid OT has anxiolytic effects in autistic rats.

QRFP administration into the medial hypothalamic nuclei improves memory in rats.

Even though several of RFamide peptides have been shown to modify memory and learning processes in different species, almost nothing is known regarding cognitive effects of recently discovered neuropeptide QRFP. Considering multiple physiological functions of QRFP, localization of QRFP-synthesizing neurons in the hypothalamus and its' widely spread binding sites within the CNS, the present study was designed to investigate the possible role of QRFP in the consolidation of spatial memory. As target area for microinjection, the medial hypothalamic area, including dorsomedial (DMN) and ventromedial (VMN) nuclei, has been chosen. At first, the effects of two doses (200 ng and 400 ng) of QRFP were investigated in Morris water maze. After that receptor antagonist BIBP3226 (equimolar amount to the effective dose of neuropeptide) was applied to elucidate whether it can prevent effects of QRFP. To reveal possible changes in anxiety level, animals were tested in Elevated plus maze. The higher dose of QRFP (400 ng) improved short-term memory consolidation in Morris water maze. Pretreatment with antagonist BIBP3226 abolished cognitive effects of QRFP. The neuropeptide did not affect anxiety level of rats. This study provides unique evidence regarding the role of QRFP in the consolidation of memory and gives the basis for further investigations of neuropeptide's cognitive effects.

Cognitive performance of the MAM-E17 schizophrenia model rats in different age-periods.

Cognitive disturbances are among the most important features of schizophrenia, and have a significant role in the outcome of the disease. However, the treatment of cognitive symptoms is poorly effective. In order to develop new therapeutic opportunities, the MAM-E17 rat model of schizophrenia can be an appropriate implement. In the present study we investigated several cognitive capabilities of MAM-treated rats using radial arm maze (RAM) task, which corresponds to the recent research directives. Because of the diachronic appearance of schizophrenia symptoms and the early appearance of cognitive deficiencies, we carried out our experiments in three different age-periods of rats, i.e. in prepuberty, late puberty and adulthood. The performance of MAM-E17 rats was similar to control rats in the acquisition phase of RAM task, except for puberty. However, after rearrangement of reward positions (in the reverse paradigm) the number of errors of MAM-treated rats was higher in each age-period. In the reverse paradigm MAM-treated groups visited more frequently those non-rewarding arms, which were previously rewarding. Our results suggest that working memory of MAM-E17 rats is impaired. This deficit depends on the difficulty of the task and on the age-period. MAM-E17 rats seem to be more sensitive in puberty in comparison to controls. Diminished behavioral flexibility was shown as well. These behavioral results observed in MAM-E17 rats were similar to those of cognitive deficiencies in schizophrenia patients. Therefore, MAM-E17 model can be a useful implement for further research aiming to improve cognition in schizophrenia.

Ventromedial prefrontal cortex is involved in preference and hedonic evaluation of tastes.

The ventromedial prefrontal cortex (vmPFC) of rats has reciprocal connections with the gustatory and the hedonic impact coding structures. The main goal of the present study was to investigate the involvement of local neurons of vmPFC and their catecholaminergic innervations in taste preference and taste reactivity test. Therefore, kainate or 6-hydroxydopamine (6-OHDA) lesions were performed in the vmPFC by iontophoretic method. In the first experiment, taste preference was tested to 250 mM and 500 mM glucose solutions over water in two-bottle choice test. In the second experiment, taste reactivity was examined to 4 concentrations of glucose solutions (250 mM, 500 mM, 750 mM and 1000 mM) and 4 concentrations of quinine solutions (0.125 mM, 0.25 mM, 1.25 mM and 2.5 mM). Our results showed, that kainate microlesion of vmPFC did not modify the preference of 250 mM and 500 mM glucose solutions in two-bottle choice test. In contrast, 6-OHDA microlesion of vmPFC resulted in increased preference to the higher concentration of glucose (500 mM) solution over water. Results of taste reactivity test showed that kainate lesion resulted in more ingestive and less rejective responses to 750 mM glucose solution and elevated rejectivity to the higher concentrations (1.25 mM and 2.5 mM) of quinine solutions. 6-OHDA lesion of vmPFC increased the number of ingestive responses to highly concentrated (500 mM, 750 mM and 1000 mM) glucose solutions and decreased the number of ingestive responses to the lower concentration (0.125 mM) of quinine solution. The present data provide evidence for the important role of vmPFC neurons and catecholaminergic innervation of the vmPFC in the regulation of hedonic evaluation of tastes and in the hedonic consummatory behavior.

Iontophoretic microlesions with kainate or 6-hydroxidopamine in ventromedial prefrontal cortex result in deficit in conditioned taste avoidance to palatable tastants.

Effects of kainate or 6-hydroxidopamine (6-OHDA) lesions in the ventromedial prefrontal cortex (vmPFC) on taste-related learning and memory processes were examined. Neurotoxins were applied by iontophoretic method to minimize the extent of lesion and the side effects. Acquisition and retention of conditioned taste avoidance (CTA) was tested to different taste stimuli (0.05 M NaCl, 0.01 M saccharin, 0.01 M citrate and 0.00025 M quinine). In the first experiment, palatability index of taste solutions with these concentrations has been determined as strongly palatable (NaCl, saccharin), weakly palatable (citrate) and weakly unpalatable (quinine) taste stimuli. In two other experiments vmPFC lesions were performed before CTA (acquisition) or after CTA (retrieval). Our results showed that both kainate and 6-OHDA microlesions of vmPFC resulted in deficit of CTA acquisition (to NaCl, saccharin and citrate) and retrieval (to NaCl and saccharin). Deficits were specific to palatable tastants, particularly those that are strongly palatable, and did not occur for unpalatable stimulus. The present data provide evidence for the important role of vmPFC neurons and catecholaminergic innervation of the vmPFC in taste related learning and memory processes.

Role of D2 dopamine receptors of the ventral pallidum in inhibitory avoidance learning.

In our present experiments, the role of D2 dopamine (DA) receptors of the ventral pallidum (VP) was investigated in one trial step-through inhibitory avoidance paradigm. Animals were shocked 3 times in the conditioning trial, with 0.5mA current for 1s. Subsequently bilateral microinjection of the D2 DA receptor agonist quinpirole was administered into the VP in three doses (0.1µg, 1.0µg or 5.0µg in 0.4µl saline). We also applied the D2 DA receptor antagonist sulpiride (0.4µg in 0.4µl saline) alone or 15min prior to the agonist treatment to elucidate whether the agonist effect was specific for the D2 DA receptors. Control animals received saline. In a supplementary experiment, it was also investigated whether application of the same conditioning method leads to the formation of short-term memory in the experimental animals. In the experiment with the D2 DA receptor agonist, only the 0.1µg quinpirole increased significantly the step-through latency during the test trials: retention was significant compared to the controls even 2 weeks after conditioning. The D2 DA receptor antagonist sulpiride pretreatment proved that the effect was due to the agonist induced activation of the D2 DA receptors of the VP. The supplementary experiment demonstrated that short-term memory is formed after conditioning in the experimental animals, supporting that the agonist enhanced memory consolidation in the first two experiments. Our results show that the activation of the D2 DA receptors in the VP facilitates memory consolidation as well as memory-retention in inhibitory avoidance paradigm.

Effects of RFamide-related peptide-1 (RFRP-1) microinjections into the central nucleus of amygdala on passive avoidance learning in rats.

The amygdaloid body (AMY) plays an important role in memory, learning and reward-related processes. RFRP-1 immunoreactive fibers and NPFF receptors were identified in the AMY, and previously we verified that RFRP-1 infused into the central nucleus of AMY (CeA) induced place preference. The aim of the present study was to examine the possible effects of RFRP-1 in the CeA on passive avoidance learning. Male Wistar rats were examined in two-compartment passive avoidance paradigm. Animals were shocked with 0.5mA current and subsequently were microinjected bilaterally with 50ng or 100ng RFRP-1 in volume of 0.4µl, or 20ng NPFF receptor antagonist RF9 (ANT) alone, or antagonist 15min before 50ng RFRP-1 treatments into the CeA. Fifty nanogram dose of RFRP-1 significantly increased the step-through latency time, the 100ng RFRP-1 and the ANT alone were ineffective. The effect of 50ng RFRP-1 was eliminated by the ANT pretreatment. Our results suggest that intraamygdaloid RFRP-1 enhances learning processes and memory in aversive situations and this effect can specifically be prevented by ANT pretreatment.

Role of ventral pallidal D2 dopamine receptors in the consolidation of spatial memory.

The role of dopamine (DA) receptors in spatial memory consolidation has been demonstrated in numerous brain regions, among others in the nucleus accumbens which innervates the ventral pallidum (VP). The VP contains both D1 and D2 DA receptors. We have recently shown that the VP D1 DA receptor activation facilitates consolidation of spatial memory in Morris water maze test. In the present study, the role of VP D2 DA receptors was investigated in the same paradigm. In the first experiment, the D2 DA receptor agonist quinpirole was administered into the VP of male Wistar rats in three doses (0.1, 1.0 or 5.0µg, respectively in 0.4µl physiological saline). In the second experiment, the D2 DA receptor antagonist sulpiride was applied to elucidate whether it can antagonise the effects of quinpirole. The antagonist (4.0µg, dissolved in 0.4µl physiological saline) was microinjected into the VP either by itself or prior to 1.0µg agonist treatment. Control animals received saline in both experiments. The two higher doses (1.0 and 5.0µg) of the agonist accelerated memory consolidation relative to controls and increased the stability of the consolidated memory against extinction. Sulpiride pretreatment antagonised the effects of quinpirole. In addition, the antagonist microinjected into the VP immediately after the second conditioning trial impaired learning functions. The present data provide evidences for the important role of VP D2 DA receptors in the consolidation and stabilization of spatial memory.

Effects of direct QRFP-26 administration into the medial hypothalamic area on food intake in rats.

The RFamide peptide family comprises a number of biologically active peptides sharing RF motif at their C-terminal end. These peptides are involved in the control of multiple physiological functions including regulation of metabolism and feeding behavior. QRFP-43 as well as its 26-aminoacid residue QRFP-26 are able to cause orexigenic effect when administered to the rodents' cerebral ventricles. QRFPs have been suggested as the endogenous ligands of the previously orphan GPR103 receptors. GPR103 receptors share amino acid identity with other receptors of neuropeptides involved in feeding (NPY, NPFF, galanin). QRFP-26 expressing neurons and binding sites are densely present in the rat medial hypothalamus (MHA), an area directly responsible for the regulation of feeding. QRFP-26 was delivered to the target area by direct intrahypothalamic microinjection, and the consumption of liquid food was measured over a 60 min period. Both doses (100 and 200 ng) significantly increased food intake. Non-specific receptor antagonist BIBP3226 eliminated the orexigenic effect caused by QRFP-26 administration. Effective doses of QRFP-26 did not modify general locomotor activity and behavioral patterns examined in the open-field test. This study is the first reporting feeding modulating effects following direct intrahypothalamic QRFP-26 administration.

Anxiolytic effect of neurotensin microinjection into the ventral pallidum.

Neurotensin (NT) acts as a neurotransmitter and neuromodulator in the central nervous system. NT is involved in reward and memory processes, drug addiction and also in the regulation of anxiety. The ventral pallidum (VP) receives neurotensinergic innervation from the ventral striatopallidal pathway originating from the nucleus accumbens. Positive reinforcing effects of NT in the VP had been shown recently, however the possible effects of NT on anxiety have not been examined yet. In our present experiments, the effects of NT on anxiety were investigated in the VP. In male Wistar rats bilateral microinjections of 100 ng or 250 ng NT were delivered in the volume of 0.4 µl into the VP, and elevated plus maze (EPM) test was performed. In another groups of animals, 35 ng NT-receptor 1 (NTR1) antagonist SR 48,692 was applied by itself, or microinjected 15 min before 100 ng NT treatment. Open field test (OPF) was also conducted. The 100 ng dose of NT had anxiolytic effect, but the 250 ng NT did not influence anxiety. The antagonist pretreatment inhibited the effect of NT, while the antagonist itself had no effect. In the OPF test there was no difference among the groups. Our present results show that microinjection of NT into the VP induces anxiolytic effect, which is specific to the NTR1 receptors because it can be eliminated by a specific NTR1 antagonist. It is also substantiated that neither the NT, nor the NTR1 antagonist in the VP influences locomotor activity.

Positive reinforcing effects of RFamide-related peptide-1 in the rat central nucleus of amygdala.

The amygdaloid body (AMY) plays an important role in memory, learning and reward-related processes. RFamide-related peptide-1 (RFRP-1) immunoreactive fibers and NPFF1 receptors were identified in the AMY, and previously we verified that neuropeptide RFRP-1 infused into the central nucleus of AMY (CeA) caused food intake decrease. The aim of the present study was to examine the possible rewarding or aversive effects of RFRP-1 in the CeA. In conditioned place preference, test male Wistar rats were microinjected bilaterally with 50 or 100ng RFRP-1 in volume of 0.4µl. In other groups of animals, 20ng NPFF receptor antagonist RF9 was applied or the antagonist was used 15min before 50ng RFRP-1 treatment. Fifty nanograms of RFRP-1 had positive reinforcing properties, while 100ng RFRP-1 had no effect. Prior treatment with NPFF receptor antagonist RF9 could block the rewarding effects of RFRP-1, while the antagonist applied alone did not influence the behavior of rats in place preference paradigm. Our results show that RFRP-1 and NPFF-1 receptors play important roles in the amygdaloid rewarding-reinforcing mechanisms.

Intraamygdaloid microinjection of RFamide-related peptide-3 decreases food intake in rats.

Some members of the RFamide peptide family are associated with feeding in rodents. For example, neuropeptide FF and prolactin-releasing peptide cause anorexigenic, while 26RFa and QRFP result in orexigenic effects. I.c.v. microinjection of RFamide-related peptide-3 (RFRP-3) facilitates feeding. Feeding related effects of RFRP-3, however, have not been studied after direct brain microinjections in rats. The central part of amygdala (CeA) is essentially involved in the regulation of feeding and body weight. RFRP-3 positive nerve cells were detected in the rat hypothalamus and RFRP-3 immunoreactive fibers were identified in the CeA. RFRP analogs bind with relatively high affinity to the NPFF1 and NPFF2 receptors (NPFF-R). RFRP-3 has potent activity for NPFF-1 that is expressed in the CeA. To evaluate the role of RFRP-3 in feeding regulation rats were microinjected with different doses of RFRP-3 and their food intake were quantified over a 60 min period. Liquid food intake of male Wistar rats was measured after bilateral intraamygdalar administration of RFRP-3 (25, 50, 100 or 200 ng/side, RFRP-3 dissolved in 0.15M sterile NaCl/0.4 µl, respectively). The 50 ng and 100 ng doses of RFRP-3 microinjections resulted in significant decrease of food intake. Twenty-five and 200 ng had no effect. Food intake decreasing effect of RFRP-3 was eliminated by NPFF-R antagonist RF9 pretreatment. In open-field test effective doses of RFRP-3 did not modify spontaneous locomotor activity and general behavior of animals did not change. Our results are the first reporting that RFRP-3 injected to the CeA resulted in a decrease of liquid food consumption. This is a receptor-linked effect because it was eliminated by NPFF-R antagonist.