Research Reports

Amelioration of cognition impairments in the valproic acid-induced animal model of autism by ciproxifan, a histamine H3-receptor antagonist

Taheri, Farahnaz^a; Esmaeilpour, Khadijeh^a,b,*; Sepehri, Gholamreza^a,*; Sheibani, Vahid^a; Shekari, Majid Asadi^a

^aNeuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran

^bPhysics and Astronomy Department, University of Waterloo, Waterloo, Ontario, Canada

Received 18 February 2022 Accepted as revised 2 January 2023.

^*Khadijeh Esmaeilpour and Gholamreza Sepehri contributed equally to the writing of this article.

Correspondence to Khadijeh Esmaeilpour, PhD, University of Waterloo, Waterloo, Ontario, Canada, E-mail: [email protected], [email protected]

Behavioural Pharmacology 34(4):p 179-196, June 2023. | DOI: 10.1097/FBP.0000000000000720

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Abstract

Introduction

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders in the nervous system, which is characterized by difficulties in social interactions, as well as verbal and nonverbal communication, and repetitive behaviors (^{Llaneza et al., 2010}). ASD is the most expensive medical condition in the USA, costing $61 billion annually, which is more than the costs of heart disease, stroke, and cancer combined (^{Buescher et al., 2014}). According to the Centers for Disease Control and Prevention, one in 68 American children suffers from ASD (^{Christensen et al., 2018}), a 10-fold increase over the last 40 years. Based on an epidemiological study, 26.4% of the rising prevalence of autism can be attributed to changes in diagnostic practices (^{King and Bearman, 2009}).

ASD behavioral symptoms typically appear between the ages of 2 and 3 years old and persist throughout life (^{Elder et al., 2017}^;^{Benger et al., 2018}). ASD is primarily diagnosed based on behavioral evaluation and parent questionnaires (^{Hennel et al. 2016}^;^{Elder et al., 2017}), as reliable biomarkers are still in the research phase (^{Varcin and Nelson, 2016}). It appears that genetic (^{De Rubeis et al., 2014}^;^{Hansen et al., 2015}) and environmental (^{Chiarotti and Venerosi, 2020}) factors influence the pathogenesis of ASD. Researchers have found that the maternal use of valproic acid (VPA) during pregnancy increases the risk of autism in humans (^{Ornoy, 2009}) and rodents (^{Sabzalizadeh et al. 2022}^;^{Haratizadeh et al. 2023}).

Animal studies have also reported social interaction deficits (^{Sabzalizadeh et al., 2022}^;^{Taheri et al., 2022}) and increased repetitive behaviors in VPA-exposed animals (^{Baronio et al., 2015}^;^{Eissa et al., 2018b}). Our previous study showed social deficits in adult VPA-exposed rats (^{Taheri et al., 2022}). Additionally, prenatal VPA caused increased anxiety in mice, as indicated by a significant decrease in the number of rearing and the frequency of entries into the center field during an open-field task (^{Yamaguchi et al., 2017}^;^{Gao et al., 2019}).

According to clinical observations, about 75% of individuals with ASD experience learning and memory problems (^{Meador et al., 2009}). The ability to recognize faces and executive functions are particularly impaired in children with ASD, which suggests recognition memory impairment (^{Golshan et al., 2019}^;^{Semino et al., 2021}). Moreover, mice prenatally exposed to VPA exhibited impairments in recognition memory (^{Hara et al., 2017}^;^{Sungur et al., 2017}). The hippocampus plays an important role in learning and memory formation (^{Miguez et al., 2015}). ASD patients (^{Meador et al., 2009}) and the VPA-induced animal model of autism (^{Gao et al., 2016}) showed remarkable changes in the structure of the hippocampus, including apoptosis, pyramidal cell loss, and spine density of pyramidal cells (^{Takuma et al., 2014}^;^{Yang et al., 2016}).

Several studies have focused on the various brain neurotransmitters such as histamine and their influence on behavior in brain disorders (^{Alhusaini et al., 2022}^;^{Taheri et al., 2022}). Histamine plays a significant role in basic physiological functions such as sleep-wake cycles, sensory and motor functions, cognition, and attention, all of which are severely affected in neuropsychiatric disorders (^{Sadek et al., 2016a}).

The H1 receptors (H1Rs), H2 receptors (H2Rs), H3 receptors (H3Rs), and H4 receptors (H4Rs) are responsible for histamine’s functions. Among these receptors, H3Rs are presynaptic autoreceptors that block the synthesis and release of histamine from histaminergic neurons and heteroreceptors that control the release of other neurotransmitters (^{Panula and Nuutinen, 2013}^;^{Panula et al., 2015}). By inhibiting H3Rs, histamine release, as well as the release of other neurotransmitters (^{Panula and Nuutinen, 2013}), is enhanced in the regions of the brain necessary for alertness and information storage (^{Sadek et al., 2016a}^,^2016b^;^{Zlomuzica et al., 2016}).

For disorders such as Alzheimer’s disease and narcolepsy, antagonists targeting H3Rs are considered promising alternative therapies (^{Sadek et al., 2016a}). Various cognitive disorders have been improved by increasing histamine in the synaptic space (^{Zlomuzica et al., 2016}). Additionally, histamine has been shown to control several aspects of behavior and arousal in animal studies (^{Ferreira et al., 2012}^;^{Sadek et al., 2016b}^;^{Eissa et al., 2018c}). H1Rs, H2Rs, and H3Rs are involved in the consolidation of object recognition memory in the histaminergic system (^{da Silveira et al., 2013}). Several diseases such as Alzheimer’s disease, sleep disorders, and autism appear to target the histaminergic system of the brain (^{Haas and Panula, 2003}).

To the best of our knowledge, no documented study has yet investigated the effects of H3R and H2R antagonists and their combination on the histological changes of neurons in the CA1 area of the hippocampus and cognitive performance in the offspring of rats exposed to VPA. As a result, the goal of the current investigation was to determine the impact of the H3R antagonist on hippocampal neuronal alterations and assess cognitive functions in the offspring of rats exposed to VPA. To ascertain the function of H2R in brain histaminergic neurotransmission and its interaction with H3R antagonist ciproxifan (CPX) in cognitive activities, the effects of the H2R antagonist famotidine (FAM) on behavioral and neuronal alterations in the hippocampus were also evaluated.

Methods

Subjects

Wistar rats were used in this experiment. The animals were housed in standard plastic cages in a room maintained at 23 ± 2°C under 12/12-h light-dark cycle. Animals had ad libitum access to food and water. The experimental procedures were conducted in accordance with guidelines relevant to the care of experimental animals, as approved by the Institutional Animal Research Ethics Committee of Kerman University of Medical Sciences (Ethics code: IR.KMU.REC.1400.059).

Pregnancy determination

Male and female rats were mated overnight. Pregnancy was identified by the existence of the spermatozoa in vaginal smear or the presence of vaginal plugs (^{Taheri et al., 2019}) the next morning, and that day was defined as embryonic day 0 (^{Taheri et al., 2018}). Pregnant rats were randomly divided into two groups. VPA: Pregnant rats received an intraperitoneal injection of 600 mg/kg VPA which was diluted with normal saline to a concentration of 200 mg/mL at embryonic day 12.5 (E12.5) (^{Taheri et al., 2022}). Saline: Pregnant rats received an intraperitoneal injection of saline at E12.5.

Drugs

Drugs used in the present study were H3R antagonist (CPX), sodium VPA, and urethane purchased from Sigma-Aldrich, USA. The pharmaceutical company, SMS Life Sciences India Limited, prepared FAM. The drugs were dissolved in normal saline, with the exception of FAM, which was dissolved in one drop of 1 M hydrochloric acid and then diluted in normal saline (^{Taheri et al., 2022}). The solutions were freshly prepared daily. H3R antagonist CPX (1 and 3 mg/kg), H2R antagonist FAM (10, 20, and 40 mg/kg), or vehicle (saline) were administered 30 min before behavioral tests (^{Taheri et al., 2022}).

Experimental groups

Offspring of both groups, VPA and Saline, were divided into seven subgroups according to treatment with Saline, CPX (1 and 3 mg/kg), FAM (10, 20, and 40 mg/kg), and a combination of effective doses of CPX and FAM (CPX3+ FAM20) (Fig. 1). One pup from each litter was used in each treatment group and totally, 98 male rats were tested during adolescence [postnatal day (PND) 48–50], and the sample size was n = 7 per condition. The timeline is shown in Fig. 2. The experiments of the current study [marble burying task (MBT), open field, novel object recognition (NOR), and Passive avoidance tests] were carried out between 8:00 a.m. and 1 p.m. in 1 day.

Fig. 1:
Animal groups arrangement. CPX, ciproxifan; FAM, famotidine; VPA, valproic acid.

Fig. 2:
Timeline diagram. VPA, valproic acid.

Behavioral assessments

Marble-burying test

MBT is an accurate reflection of repetitive digging behavior (^{Thomas et al., 2009}). Briefly, cages (26 cm × 48 cm × 20 cm) were filled with fresh, unscented rat bedding material to a depth of 5 cm, and the bedding surface was leveled by placing another cage of the same size onto the surface of the bedding. For habituation, each rat was added individually. The rat was removed after 10 min, and 20 glass marbles (15 mm diameter) were arranged equidistantly in a 4 × 5 arrangement on the bedding surface. For 30 min, each rat was allowed to explore its designated test cage. We recorded the number of marbles buried (more than 50% of marble was covered by the bedding) (^{Kim et al., 2014}). During this test, the number of marbles buried serves as a proxy measure of rat digging behavior.

Open-field test

The open-field test is used to evaluate exploratory, locomotor activity, and anxiety-like behaviors as well as repetitive behaviors in rodents (^{Servadio et al., 2015}). The used apparatus was made up of Plexiglas (90 × 90 × 50 cm) with high walls and a dark floor. The box was divided into 16 small squares by imaginary lines in the Noldus Ethovision system; therefore, the mice could spend time in both peripheral and central areas. The animal was gently placed in the center of the apparatus and its locomotor and anxiety-like behaviors were recorded for 5 min using an automatic video camera fixed above the apparatus. Total movement distance and the velocity of the movement were measured to evaluate locomotor activity. In order to evaluate anxiety-like behavior, the time spent in the inner zone (central area) was measured (Fig. 3). Furthermore, the open-field test can be used to determine the amount of repetitive and monotonous activities (^{Servadio et al., 2015}). Animal movements, such as wiping and licking the face and body, were recorded continuously and consecutively (^{Malkova et al., 2012}). This level of movement was considered repetitive and monotonous in an animal model of autism (^{McFarlane et al., 2008}). It has been shown that many animal models of autism exhibit stereotypical and repetitive movement parameters as well as anxiety-like behaviors in an open field (^{Moy et al., 2008}). To avoid an odor signature, the apparatus was cleaned with 70% ethanol, and the test was performed in a light-controlled environment. A video camera on top of the field recorded these elements, and the animal’s performance was recorded by the Noldus Ethovision system, version 7.1, Noldus Company, Netherland. The number of grooming behavior was recorded by the experimenter (^{Taheri et al., 2020}).

Novel object recognition test

The NOR task evaluates the rodents’ ability to recognize a novel object in the environment (Fig. 4). The task procedure consists of three phases: habituation, familiarization (training), and test phases. In the habituation phase, each rat is allowed to freely explore an empty wooden (60 × 60 × 40 cm) arena for 10 min. Twenty-four hours after the habituation, animals were allowed to explore two identical objects, at the same location inside the box for 5 min (training phase) (Fig. 4a). After a retention interval of 15 min, during 3-min test phase, rat is returned to arena with two objects, one is familiar and other is novel (Fig. 4b).

The location of novel versus familiar objects is counterbalanced between trials. The rats spend more time exploring the novel object during the first few minutes of the test phase, and when this bias is observed, the rats can recall the sample object (^{Sivakumaran et al., 2018}). In the training and test phases, animals that did not explore any of the objects were excluded from the analysis (^{Languille et al., 2015}).

Objects were randomly changed to avoid the natural preference of animals and both objects and boxes were cleaned with 70% ethanol before the trial. During the test phase, sniffing or touching the object at a distance of less than 2 cm was quantified by the camera. Finally, a discrimination ratio (recognition index) was defined as the ratio of time spent exploring each object to the total time spent exploring both objects multiplied by 100 in the training and test phases (^{Taheri et al., 2020}).

Passive avoidance test

The Passive avoidance task is a fear-aggravated test used to evaluate associative learning and memory in rodents. The animal learns to avoid an environment in which a prior aversive stimulus has been delivered. Shuttle-box device with dimensions of 100 [L] × 25[W] × 25[H] (cm) consisting of two compartments (light and dark) separated by a door was used. Each animal was first habituated to the test equipment by placing it in the light compartment for 5 min, the animal was allowed to move to the dark chapter, and then it was returned to the home cage. This process was repeated once and if an animal failed to move into the dark compartment, it was removed from the study. Finally, 2 h later, the animal was placed into the light compartment, the door was opened after 10 s and, on entering the dark compartment, it was given an electric shock (50 Hz, 0.5 mA, 2 s; via wires embedded in the dark chamber floor). This final part of the process was repeated at 2 min intervals until the animal learned to avoid the dark compartment (remained in the light compartment for at least 120 s) and the number of shocks required for learning was recorded. The assessment phase of the test was undertaken 24 h after the learning phase. The animal was placed in the light chamber (door closed) and, after 10 s, the door was opened, and the time taken by the animal to enter the dark chamber was recorded as the step-through latency (STL). The total time spent in the dark compartment during a period of 5 min after door opening was also recorded (^{Rehman et al., 2020}).

The behavioral tests were performed 30 min after the injection of saline or different doses of CPX and FAM.

Histology: hematoxylin and eosin

After the end of the behavioral tests, all of the groups were anesthetized with carbon dioxide and then sacrificed. The brain was immediately fixed in 10% buffered formalin and processed according to a histological method. Paraffinized brain tissues were cut into 8-μm sections using a semi-automated microtome, and the sections were stained with hematoxylin and eosin (Sigma, Missouri, USA). For assessment of neuronal counting, stained sections of the hippocampal CA1 area were evaluated. Neurons were counted in three microscopic fields at 400X magnification, and neurons with visible round nucleus, prominent nucleolus, and intact cytoplasm were counted as intact neurons.

Statistical analysis

To evaluate the effect of H3 antagonist on measured parameters, two-way analysis of variance (ANOVA) was used to compare the mean differences between groups in behavioral tests (MBT, open field, NOR, and Passive avoidance). To analyze the histological results, one-way ANOVA was used. If there was statistical significance between the groups, Turkey’s multiple-comparison was performed as a post-hoc. The data were presented as mean ± SEM and differences among groups were considered significant at a P < 0.05. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, California, USA).

Results

Effect of valproic acid on crooked-tail phenotype

Genetic mutations, malnutrition, and teratogen exposure during gestation can cause neural tube defects (NTD) (^{Kaufman, 2004}). Previous studies determined that VPA-induced crooked tail phenotypes, a mild form of NTD, and autistic-like behavior impairments in mice and rats.

(^{Ornoy, 2009}^;^{Kim et al., 2011}). VPA induced the crooked-tail phenotype in all rats in the current study (Fig. 5).

Fig. 5:
Neural tube defects by VPA exposure. Malformations of tail structure occurred in the VPA-exposed group. VPA, valproic acid.

Stereotyped repetitive behavior test

Effects of H3 antagonist (ciproxifan) on stereotyped repetitive behavior in valproic acid-exposed rats in marble-burying test

The MBT test was used to evaluate repetitive behavior. MBT behavioral is an accurate reflection of repetitive digging behavior. Two-way ANOVA showed a significant difference among groups in repetitive behavior [F (2, 36) = 4.372]. In the VPA + Saline group, repetitive behaviors were significantly higher than in the Saline + Saline group. Indeed, the VPA + Saline group buried significantly more marbles than the Saline + Saline group (P < 0.01). In the VPA + CPX3 group, CPX3 treatment significantly decreased the elevated percentage of marbles buried compared to the VPA + Saline group (P < 0.01) (Fig. 6a).

Fig. 6:
Effects of CPX, FAM, and combination of (CPX and FAM) on stereotyped repetitive behavior in the VPA-exposed rats in MBT. (a) VPA-exposed rats buried significantly more marbles compared to the saline-exposed rats. After treatment with CPX, the elevated percentage of marbles buried significantly decreased in the VPA + CPX3 group compared to the VPA + Saline group. (b) FAM had no effect on the percentage of buried marbles. (c) Combination of CPX and FAM had no effect on the percentage of buried marbles. Two-way ANOVA was used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05,**P < 0.01 vs. Saline + Saline, ##P < 0.01 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.

Effects of H2 antagonist (famotidine) on stereotyped repetitive behavior in valproic acid-exposed rats in marble-burying test

We found no significant difference between the VPA + Saline group and the VPA + FAM10, VPA + FAM20, and VPA + FAM40 groups in the MBT test. FAM did not affect the percentage of marbles buried (Fig. 6b).

Effects of combination of H2 H3 antagonists (ciproxifan and famotidine) on stereotyped repetitive behavior in valproic acid-exposed rats in marble burying task

ANOVA analysis showed no significant difference among groups in the percentage of marbles buried after treatment with CPX and FAM (Fig. 6c).